Exploiting Bacterial Pathways for BBB Crossing with PLGA Nanoparticles Modified with a Mutated Form of Diphtheria Toxin (CRM197): In Vivo Experiments.

Drugs can be targeted to the brain using polymeric nanoparticles (NPs) engineered on their surface with ligands able to allow crossing of the blood-brain barrier (BBB). This article aims to investigate the BBB crossing efficiency of polymeric poly lactide-co-glycolide (PLGA) NPs modified with a mutated form of diphtheria toxin (CRM197) in comparison with the results previously obtained using PLGA NPs modified with a glycopeptide (g7-NPs). Different kinds of NPs, covalently coupled PLGA with different fluorescent probes (DY405, rhodamine-B base and DY675) and different ligands (g7 and CRM197) were tested in vivo to assess their behavior and trafficking. The results highlighted the possibility to distinguish the different kinds of simultaneously administered NPs and to emphasize that CRM-197 modified NPs and g7-NPs can cross the BBB at a similar extent. The analysis of BBB crossing and of the neuronal tropism of CRM197 modified NPs, along with their BBB crossing pathways were also developed. In vivo pharmacological studies performed on CRM197 engineered NPs, loaded with loperamide, underlined their ability as drug carriers to the CNS.

Anticancer drug-loaded quantum dots engineered polymeric nanoparticles: Diagnosis/therapy combined approach.

Primary Effusion Lymphoma (PEL) is an HHV-8-related non Hodgkin lymphoma localized in body cavities (as pleural, peritoneal and pericardial) presenting lymphomatous effusion that, until now, lack of an effective therapy. Curcumin was reported to display pro-apoptotic effect via the inhibition of the JAK/STAT pathway, that is overexpressed in PEL cells, as consequence of virus infection. The administration of curcumin is severely restricted by its physicochemical properties, mainly its low solubility in biological fluid and consequently low bioavailability. Encapsulation into biocompatible and biodegradable PLGA nanoparticles (NPs) could be a strategy to overcome biological limits of curcumin, offering a valuable step forward for its clinical application. In this study we described single-emulsion process for curcumin loading into NPs (encapsulation efficiency about 35%). We applied a post-formulation strategy (NHS/EDC reaction) to decorate the surface of the curcumin-loaded NPs with quantum dots (QDs) as imaging agents (QDs-NPs-Cur, 24pmol of QDs per 100mg of NPs) obtaining tools useful for possible application in theranostic approach. Bifunctionalized NPs were tested in vitro on two PEL's cell line (BCBL-1 and HBL-6). The efficacy of the treatment was evaluated by cytofluorimetric assay by measuring both cell viability and cell density. We found that the NPs significantly improve the cellular effect of curcumin (respect to free drug). Moreover, by means of confocal microscopy, both the localization of bifunctional NPs and of the released drug were easily detectable. Thus, we conclude that the delivery of curcumin using bifunctional traceable NPs is a promising future approach for the diagnosis and the treatment of PEL.

Apoferritin nanocage as streptomycin drug reservoir: Technological optimization of a new drug delivery system.

The aim of this study is to formulate and characterize streptomycin-loaded apoferritin nanoparticles (ApoStrep NPs) for their potential therapeutic use in bacterial resistant infections (i.e. tuberculosis). ApoStrep NPs were prepared by disassembly/reassembly process via pH method and changing apoferritin/drug molar ratio, purified by dialyses process also associated with gel filtration chromatography and characterized in their chemico-physical and technological parameters as yield, size distribution, polidispersivity, morphology, internal structure, zeta potential and loading efficacy. The results showed that spherical reproducible NPs could be obtained by using apoferritin/drug molar ratio lower than 1:25 and purification based on the combination of dialysis and gel filtration chromatography. Photon correlation spectroscopy, Uv-visible detection and electron microscopy showed the maintenance of the native apoferritin chemico-physical properties and structure. When formulated with apoferritin/drug 1:10 and 1:25 molar ratio, ApoStrep NPs showed remarkable encapsulation efficacy (35% and 28%, respectively) along with kinetic profile of drug delivery, approximately 15% at 37 °C in 72h, as evidenced by "in vitro" release experiments.

Electrocardiographic and pathological studies of the heart in experimental guinea pig leukemia.

Electrocardiographic tracings were performed on 26 strain 2 guinea pigs in which leukemia was induced by inoculation of L2C leukemic cells. All inoculated animals developed leukemia. In terminal phases of this disease significant electrocardiographic changes were observed in 20 of the 26 animals; in 5 animals the electrocardiograms were normal and in 1 guinea pig the changes were borderline. The most significant changes consisted of the onset of a Q-wave or a T-wave inversion or both. Pathological examination of the heart removed in the terminal phase of the disease revealed infiltration of the endocardium and epicardium and in the capillaries of the myocardium. Such areas of infiltration, when viewed with the electron microscope, revealed the presence of leukemic cells within the lumen of capillaries and in areas immediately surrounding the capillaries. Infiltration of myocardial fibers was not observed. The electrocardiographic changes observed in guinea pig leukemia may be related to the leukemic infiltration of myocardial capillaries and the resulting anoxia.

EXPLOITING THE VERSATILITY OF CHOLESTEROL IN NANOPARTICLES FORMULATION.

The biocompatibility of polymers, lipids and surfactants used to formulate is crucial for the safe and sustainable development of nanocarriers (nanoparticles, liposomes, micelles, and other nanocarriers). In this study, Cholesterol (Chol), a typical biocompatible component of liposomal systems, was formulated in Chol-based solid nanoparticles (NPs) stabilized by the action of surfactant and without the help of any other formulative component. Parameters as type (Solutol HS 15, cholic acid sodium salt, poly vinyl alcohol and Pluronic-F68), concentration (0.2; 0.5 and 1% w/v) of surfactant and working temperature (r.t. and 45°C) were optimized and all samples characterized in terms of size, zeta potential, composition, thermal behavior and structure. Results demonstrated that only Pluronic-F68 (0.5% w/v) favors the organization of Chol chains in structured NPs with mean diameter less than 400nm. Moreover, we demonstrated the pivotal role of working temperature on surfactant aggregation state/architecture/stability of Chol-based nanoparticles. At room temperature, Pluronic-F68 exists in solution as individual coils. In this condition, nanoprecipitation of Chol formed the less stable NPs with a 14±3% (w/w) of Pluronic-F68 prevalently on surface (NP-Chol/0.5). On the contrary, working near the critical micelle temperature (CMT) of surfactant (45°C), Chol precipitates with Pluronic-F68 (9±5% w/w) in a compact stable matricial structure (NP-Chol/0.5-45). In vitro studies highlight the low toxicity and the affinity of NP-Chol/0.5-45 for neuronal cells suggesting their potential applicability in pathologies with a demonstrated alteration of neuronal plasticity and synaptic communication (i.e. Huntington's disease).

Nutlin-3 loaded nanocarriers: Preparation, characterization and in vitro antineoplastic effect against primary effusion lymphoma.

In this investigation, Nutlin-3 (Nut3), a novel antitumor drug with low water solubility (<0.1mg/L at 25°C), was loaded into liposomes (Lipo-Nut3), polymeric nanoparticles (NPs-Nut3) and nanoparticles engineered with an antibody direct against Syndecan-1/CD 138 (Syn-NPs-Nut3) to obtain carriers targeted to PEL (primary effusion lymphoma). The physicochemical properties of these carriers were determined. Atomic force microscopy showed that all the particles were well formed and spherical in shape. The presence of the antibody on surface led to a significant increase of mean diameter (280 ± 63 nm), PDI (0.3) and the shift of zeta potential towards neutrality (-1 mV). The entrapment efficiency of Lipo-Nut3, NPs-Nut3 and Syn-NPs-Nut3 was 30, 52 and 29%, and drug loading was 1.4, 4.5 and 2.6%, respectively. By performing cytofluorimetric analyses and bromodeoxyuridine (BrdU) assay, the efficacy of nanocarriers to deliver the antineoplastic drug into a PEL cell line namely BCBL-1 (immortalized body cavity B-cell lymphoma) was investigated. Two days after the treatment with 20 µM of Syn-NPs-Nut3, the cell density decreased at about 60% while the cell viability decreased at 56% only 5 days after transfection, when compared with untreated cells. A cell cycle arrest was observed with a significant decrease of cells in S-phase and increasing of apoptotic cell, if compared with untreated control. These results confirms the potential of nanocarriers approaches to deliver antitumor drug with unfavorable chemico-physical properties. Moreover, this study strongly suggests that Syn-NPs-Nut3 can be a valuable drug carrier system for the treatment of PEL lymphoma.

Serological screening for the prevention of transfusion-acquired cytomegalovirus infection.

The presence of antibodies against cytomegalovirus (CMV)-induced immediate-early antigens (IEA), early antigens (EA) and late antigens (LA) was sought in 500 healthy blood donors. Antibodies to late antigens were detected in 76% and antibodies to immediate-early and early antigens were detected in 9.6% and 10.2%, respectively. The incidence of antibodies to CMV-induced immediate-early and early antigens increased with age, was higher in females than in males and was related to high values for antibodies against CMV late antigens. Since antibodies to CMV immediate-early and early antigens are significantly associated with active CMV infection, criteria for choosing blood samples unlikely to transmit infective CMV are evaluated.

QED v 1.0: a software package for quantitative electron diffraction data treatment.

A new software package for quantitative electron diffraction data treatment of unknown structures is described. No "a priori" information is required by the package which is able to perform in successive steps the 2-D indexing of digitised diffraction patterns, the extraction of the intensity of the collected reflections and the 3-D indexing of all recorded patterns, giving as results the lattice parameters of the investigated structure and a series of data files (one for each diffraction pattern) containing the measured intensities and the relative e.s.d.s of the 3-D indexed reflections. The software package is mainly conceived for the treatment of diffraction patterns taken with a Gatan CCD Slow-Scan Camera, but it can also deal with generic digitised plates. The program is designed to extract intensity data suitable for structure solution techniques in electron crystallography. The integration routine is optimised for a correct background evaluation, a necessary condition to deal with weak spots of irregular shape and an intensity just above the background.

Functional assessment of cellular non-specific and specific immunity in selected healthy elderly.

Thirteen healthy elderly were selected according to a simplified SENIEUR admission protocol including clinical, hematological and biochemical parameters. The goal of this protocol was to limit the influence of diseases and/or medications on the assessment of immune functions in the elderly. Plasma zinc levels of healthy elderly were comparable to those of young subjects. Cellular nonspecific immunity was determined by measuring chemiluminescence (CL) of peripheral blood granulocytes activated by opsonized zymosan particles. CL of granulocytes from healthy elderly was delayed in comparison to that of young controls when autologous serum was used. Lymphocyte proliferation induced by phytohemagglutinin-P (PHA-P) or zinc chloride (ZnCl(2)) in a serum free medium was lower in the elderly than in young controls. Preincubation of lymphocytes with ZnCl(2) before PHA-P stimulation did not restore the impaired proliferative activity of cells from old donors.

Nanotechnology and Alzheimer's disease: what has been done and what to do.

Up to date, Alzheimer's Disease (AD) is considered as an "urgency" for public health, since it represents one of the most dramatic causes of death in adults. The drugs currently used for AD are only symptomatic, thus not curing the pathology, but only trying to slow or delay the progression of the pathology. Moreover, there is a total lack of early identification, with only "probable'' or ''possible'' diagnosis of AD patients. With this review, we aimed to individuate and to highlight the most promising approaches for AD therapy and diagnosis. In this view, at the cutting-edge of innovation, nanocarriers as polymeric nanoparticles, liposomes, nanoassembly and dendrimers, have been studied and investigated in order to ameliorate the detection (in vitro and in vivo) and/or the therapeutic options in AD. In this review, the most outstanding nanomedicine-driven approaches in AD imaging/detection and treatments are summarized in order to help in individuating values and criticisms. Moreover, an overview of one of the most innovative strategies in AD management, namely theranostic nanomedicine, is reported and commented.

Neurotrophic factors and neurodegenerative diseases: a delivery issue.

Neurotrophic factors (NTFs) represent one of the most stimulating challenge in neurodegenerative diseases, due to their potential in neurorestoring and neuroprotection. Despite the large number of proofs-of-concept and evidences of their activity, most of the clinical trials, mainly regarding Parkinson's disease and Alzheimer's disease, demonstrated several failures of the therapeutic intervention. A large number of researches were conducted on this hot topic of neuroscience, clearly evidencing the advantages of NTF approach, but evidencing the major limitations in its application. The inability in crossing the blood-brain barrier and the lack of selectivity actually represent some of the most highlighted limits of NTFs-based therapy. In this review, beside an overview of NTF activity versus the main neuropathological disorders, a summary of the most relevant approaches, from invasive to noninvasive strategies, applied for improving NTF delivery to the central nervous systems is critically considered and evaluated.

Cidofovir-loaded liposomes: an intro-study using BCBL-1 cell line as a model for primary effusion lymphoma.

Cidofovir (HPMPC) was recently reported to exert a valuable antineoplastic activity against primary effusion lymphoma (PEL), a B-cell neoplasm associated with Human Herpesvirus-8 (HHV-8) infection. In this study, we developed and characterized liposomes encapsulating HPMPC to increase drug efficacy reducing the administered dose and the related toxicity, which actually hamper its clinical therapeutic use in patients affected with PEL. The liposomes, obtained using different formulations of neutral and cationic lipids, were analyzed by microscopical (AFM) and spectroscopical (PCS and NMR) techniques. Using an in vitro model of PEL (BCBL-1 cell line), the carrier toxicity and the antineoplastic efficacy of liposomes were evaluated by flow cytometry applying apoptosis and cell death analysis. The in vitro study showed the applicability of the liposomes within a restricted range of lipidic concentrations according to the lipids used during the preparation. The moderate increases in the percentage of apoptotic/necrotic cells suggests that liposomal delivery allows the release of HPMPC into BCBL-1 cells enabling an unexpected antineoplastic activity of this drug even at lower doses.

Flow cytometry: a tool in immunohematology for D+W (Du) antigen evaluation?

We performed a flow-cytometric analysis of the expression of D antigen in D+W samples (previously termed Du). We also analysed a series of D-positive and D-negative (cde phenotype) samples to obtain positive and negative controls, respectively. The evaluation was carried out by immunofluorescence and the intensity of positivity was expressed as mean channel value (MCV) of fluorescence. Results demonstrated that D+W samples have lower expression (less than 1 log) than D-positive cases (p > 0.001, Student t test), while cde samples show the same MCVs as negative controls. Moreover, it was also possible to set a grading of D antigen expression and to analyze cases difficult to assess by agglutination only.

Synthesis and structure of new bronchospasmolytic agents. I.

The crystal structures of two phenylethanolamines showing bronchospasmolytic activity have been determined at room temperature [293 (2) K]. Crystal data are as follows: 11-morpholinotricyclo[6.3.0.0(2,7)]undeca-2,4,6-trien-l-ol (3), C15H19NO2, M(r) = 245.3, triclinic, P1, a = 10.360 (5), b = 12.169 (5), c = 12.488 (4) A, alpha = 95.14 (10), beta = 108.49 (12), gamma = 114.69 (5) degrees, V = 1311 (2) A3, Z = 4, Dx = 1.243 Mg m-3, Cu K alpha 1 radiation, lambda = 1.540562 A, mu = 0.618 mm-1, F(000) = 528, R = 0.0537 for 3009 observed reflections; 4-morpholino-1,2-benzocyclononen-3-ol monohydrate (5), C17H25NO2.H2O, M(r) = 293.4, monoclinic, P2(1)/c, a = 10.063 (9), b = 19.398 (5), c = 8.670 (5) A, beta = 110.56 (1) degree, V = 1585 (2) A3, Z = 4, Dx = 1.230 Mg m-3, Mo K alpha 1 radiation, lambda = 0.709300 A, mu = 0.0778 mm-1, F(000) = 640, R = 0.0376 for 1407 observed reflections. The stereochemistry of compound (3) is found to be 'all cis', which allows the mechanism of formation of these compounds to be interpreted. The various aspects of the conformations of these molecules are discussed.

Cell adhesion molecules CD11a and CD18 in blood monocytes in old age and the consequences for immunological dysfunction. Preliminary results.

Adhesion molecules, such as leukocyte-function-associated antigen (LFA-1 or CD11a/CD18), intercellular adhesion molecule 1 (ICAM-1 or CD54) and Hermes antigen (HCAM or CD44), have important roles in many adhesive interactions involving cells of the immune system. Since it has been shown that many immunological alterations were present in aged subjects, we studied the expression and density of these molecules on peripheral blood lymphocytes and monocytes from healthy old subjects. A decrease in monocyte subpopulations bearing CD11a/CD18 and an increase in CD11a/CD18 and and CD44 antigen density on lymphocytes and on monocytes, respectively, were observed. These changes might be an event in the mechanism leading to the decreased lymphocyte proliferative response in vitro and to other immunological dysfunctions reported in old subjects.

Analysis of natural killer cells in patients with idiopathic autoimmune hemolytic anemia.

A functional and phenotypic analysis of the circulating natural killer (NK) cell population was carried out in 9 patients with idiopathic autoimmune hemolytic anemia (IAHA). The NK-cell activity, assessed by a sensitive method based on the inhibition of target clone growth in plasma semisolid medium, was markedly decreased in all patients as compared with normal controls and was not restored by stimulation of the cells with recombinant alpha interferon (alpha-IFN). Analysis of peripheral blood phenotypic markers showed that cells bearing Leu7 and CD16 antigens numbered in the normal range. These findings suggest that IAHA patients exhibit a functional impairment of the NK compartment.

Mixed lymphocyte reactions evaluated by means of bromodeoxyuridine incorporation.

The mixed lymphocyte reactions are usually performed by the uptake of 3H thymidine (3H TdR) in the study of histocompatibility for allogeneic bone marrow transplantation. Bromodeoxyuridine (BrdUrd), an analogue of thymidine, can be used as an alternative marker of proliferation. In this study we compared the evaluation of the proliferative activity of alloreactive lymphocytes in MLR by 3H TdR and BrdUrd incorporation in flow cytometry. The results show that BrdUrd is able to recognize proliferative activity at least 24-48 hours before 3H TdR, and allows discrimination of proliferation of a few cells despite its proximity to the autologous controls. However, the large quantity of cells needed for every combination is presently a disadvantage of the method.

Detection of platelet-associated antibodies by flow cytometry in hematological autoimmune disorders.

We describe our experience in the evaluation of platelet-associated immunoglobulins (PAIg) by flow cytometry in comparison to solid-phase assay in patients affected by idiopathic thrombocytopenic purpura and by Evans syndrome. Results show that the analysis of PAIg by flow cytometry is easy and reliable and correlates well with data obtained by the solid-phase technique. In addition, flow cytometry allows the evaluation of samples containing small numbers of platelets (< 20,000/mm3); the analysis is objective, not influenced by personal experience. Moreover, flow cytometry appears simple enough to be performed in a routine laboratory, and data might be retrieved to perform batch analysis. Our results appear to indicate that PAIg flow cytometry might be a sensitive tool for the evaluation of patients with autoimmune thrombocytopenia.