Evaluation of cellular uptake and intracellular trafficking as determining factors of gene expression for amino acid-substituted gemini surfactant-based DNA nanoparticles

<p>Abstract</p> <p>Background</p> <p>Gene transfer using non-viral vectors offers a non-immunogenic and safe method of gene delivery. Cellular uptake and intracellular trafficking of the nanoparticles can impact on the transfection efficiency of these vectors. Therefore, understanding the physicochemical properties that may influence the cellular uptake and the intracellular trafficking can aid the design of more efficient non-viral gene delivery systems. Recently, we developed novel amino acid-substituted gemini surfactants that showed higher transfection efficiency than their parent compound. In this study, we evaluated the mechanism of cellular uptake of the plasmid/gemini surfactant/helper lipid nanoparticles and their effect on the transfection efficiency.</p> <p>Results</p> <p>Nanoparticles were incubated with Sf 1 Ep cells in the presence of different endocytic inhibitors and gene expression (interferon-γ) was measured using ELISA. Clathrin-mediated and caveolae-mediated uptake were found to be equally contributing to cellular internalization of both P/12-7NH-12/L (parent gemini surfactant) and P/12-7NGK-12/L (amino acid-substituted gemini surfactant) nanoparticles. The plasmid and the helper lipid were fluorescently tagged to track the nanoparticles inside the cells, using confocal laser scanning microscopy. Transmission electron microscopy images showed that the P/12-7NGK-12/L particles were cylindrical while the P/12-7NH-12/L particles were spherical which may influence the cellular uptake behaviour of these particles. Dye exclusion assay and pH-titration of the nanoparticles suggested that high buffering capacity, pH-dependent increase in particle size and balanced DNA binding properties may be contributing to a more efficient endosomal escape of P/12-7NGK-12/L compared to the P/12-7NH-12/L nanoparticles, leading to higher gene expression.</p> <p>Conclusion</p> <p>Amino-acid substitution in the spacer of gemini surfactant did not alter the cellular uptake pathway, showing similar pattern to the unsubstituted parent gemini surfactant. Glycyl-lysine substitution in the gemini spacer improved buffering capacity and imparted a pH-dependent increase of particle size. This property conferred to the P/12-7NGK-12/L nanoparticles the ability to escape efficiently from clathrin-mediated endosomes. Balanced binding properties (protection and release) of the 12-7NGK-12 in the presence of polyanions could contribute to the facile release of the nanoparticles internalized via caveolae-mediated uptake. A more efficient endosomal escape of the P/12-7NGK-12/L nanoparticles lead to higher gene expression compared to the parent gemini surfactant.</p

Design and Evaluation of Gemini Surfactant-Based Lipoplexes Modified with Cell-Binding Peptide for Targeted Gene Therapy

Purpose Achieving successful gene therapy requires delivery of a gene vector specifically to the targeted tissue with efficient expression and a good safety profile. The objective of this work was to develop, characterize and determine if a novel gemini surfactant-based lipoplex systems, modified with a cancer-targeting peptide p18-4, could serve this role. Methods The targeting peptide p18-4 was either chemically coupled to a gemini surfactant backbone or physically co-formulated with the lipoplexes. The influence of targeting ligand and formulation strategies on essential physicochemical properties of the lipoplexes was evaluated by dynamic light scattering and small angle X-ray scattering techniques. In vitro transfection activity and cellular toxicity of lipoplexes were assessed in a model human melanoma cell line. Results All lipoplexes zeta potential and particle size were optimal for cellular uptake and physical stability of the system. The lipoplexes adopted an inverted-hexagonal lipid arrangement. The lipoplexes modified with the peptide showed no significant changes in physicochemical properties or lipoplex assembly. The modification of the lipoplexes with the targeting peptide significantly enhanced protein expression 2-6 fold compared to non-modified lipoplexes. In addition, p18-4 modified lipoplexes significantly improved the safety of the lipoplexes. The ability of the p18-4 modified lipoplexes to selectively express the model protein was confirmed by using healthy human epidermal keratinocytes (HEKa). Conclusion The gemini surfactant-based lipoplexes modified with p18-4 peptide showed significantly higher efficiency and safety compared to the system that did not contain a cancer targeting peptide and provided evidence for their potential application to achieve targeted melanoma gene therapy

Computed Tomography Imaging of Primary Lung Cancer in Mice Using a Liposomal-Iodinated Contrast Agent

To investigate the utility of a liposomal-iodinated nanoparticle contrast agent and computed tomography (CT) imaging for characterization of primary nodules in genetically engineered mouse models of non-small cell lung cancer.Primary lung cancers with mutations in K-ras alone (Kras(LA1)) or in combination with p53 (LSL-Kras(G12D);p53(FL/FL)) were generated. A liposomal-iodine contrast agent containing 120 mg Iodine/mL was administered systemically at a dose of 16 µl/gm body weight. Longitudinal micro-CT imaging with cardio-respiratory gating was performed pre-contrast and at 0 hr, day 3, and day 7 post-contrast administration. CT-derived nodule sizes were used to assess tumor growth. Signal attenuation was measured in individual nodules to study dynamic enhancement of lung nodules.A good correlation was seen between volume and diameter-based assessment of nodules (R(2)>0.8) for both lung cancer models. The LSL-Kras(G12D);p53(FL/FL) model showed rapid growth as demonstrated by systemically higher volume changes compared to the lung nodules in Kras(LA1) mice (p<0.05). Early phase imaging using the nanoparticle contrast agent enabled visualization of nodule blood supply. Delayed-phase imaging demonstrated significant differential signal enhancement in the lung nodules of LSL-Kras(G12D);p53(FL/FL) mice compared to nodules in Kras(LA1) mice (p<0.05) indicating higher uptake and accumulation of the nanoparticle contrast agent in rapidly growing nodules.The nanoparticle iodinated contrast agent enabled visualization of blood supply to the nodules during the early-phase imaging. Delayed-phase imaging enabled characterization of slow growing and rapidly growing nodules based on signal enhancement. The use of this agent could facilitate early detection and diagnosis of pulmonary lesions as well as have implications on treatment response and monitoring

Quality control of plant food supplements

It is essential to guarantee the safety of unprocessed plants and food supplements if consumers' health is to be protected. Although botanicals and their preparations are regulated at EU level, at least in part, there is still considerable discretion at national level, and Member States may choose to classify a product either as a food supplement or as a drug. Accurate data concerning the finished products and the plant used as the starting point are of major importance if risks and safety are to be properly assessed, but in addition standardized criteria for herbal preparation must be laid down and respected by researchers and manufacturers. Physiologically active as well as potentially toxic constituents need to be identified, and suitable analytical methods for their measurement specified, particularly in view of the increasing incidence of economically motivated adulteration of herbal raw materials and extracts. It remains the duty of food operators to keep up with the scientific literature and to provide sufficient information to enable the adaptation of specifications, sampling schemes and analytical methods to a fast-changing environment

Primary and secondary prevention to effectively reduce the risk of bisphosphonate-related osteonecrosis of the jaw in patients with bone metastases .

Background Bone is one of the most frequent sites of metastasis in patients with advanced cancer. Nearly all patients with myeloma, 65–75% of patients with prostate or breast cancer, and 30–40% of patients with lung cancer or other solid tumors, eventually develop bone metastases. Bisphosphonates (BP), particularly zoledronic acid and denosumab, were demonstrated to effectively reduce skeletal complications in patients with bone metastases. However, bisphosphonate-related osteonecrosis of the jaw (BRONJ) can occur spontaneously, favored by dental extraction, dental implant surgery, or denture wearing. The purpose of this study was to underline the role of dental prevention as an effective tool to reduce the risk of BRONJ. Material and methods BRONJ was identified with the standardized query “osteonecrosis” among all data from patients treated at Modena Cancer Center from 2005 to 2016. For each case, demographic and medical information were analyzed, as well as data about notification (year of occurrence, outcome), type and duration of BP exposure, and associated risk factors (dento-alveolar surgery, chemotherapy, antiangiogenics). Data were differently analyzed taking into account the implementation of a Dental Prevention Service in patients who are candidates for BP therapy.Results Among 1663 patients treated with BP, 63 cases of BRONJ were identified (3.8%). 44 female and 19 men with a median age of 69 years (range 47-90 years), have been treated with BP for bone metastases from breast cancer (54%), hematologic malignancy (21%), prostate cancer (13%), renal cancer (5%), lung cancer (2%) and other tumors (5%). 15 maxillae and 48 mandibles were involved. The trigger event was a dental extraction in 29% of the cases, being spontaneously the other 71%. The median time to BRONJ was 28 months (range 1-89.1 months) from the first dose of BP, and 25 was the mean number of BP doses administered before BRONJ. Overall, a preliminary odontoiatric evaluation was performed in only 14 cases (22%). All but one of these dentistry opinions were obtained after 2010 when the Dental Prevention Service was created, which is a drop out of the risk of BRONJ from 4.1 to 1.9%. Conclusions. Prevention of the BRONJ is critical in in bone metastatic patients. The incidence of BRONJ over time can drop to 1.9% when primary and secondary prevention measures are implemented in routine clinical practice

Preterm birth: causes and consequences

Department of Obstetrics and Gynecology, Nicolae Testemitanu State Medical and Pharmaceutical University, Congresul III al Medicilor de Familie din Republica Moldova 17–18 mai, 2012, Chişinău, Republica MoldovaMajor importance is now given to prematurity, this explains still high incidence of preterm birth worldwide, which on average is 10%. Our aim was to assess risk factors of preterm birth complicated by abruptio placentae and postpartum endometritis occurred during 2010 in our clinic. The article was based on analysis of 190 medical records with preterm birth. The study established maternal risk factors of preterm birth: spontaneous and medical abortions, previous premature births and non-specific genital infections and others. As risk factors for development of abruptio placentae in preterm birth were designated: high blood pressure, extreme values of age, premature rupture of membranes, multiparity В настоящее время большое значение уделяется преждевременности, это объясняется всё ещё высокой частотой преждевременных родов во всём мире, которые составляют в среднем 10%. Нашей целью было оценить факторы риска преждевременных родов, осложнённые преждевременной отслойкой нормально расположенной плаценты и послеродовым эндометритом, которые произошли в течение 2010 в нашей клинике. Статья была основана на анализе 190 медицинских историй преждевременных родов. Исследование установило материнские факторы риска преждевременных родов: спонтанные и медицинские аборты, преждевременные роды в анамнезе, неспецифическая генитальная инфекция и другое. Были указаны факторы риска развития преждевременной отслойки нормально расположенной плаценты при преждевременных родах: высокое артериальное давление, критические цифры возраста, преждевременный разрыв плодных оболочек, многорожавшие

Identifying Human Disease Genes through Cross-Species Gene Mapping of Evolutionary Conserved Processes

Understanding complex networks that modulate development in humans is hampered by genetic and phenotypic heterogeneity within and between populations. Here we present a method that exploits natural variation in highly diverse mouse genetic reference panels in which genetic and environmental factors can be tightly controlled. The aim of our study is to test a cross-species genetic mapping strategy, which compares data of gene mapping in human patients with functional data obtained by QTL mapping in recombinant inbred mouse strains in order to prioritize human disease candidate genes.We exploit evolutionary conservation of developmental phenotypes to discover gene variants that influence brain development in humans. We studied corpus callosum volume in a recombinant inbred mouse panel (C57BL/6J×DBA/2J, BXD strains) using high-field strength MRI technology. We aligned mouse mapping results for this neuro-anatomical phenotype with genetic data from patients with abnormal corpus callosum (ACC) development.).This approach that exploits highly diverse mouse strains provides an efficient and effective translational bridge to study the etiology of human developmental disorders, such as autism and schizophrenia

Plant Food Supplements with Anti-Inflammatory Properties: A Systematic Review (II)

The aim of this systematic review is to summarize the evidence for or against the efficacy of plant food supplements (PFS) for coping inflammatory conditions by considering epidemiological and human intervention studies. The review considers six botanical species commonly used as food supplements/medicinals: Urtica dioica L., Symphytum officinalis L., Calendula officinalis L., Curcuma longa L., Boswellia serrata Roxb., and Harpagophytum procumbens L. The search retrieved 579 publications. By removing the duplicates and applying the inclusion/exclusion criteria, the final number of papers was 47. No epidemiological data were found. The bibliographic search found no paper regarding the anti-inflammatory effects of Calendula officinalis L. and Symphytum officinalis L. by oral use. In spite of the long-term traditional use for inflammatory disorders, Curcuma longa L. and Harpagophytum procumbens L. warrant further investigation, whereas the efficacy of Urtica dioica L, even if the available data on hard endpoints are promising, requires other trials. Boswellia serrata Roxb. was found to be the most promising, since it shows the best efficacy for the treatment of pain/inflammatory conditions. In conclusion, it is advisable to conduct further studies with more homogeneous population and larger number of subjects by avoiding the heterogeneity of the herbal preparations considered

Features of Muon Arrival Time Distributions of High Energy EAS at Large Distances From the Shower Axis

In view of the current efforts to extend the KASCADE experiment (KASCADE-Grande) for observations of Extensive Air Showers (EAS) of primary energies up to 1 EeV, the features of muon arrival time distributions and their correlations with other observable EAS quantities have been scrutinised on basis of high-energy EAS, simulated with the Monte Carlo code CORSIKA and using in general the QGSJET model as generator. Methodically various correlations of adequately defined arrival time parameters with other EAS parameters have been investigated by invoking non-parametric methods for the analysis of multivariate distributions, studying the classification and misclassification probabilities of various observable sets. It turns out that adding the arrival time information and the multiplicity of muons spanning the observed time distributions has distinct effects improving the mass discrimination. A further outcome of the studies is the feature that for the considered ranges of primary energies and of distances from the shower axis the discrimination power of global arrival time distributions referring to the arrival time of the shower core is only marginally enhanced as compared to local distributions referring to the arrival of the locally first muon.Comment: 24 pages, Journal Physics G accepte