Transgene expression in mice of the Opa1 mitochondrial transmembrane protein through bicontinuous cubic lipoplexes containing gemini imidazolium surfactants.

BACKGROUND: Lipoplexes are non-viral vectors based on cationic lipids used to deliver DNA into cells, also known as lipofection. The positively charge of the hydrophilic head-group provides the cationic lipids the ability to condensate the negatively charged DNA into structured complexes. The polar head can carry a large variety of chemical groups including amines as well as guanidino or imidazole groups. In particular, gemini cationic lipids consist of two positive polar heads linked by a spacer with different length. As for the hydrophobic aliphatic chains, they can be unsaturated or saturated and are connected to the polar head-groups. Many other chemical components can be included in the formulation of lipoplexes to improve their transfection efficiency, which often relies on their structural features. Varying these components can drastically change the arrangement of DNA molecules within the lamellar, hexagonal or cubic phases that are provided by the lipid matrix. Lipofection is widely used to deliver genetic material in cell culture experiments but the simpler formulations exhibit major drawbacks related to low transfection, low specificity, low circulation half-life and toxicity when scaled up to in vivo experiments. RESULTS: So far, we have explored in cell cultures the transfection ability of lipoplexes based on gemini cationic lipids that consist of two C16 alkyl chains and two imidazolium polar head-groups linked with a polyoxyethylene spacer, (C16Im)2(C4O). Here, PEGylated lipids have been introduced to the lipoplex formulation and the transgene expression of the Opa1 mitochondrial transmembrane protein in mice was assessed. The addition of PEG on the surface of the lipid mixed resulted in the formation of Ia3d bicontinuous cubic phases as determined by small angle X-ray scattering. After a single intramuscular administration, the cubic lipoplexes were accumulated in tissues with tight endothelial barriers such as brain, heart, and lungs for at least 48 h. The transgene expression of Opa1 in those organs was identified by western blotting or RNA expression analysis through quantitative polymerase chain reaction. CONCLUSIONS: The expression reported here is sufficient in magnitude, duration and toxicity to consolidate the bicontinuous cubic structures formed by (C16Im)2(C4O)-based lipoplexes as valuable therapeutic agents in the field of gene delivery.

MKC-Quatsomes: a stable nanovesicle platform for bio-imaging and drug-delivery applications.

Quatsomes are outstanding new lipid-based nanovesicles that are highly homogeneous and stable in different media for years, but the composition must be carefully chosen to avoid any potentially toxic side effects in in vivo applications. To this end, we have developed and studied a novel type of Quatsomes composed of cholesterol and myristalkonium chloride (MKC), the latter being extensively used as antimicrobial preservative in many ophthalmic and parenteral formulations on the EU and USA market. We have synthesized these novel MKC-Quatsomes in different media that are suitable for parenteral administration, and confirmed their stability in these media for 18 months, as well as the stability in human serum for 24 hours. Biodistribution assays were performed after intravenous injection of fluorescently labeled MKC-Quatsomes in live mice bearing xenografted colorectal tumors, showing nanovesicle accumulation in tumors, liver, spleen, and kidneys. No histological alteration or toxicity was observed in any of these organs.

Gemini-Based Lipoplexes Complement the Mitochondrial Phenotype in MFN1-Knockout Mouse Embryonic Fibroblasts.

Mitochondria form a dynamic network of constantly dividing and fusing organelles. The balance between these antagonistic processes is crucial for normal cellular function and requires the action of specialized proteins. The mitochondrial membrane proteins mitofusin 1 (Mfn1) and mitofusin 2 (Mfn2) are responsible for the fusion of the outer membrane of adjacent mitochondria. Mutations within Mfn1 or Mfn2 impair mitochondrial fusion and lead to some severe mitochondrial dysfunctions and mitochondrial diseases (MDs). A characteristic phenotype of cells carrying defective Mfn1 or Mfn2 is the presence of a highly fragmented mitochondrial network. Here, we use a biocompatible mixture of lipids, consisting on synthetic gemini cationic lipids (GCLs) and the zwitterionic phospholipid (DOPE), to complex, transport, and deliver intact copies of MFN1 gene into MFN1-Knockout mouse embryonic fibroblasts (MFN1-KO MEFs). We demonstrate that the GCL/DOPE-DNA lipoplexes are able to introduce the intact MFN1 gene into the cells and ectopically produce functional Mfn1. A four-fold increase of the Mfn1 levels is necessary to revert the MFN1-KO phenotype and to partially restore a mitochondrial network. This phenotype complementation was correlated with the transfection of GCL/DOPE-MFN1 lipoplexes that exhibited a high proportion of highly packaged hexagonal phase. GCL/DOPE-DNA lipoplexes are formulated as efficient therapeutic agents against MDs.

pH-triggered endosomal escape of pore-forming Listeriolysin O toxin-coated gold nanoparticles.

BACKGROUND: A major bottleneck in drug delivery is the breakdown and degradation of the delivery system through the endosomal/lysosomal network of the host cell, hampering the correct delivery of the drug of interest. In nature, the bacterial pathogen Listeria monocytogenes has developed a strategy to secrete Listeriolysin O (LLO) toxin as a tool to escape the eukaryotic lysosomal system upon infection, allowing it to grow and proliferate unharmed inside the host cell. RESULTS: As a "proof of concept", we present here the use of purified His-LLO H311A mutant protein and its conjugation on the surface of gold nanoparticles to promote the lysosomal escape of 40 nm-sized nanoparticles in mouse embryonic fibroblasts. Surface immobilization of LLO was achieved after specific functionalization of the nanoparticles with nitrile acetic acid, enabling the specific binding of histidine-tagged proteins. CONCLUSIONS: Endosomal acidification leads to release of the LLO protein from the nanoparticle surface and its self-assembly into a 300 Å pore that perforates the endosomal/lysosomal membrane, enabling the escape of nanoparticles.

Lipid-peptide bioconjugation through pyridyl disulfide reaction chemistry and its application in cell targeting and drug delivery.

BACKGROUND: The design of efficient drug delivery vectors requires versatile formulations able to simultaneously direct a multitude of molecular targets and to bypass the endosomal recycling pathway of cells. Liposomal-based vectors need the decoration of the lipid surface with specific peptides to fulfill the functional requirements. The unspecific binding of peptides to the lipid surface is often accompanied with uncontrolled formulations and thus preventing the molecular mechanisms of a successful therapy. RESULTS: We present a simple synthesis pathway to anchor cysteine-terminal peptides to thiol-reactive lipids for adequate and quantitative liposomal formulations. As a proof of concept, we have synthesized two different lipopeptides based on (a) the truncated Fibroblast Growth Factor (tbFGF) for cell targeting and (b) the pH sensitive and fusogenic GALA peptide for endosomal scape. CONCLUSIONS: The incorporation of these two lipopeptides in the liposomal formulation improves the fibroblast cell targeting and promotes the direct delivery of cargo molecules to the cytoplasm of the cell.

Gadolinium-Decorated Silica Microspheres as Redox-Responsive MRI Probes for Applications in Cell Therapy Follow-Up.

The redox microenvironment within a cell graft can be considered as an indicator to assess whether the graft is metabolically active or hypoxic. We present a redox-responsive MRI probe based on porous silica microparticles whose surface has been decorated with a Gd-chelate through a disulphide bridge. Such microparticles are designed to be interspersed with therapeutic cells within a biocompatible hydrogel. The onset of reducing conditions within the hydrogel is paralleled by an increased clearance of Gd, that can be detected by MRI.

Effects of a delocalizable cation on the headgroup of gemini lipids on the lipoplex-type nanoaggregates directly formed from plasmid DNA.

Lipoplex-type nanoaggregates prepared from pEGFP-C3 plasmid DNA (pDNA) and mixed liposomes, with a gemini cationic lipid (CL) [1,2-bis(hexadecyl imidazolium) alkanes], referred as (C16Im)2Cn (where Cn is the alkane spacer length, n = 2, 3, 5, or 12, between the imidazolium heads) and DOPE zwitterionic lipid, have been analyzed by zeta potential, gel electrophoresis, SAXS, cryo-TEM, fluorescence anisotropy, transfection efficiency, fluorescence confocal microscopy, and cell viability/cytotoxicity experiments to establish a structure-biological activity relationship. The study, carried out at several mixed liposome compositions, α, and effective charge ratios, ρeff, of the lipoplex, demonstrates that the transfection of pDNA using CLs initially requires the determination of the effective charge of both. The electrochemical study confirms that CLs with a delocalizable positive charge in their headgroups yield an effective positive charge that is 90% of their expected nominal one, while pDNA is compacted yielding an effective negative charge which is only 10-25% than that of the linear DNA. SAXS diffractograms show that lipoplexes formed by CLs with shorter spacer (n = 2, 3, or 5) present three lamellar structures, two of them in coexistence, while those formed by CL with longest spacer (n = 12) present two additional inverted hexagonal structures. Cryo-TEM micrographs show nanoaggregates with two multilamellar structures, a cluster-type (at low α value) and a fingerprint-type, that coexist with the cluster-type at moderate α composition. The optimized transfection efficiency (TE) of pDNA, in HEK293T, HeLa, and H1299 cells was higher using lipoplexes containing gemini CLs with shorter spacers at low α value. Each lipid formulation did not show any significant levels of toxicity, the reported lipoplexes being adequate DNA vectors for gene therapy and considerably better than both Lipofectamine 2000 and CLs of the 1,2-bis(hexadecyl ammnoniun) alkane series, recently reported.

Polar ammoniostyryls easily converting a clickable lipophilic BODIPY in an advanced plasma membrane probe.

A very simple, small and symmetric, but highly bright, photostable and functionalizable molecular probe for plasma membrane (PM) has been developed from an accessible, lipophilic and clickable organic dye based on BODIPY. To this aim, two lateral polar ammoniostyryl groups were easily linked to increase the amphiphilicity of the probe and thus its lipid membrane partitioning. Compared to the BODIPY precursor, the transversal diffusion across lipid bilayers of the ammoniostyryled BODIPY probe was highly reduced, as evidenced by fluorescence confocal microscopy on model membranes built up as giant unilamellar vesicles (GUVs). Moreover, the ammoniostyryl groups endow the new BODIPY probe with the ability to optically work (excitation and emission) in the bioimaging-useful red region, as shown by staining of the plasma membrane of living mouse embryonic fibroblasts (MEFs). Upon incubation, this fluorescent probe rapidly entered the cell through the endosomal pathway. By blocking the endocytic trafficking at 4 °C, the probe was confined within the PM of MEFs. Our experiments show the developed ammoniostyrylated BODIPY as a suitable PM fluorescent probe, and confirm the synthetic approach for advancing PM probes, imaging and science.

How does the spacer length of cationic gemini lipids influence the lipoplex formation with plasmid DNA? Physicochemical and biochemical characterizations and their relevance in gene therapy.

Lipoplexes formed by the pEGFP-C3 plasmid DNA (pDNA) and lipid mixtures containing cationic gemini surfactant of the 1,2-bis(hexadecyl dimethyl ammonium) alkanes family referred to as C16CnC16, where n=2, 3, 5, or 12, and the zwitterionic helper lipid, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) have been studied from a wide variety of physical, chemical, and biological standpoints. The study has been carried out using several experimental methods, such as zeta potential, gel electrophoresis, small-angle X-ray scattering (SAXS), cryo-TEM, gene transfection, cell viability/cytotoxicity, and confocal fluorescence microscopy. As reported recently in a communication (J. Am. Chem. Soc. 2011, 133, 18014), the detailed physicochemical and biological studies confirm that, in the presence of the studied series lipid mixtures, plasmid DNA is compacted with a large number of its associated Na+ counterions. This in turn yields a much lower effective negative charge, qpDNA−, a value that has been experimentally obtained for each mixed lipid mixture. Consequently, the cationic lipid (CL) complexes prepared with pDNA and CL/DOPE mixtures to be used in gene transfection require significantly less amount of CL than the one estimated assuming a value of qDNA−=−2. This drives to a considerably lower cytotoxicity of the gene vector. Depending on the CL molar composition, α, of the lipid mixture, and the effective charge ratio of the lipoplex, ρeff, the reported SAXS data indicate the presence of two or three structures in the same lipoplex, one in the DOPE-rich region, other in the CL-rich region, and another one present at any CL composition. Cryo-TEMand SAXS studies with C16CnC16/DOPE-pDNA lipoplexes indicate that pDNA is localized between the mixed lipid bilayers of lamellar structures within a monolayer of ∼2 nm. This is consistent with a highly compacted supercoiled pDNA conformation compared with that of linear DNA. Transfection studies were carried out with HEK293T, HeLa, CHO, U343, and H460 cells. The α and ρeff values for each lipid mixture were optimized on HEK293T cells for transfection, and using these values, the remaining cells were also transfected in absence (-FBS-FBS) and presence (-FBS+FBS) of serum. The transfection efficiency was higher with the CLs of shorter gemini spacers (n=2 or 3). Each formulation expressed GFP on pDNA transfection and confocal fluorescence microscopy corroborated the results. C16C2C16/DOPE mixtures were the most efficient toward transfection among all the lipid mixtures and, in presence of serum, even better than the Lipofectamine2000, a commercial transfecting agent. Each lipid combination was safe and did not show any significant levels of toxicity. Probably, the presence of two coexisting lamellar structures in lipoplexes synergizes the transfection efficiency of the lipid mixtures which are plentiful in the lipoplexes formed by CLs with short spacer (n=2, 3) than those with the long spacer (n=5, 12).

Tunable gold nanorod/NAO conjugates for selective drug delivery in mitochondria-targeted cancer therapy.

Nonyl acridine orange (NAO) is a lipophilic and positively charged molecule widely used as a mitochondrial fluorescent probe. NAO is cytotoxic at micromolar concentration and might be potentially used as a mitochondria-targeted drug for cancer therapy. However, the use of NAO under in vivo conditions would be compromised by the unspecific interactions with off-target cells and negatively charged proteins present in the bloodstream. To tackle this limitation, we have synthesized NAO analogues carrying an imidazole group for their specific binding to nitrilotriacetic (NTA) functionalized gold nanorods (AuNRs). We demonstrate that AuNRs provide 104 binding sites and a controlled delivery under acidic conditions. Upon incubation with mouse embryonic fibroblasts, the endosomal acidic environment releases the NAO analogues from AuNRs, as visualized through the staining of the mitochondrial network. The addition of the monoclonal antibody Cetuximab to the conjugates enhanced their uptake within lung cancer cells and the conjugates were cytotoxic at subnanomolar concentrations (c50 ≈ 0.06 nM). Moreover, the specific interactions of Cetuximab with the epidermal growth factor receptor (EGFR) provided a specific targeting of EGFR-expressing lung cancer cells. After intravenous administration in patient-derived xenografts (PDX) mouse models, the conjugates reduced the progression of EGFR-positive tumors. Overall, the NAO-AuNRs provide a promising strategy to realize membrane mitochondria-targeted conjugates for lung cancer therapy.

Adipokines Profile and Inflammation Biomarkers in Prepubertal Population with Obesity and Healthy Metabolic State.

(1) Background and aims: Obesity and high body max index (BMI) have been linked to elevated levels of inflammation serum markers such as C-reactive protein (CRP), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha), adiponectin, and resistin. It has been described that adipose tissue presents a high production and secretion of these diverse pro-inflammatory molecules, which may have local effects on the physiology of the fat cell and also systemic effects on other organs. Our aim was to evaluate the impact that lifestyle modifications, following a Mediterranean Diet (MedDiet) program and physical activity (PA) training, would have on inflammatory biomarkers in a metabolically healthy prepubertal population with obesity (MHOPp) from Malaga (Andalusia, Spain). (2) Methods: 144 MHOPp subjects (aged 5-9 years) were included in this study as they met ≤1 of the following criteria: waist circumference and blood pressure ≥ 90 percentile, triglycerides > 90 mg/dL, high-density lipoprotein cholesterol (HDL-c) < 40 mg/dL, or impaired fasting glucose (≥100 md/dL). Selected subjects followed a personalized intensive lifestyle modification. Anthropometric measurements, inflammation biomarkers, and adipokine profile were analyzed after 12 and 24 months of intervention. (3) Results: 144 MHOPp participants (75 boys-52% and 69 girls-48%; p = 0.62), who were 7.8 ± 1.4 years old and had a BMI 24.6 ± 3.3 kg/m2, were included in the study. After 24 months of MedDiet and daily PA, a significant decrease in body weight (-0.5 ± 0.2 SD units; p < 0.0001) and BMI (-0.7 ± 0.2 SD units; p < 0.0001) was observed in the total population with respect to baseline. Serum inflammatory biomarkers (IL-6, TNF-alpha, and CRP) after 24 months of intervention were significantly reduced. Adipokine profile (adiponectin and resistin) did not improve with the intervention, as adiponectin levels significantly decreased and resistin levels increased in all the population. Inflammatory biomarkers and adipokine profile had a significant correlation with anthropometric parameters, body composition, and physical activity. (4) Conclusions: After 24 months of lifestyle modification, our MHOPp reduced their Z-score of BMI, leading to an improvement of inflammatory biomarkers but inducing deterioration in the adipokine profile, which does not improve with MedDiet and physical activity intervention. An adequate education within the family about healthier habits is necessary to prevent and reduce an excessive increase in obesity in childhood.

Metabolically healthy obesity: Inflammatory biomarkers and adipokines in elderly population.

BACKGROUND AND AIMS: Obesity is linked to elevated levels of inflammatory serum markers such as C-reactive protein (CRP), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNFa). Adiponectin and resistin are adipokines related to obesity. It has been described that adipose tissue presents a high production and secretion of these diverse pro-inflammatory molecules, which may have local effects on the physiology of fat cells as well as systemic effects on other organs. Our aim was to evaluate the impact that lifestyle modifications, by following a Mediterranean Diet (MedDiet) program and physical activity (PA) training, would have on inflammatory biomarkers and adipokine profile in a Metabolically Healthy Obese (MHO) elderly population from Malaga (Andalusia, Spain). SUBJETCS AND METHODS: Subjects aged ≥65 years (65 to 87 years old) with obesity (BMI ≥30 kg/m2) were included in this study if they met ≤1 of the following criteria: systolic blood pressure ≥130 mmHg and/or diastolic blood pressure ≥ 85 mmHg; triglycerides ≥150 mg/dL; HDL-C <40mg/dL in men and <50mg/dL women; and fasting blood glucose ≥100mg/dL. Selected subjects underwent a personalized intensive lifestyle modification. Anthropometric measurements, PA, MedDiet adherence, analytical parameters, and inflammatory biomarkers were analyzed after 12 months of intervention. RESULTS: 166 MHO elderly subjects, 40 (24.1%) male and 126 (75.9%) female (p < 0.0001), aged 71.7±5.2 years old (65 to 87 years old) were included in the study. After 12 months of intervention, only the waist circumference was significantly reduced in all the population (-2.5 cm, p<0.0001), although weight and BMI were maintained. MedDiet adherence increased significantly (p<0.001), but all intensity levels of PA decreased significantly (p<0.001). Concerning inflammatory biomarkers, only TNFa serum increased their levels after the intervention (p<0.001). Regarding the adipokine profile, adiponectin concentrations experienced a significant increment (p<0.001); besides, resistin concentrations decreased significantly (p<0.001). In this sense, only TNFa, adiponectin, and resistin correlated with PA. Adiponectin also correlates with insulin, triglycerides and HDL-c in baseline conditions and after 12 months of intervention; CRP, IL-6, TNFa, adiponectin, and resistin concentrations correlated with anthropometric parameters and some intensities of PA. In addition, adiponectin levels correlates with insulin, triglycerides and HDL-c. In baseline conditions, resistin levels correlated positively with TNFa (p = 0.01) and CRP (p<0.0001) levels. TNFa and IL-6 correlated positively with CRP (p = 0.03 and p<0.0001, respectively). After 12 months of intervention, only IL-6 correlated positively with CRP (p = 0.006). In addition, adipokines levels correlated positively during the process of lifestyle modification. However, during this process, only IL-6 correlated positively with itself (p<0.0001) and with CRP (p = 0.03). CONCLUSION: Healthy aging is a multifactorial biological process in which lifestyle is essential. The presence of obesity in elderly metabolically healthy population is not a problem necessarily. Elderly MHO population who eat a MedDiet and practice regularly PA are capable to modulate their production of inflammatory cytokines (CRP, IL-6, TNFa) and adipokines profile (adiponectin, resistin), preventing other metabolic disorders.

A healthy lifestyle is associated with lower arterial stiffness in a metabolically healthy elderly population with overweight or obesity.

BACKGROUND AND AIMS: Arterial stiffness is linked with the development of cardiovascular and noncardiovascular diseases. In clinical practice, measurement of carotid-femoral pulse wave velocity (cf-PWV) has become a widely used study for the assessment of cardiovascular risk in elderly population. Our aim was to evaluate whether maintaining a healthy life, based on Mediterranean Diet (MedDiet) and regular practice of physical activity, are associated with arterial stiffness in an elderly, metabolically healthy with overweight or obesity (MHOe) population. INDIVIDUALS AND METHODS: A transversal, analytical-descriptive study in MHOe population (aged ≥65 years) with a BMI at least 27 kg/ m 2 who had one or less of the following cardiometabolic disorders: fasting plasma glucose at least 100 mg/dl, blood pressure at least 135/85 mmHg (or the use of blood pressure-lowering agents), low high-density lipoprotein (HDL) cholesterol (≤ 40 mg/dl for men, ≤50 mg/dl for women) or triglycerides at least 150 mg/dl (or the use of lipid-lowering therapies) was conducted. Blood pressure, height, weight, BMI, waist to hip ratio (WHR), practice of physical activity, MedDiet adherence and food intake along with cf-PWV were analysed. RESULTS: One hundred and fifty-eight MHOe individuals (age: 72.2 ±â€Š5.0 years, BMI: 31.6 ±â€Š3.8 kg/m 2 ) were recruited. One hundred and nine of them were younger than 75 years of age (young-old, age: 69.3 ±â€Š2.8 years and BMI: 32.0 ±â€Š3.9 kg/m 2 ) and 49 of them aged 75 years or older (old-old, age: 78.1 ±â€Š2.9 years and BMI: 30.7 ±â€Š3.6 kg/m 2 ). All population showed a strong adherence to the Med Diet due major consumption of homemade meal, olive oil and lean meats. In addition, they presented an important practice of all intensities of physical activity. Young-old individuals had a cf-PWV of 9.7 ±â€Š2.2 m/s and old-old individuals had a cf-PWV of 11.1 ±â€Š4.4 m/s. In all populations, a negative correlation between cf-PWV and BMI ( r  = -0.17, P  = 0.04) and a positive correlation with WHR in men ( r  = 0.18, P  = 0.03) was found. WHR shows a significantly positive correlation with the cf-PWV values in old-old women participants ( r  = 0.41, P  = 0.008). On the other side, only vigorous physical activity showed a negative correlation with cf-PWV in all population and in young-old individuals ( r  = -0.20; P  = 0.02 and r  = -0.22; P  = 0.03, respectively). CONCLUSION: Healthy lifestyle habits based on MedDiet adherence and regular practice of physical activity are associated with lower arterial stiffness in a metabolically healthy population with overweight or obesity older than 65 years compared with data from other elderly populations previously reported in the literature.

Why is less cationic lipid required to prepare lipoplexes from plasmid DNA than linear DNA in gene therapy?

The most important objective of the present study was to explain why cationic lipid (CL)-mediated delivery of plasmid DNA (pDNA) is better than that of linear DNA in gene therapy, a question that, until now, has remained unanswered. Herein for the first time we experimentally show that for different types of CLs, pDNA, in contrast to linear DNA, is compacted with a large amount of its counterions, yielding a lower effective negative charge. This feature has been confirmed through a number of physicochemical and biochemical investigations. This is significant for both in vitro and in vivo transfection studies. For an effective DNA transfection, the lower the amount of the CL, the lower is the cytotoxicity. The study also points out that it is absolutely necessary to consider both effective charge ratios between CL and pDNA and effective pDNA charges, which can be determined from physicochemical experiments.

Statin Therapy in Very Old Patients: Lights and Shadows.

Atherosclerotic cardiovascular diseases (ASCVD) are the leading cause of death worldwide. High levels of total cholesterol-and of low-density lipoprotein cholesterol in particular-are one of the main risk factors associated with ASCVD. Statins are first-line treatment for hypercholesterolemia and have been proven to reduce major vascular events in adults with and without underlying ASCVD. Findings in the literature show that statins reduce coronary and cerebrovascular morbidity and mortality in middle-aged people, but their benefits in older adults are not as well-established, especially in primary prevention. Furthermore, many particularities must be considered regarding their use in old subjects, such as age-related changes in pharmacokinetics and pharmacodynamics, comorbidities, polypharmacy, and frailty, which decrease the safety and efficacy of statins in this population. Myopathy and a possible higher risk of falling along with cognitive decline are classic concerns for physicians when considering statin use in the very old. Additionally, some studies suggest that the relative risk for coronary events and cardiovascular mortality associated with high levels of cholesterol decreases after age 70, making the role of statins unclear. On the other hand, ASCVD are one of the most important causes of disability in old subjects, so cardiovascular prevention is of particular interest in this population in order to preserve functional status. This review aims to gather the current available evidence on the efficacy and safety of statin use in very old patients in both primary and secondary prevention.

Lifestyle Modification Program on a Metabolically Healthy Elderly Population with Overweight/Obesity, Young-Old vs. Old-Old. CONSEQUENCES of COVID-19 Lockdown in This Program.

The SARS-CoV-2 pandemic led to lockdowns, which affected the elderly, a high-risk group. Lockdown may lead to weight gain due to increased food intake and reduced physical activity (PA). Our study aimed to analyze the impact of a 12-month lifestyle intervention on a metabolically healthy overweight/obese elderly (MHOe) population and how the lockdown by COVID-19 affected this program. Methods: MHOe participants (65-87 years) were recruited to participate in a lifestyle modification intervention based on the Mediterranean diet (MedDiet) and regular PA. Participants were classified into two groups: young-old (<75 years) or old-old (≥75 years). Anthropometric and clinical characteristics, energy intake, and energy expenditure were analyzed at baseline and after 12 months of intervention. Results: The final sample included 158 MHOe participants of both sexes (age: 72.21 ± 5.04 years, BMI: 31.56 ± 3.82 kg/m2): 109 young-old (age: 69.26 ± 2.83 years, BMI: 32.0 ± 3.85 kg/m2) and 49 old-old (age: 78.06 ± 2.88 years, BMI: 30.67 ± 3.64 kg/m2). After 12 months of intervention and despite lockdown, the young-old group increased MedDiet adherence (+1 point), but both groups drastically decreased daily PA, especially old-old participants. Fat mass significantly declined in the total population and the young-old. Depression significantly increased (26.9% vs. 21.0%, p < 0.0001), especially in the old-old (36.7% vs. 22.0%, p < 0.0001). No significant changes were found in the glycemic or lipid profile. Conclusions: This study indicates that ongoing MedDiet intake and regular PA can be considered preventative treatment for metabolic diseases in MHOe subjects. However, mental health worsened during the study and should be addressed in elderly individuals.

Experimental and theoretical approach to the sodium decanoate-dodecanoate mixed surfactant system in aqueous solution.

The mixed system consisting of two anionic surfactants of identical headgroups but with 10 and 12 carbon atoms on the hydrophobic tail, sodium decanoate (C(10)Na) and sodium dodecanoate (C(12)Na), has been studied in aqueous solution at 298.15 K by means of conductivity and fluorescence spectroscopy experiments and from a theoretical point of view. The monomeric and micellar phases of the mixed aggregates were analyzed through the experimental determination of the total critical micelle concentration, cmc*, the degree of ionization of the mixed micelle, beta, and the total aggregation number, N*. Results indicate that, compared to the ideal behavior, the mixed system with two anionic surfactants differing only in two methylenes in the hydrophobic tail shows a negative deviation in the cmc* and a positive one in N*. Pure surfactants (C(10)Na and C(12)Na) form spherical micelles, but mixed micelles must aggregate with a rodlike shape to allow more surfactant molecules than expected. In addition, rodlike micelles result in more compacted aggregation (i.e., less area per polar head). From the experimental data in this work, several theoretical models for mixed surfactant systems have been checked: Rubingh's model predicts lower deviations from ideality than Motomura's model. The stability of the micelles has been analyzed by computing the standard Gibbs energy of micelle formation, Delta G(mic,0), of pure and mixed micelles. Results of this work reinforce the feature that mixed systems formed by alkylsurfactants with the same polar head that differ in the hydrocarbon length, usually admitted as roughly ideal systems, may show nonideal behavior. This deviation, being mostly related to the difference in the chain length, Delta n(c), between surfactants can be analyzed only when very accurate experimental techniques as well as adequate theoretical models are used.

Effect of lipid composition on the structure and theoretical phase diagrams of DC-Chol/DOPE-DNA lipoplexes.

Lipoplexes constituted by calf-thymus DNA (CT-DNA) and mixed cationic liposomes consisting of varying proportions of the cationic lipid 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Chol) and the zwitterionic lipid, 1,2-dioleoyl-sn-glycero-3-phosphoetanolamine (DOPE) have been analyzed by means of electrophoretic mobility, SAXS, and fluorescence anisotropy experiments, as well as by theoretically calculated phase diagrams. Both experimental and theoretical studies have been run at several liposome and lipoplex compositions, defined in terms of cationic lipid molar fraction, α, and either the mass or charge ratios of the lipoplex, respectively. The experimental electrochemical results indicate that DC-Chol/DOPE liposomes, with a mean hydrodynamic diameter of around (120 ± 10) nm, compact and condense DNA fragments at their cationic surfaces by means of a strong entropically driven electrostatic interaction. Furthermore, the positive charges of cationic liposomes are compensated by the negative charges of DNA phosphate groups at the isoneutrality L/D ratio, (L/D)(ϕ), which decreases with the cationic lipid content of the mixed liposome, for a given DNA concentration. This inversion of sign process has been also studied by means of the phase diagrams calculated with the theoretical model, which confirms all the experimental results. SAXS diffractograms, run at several lipoplex compositions, reveal that, irrespectively of the lipoplex charge ratio, DC-Chol/DOPE-DNA lipoplexes show a lamellar structure, L(α), when the cationic lipid content on the mixed liposomes α ≥ 0.4, while for a lower content (α = 0.2) the lipoplexes show an inverted hexagonal structure, H(II), usually related with improved cell transfection efficiency. A similar conclusion is reached from fluorescence anisotropy results, which indicate that the fluidity on liposome and lipoplexes membrane, also related with better transfection results, increases as long as the cationic lipid content decreases.

Supramolecular zippers elicit interbilayer adhesion of membranes producing cell death.

BACKGROUND: The fluorescent dye 10-N-nonyl acridine orange (NAO) is widely used as a mitochondrial marker. NAO was reported to have cytotoxic effects in cultured eukaryotic cells when incubated at high concentrations. Although the biochemical response of NAO-induced toxicity has been well identified, the underlying molecular mechanism has not yet been explored in detail. METHODS: We use optical techniques, including fluorescence confocal microscopy and lifetime imaging microscopy (FLIM) both in model membranes built up as giant unilamellar vesicles (GUVs) and cultured cells. These experiments are complemented with computational studies to unravel the molecular mechanism that makes NAO cytotoxic. RESULTS: We have obtained direct evidence that NAO promotes strong membrane adhesion of negatively charged vesicles. The attractive forces are derived from van der Waals interactions between anti-parallel H-dimers of NAO molecules from opposing bilayers. Semi-empirical calculations have confirmed the supramolecular scenario by which anti-parallel NAO molecules form a zipper of bonds at the contact region. The membrane remodeling effect of NAO, as well as the formation of H-dimers, was also confirmed in cultured fibroblasts, as shown by the ultrastructure alteration of the mitochondrial cristae. CONCLUSIONS: We conclude that membrane adhesion induced by NAO stacking accounts for the supramolecular basis of its cytotoxicity. GENERAL SIGNIFICANCE: Mitochondria are a potential target for cancer and gene therapies. The alteration of the mitochondrial structure by membrane remodeling agents able to form supramolecular assemblies via adhesion properties could be envisaged as a new therapeutic strategy.

Selective functionalization of mesoporous silica nanoparticles with ibuprofen and Gd(III) chelates: a new probe for potential theranostic applications.

Organo-modified mesoporous silica nanoparticles, loaded with ibuprofen into the pores and functionalized on the external surface with a stable Gd(iii)-DOTA-monoamide chelate, were prepared and explored as potential theranostic probes.