Nanometric Vesicles Research Articles

First study on the release of a natural antimicrobial agent, estragole, from freeze-dried delivery systems based on cyclodextrins and liposomes

Estragole is considered as a promising candidate for food preservation due to its antioxidant and antimicrobial properties. However, its sensitivity to light and oxygen, its volatility and hydrophobicity prevent its large application. In order to increase the stability of estragole, conventional liposomes (CL) and drug-in-cyclodextrin-in-liposomes (DCL) were prepared by the ethanol-injection method using Phospholipon 90H in combination with cholesterol and then freeze-dried using hydroxypropyl-ß-cyclodextrin (HP-ß-CD) as a cryoprotectant. Fresh and reconstituted vesicles demonstrated nanometric vesicles size, spherical shape and negative surface charge. Moreover, all suspensions were homogeneous. Compared to CL, DCL improved estragole encapsulation efficiency, and conserved the same loading rate during freeze-drying. Freeze-dried CL and DCL retained estragole more efficiently compared to the reconstituted ones, as evidenced by release study performed by multiple headspace extraction. Finally, we demonstrated, for the first time, that HP-ß-CD did not affect the membrane fluidity of fresh and reconstituted Phospholipon 90H/cholesterol CL and DCL.

Comparative study of liposomes, ethosomes and transfersomes as carriers for enhancing the transdermal delivery of diflunisal: In vitro and in vivo evaluation

The current study aimed to develop an effective transdermal nanovesicular carrier of diflunisal that provides enhanced delivery through the skin. Two types of nanovesicles, ethosomes and transfersomes, were investigated and compared to conventional liposomes. Ethosomes with variable ethanol contents (10, 30 and 50%) and transfersomes using different edge activators, including sodium deoxycholate, sodium cholate and sodium taurocholate, were prepared and characterized. The obtained vesicles revealed good entrapment efficiencies (46.73–65.99%), nanometric vesicle sizes (453.10–796.80 nm) and negative zeta potential values (−45.40 to −86.90 mV). Ethosomes with 30% ethanol and sodium deoxycholate-containing transfersomes were incorporated into hydrogels to evaluate their in vitro release and permeation patterns. Nanovesicular hydrogels exhibited more sustained diflunisal release than did corresponding dispersions. Compared to liposomal hydrogel, both carriers proved the superiority of diflunisal permeation and flux across the skin. Confocal laser scanning microscopy showed improved penetration of rhodamine-loaded nanovesicles through skin layers with a wider distribution and higher fluorescence intensity. Compared to liposomes, selected nanovesicles exhibited remarkable antinociceptive and anti-inflammatory effects manifested by significant reduction in number of writhings and significantly higher inhibition of paw oedema. Hence, the developed nanovesicles could be considered promising carriers for transdermal delivery of diflunisal for pain and inflammation management.

Doxorubicin Expands in Vivo Secretion of Circulating Exosome in Mice.

Modulation of tumor immunity is a known factor in the antitumor activity of many chemotherapeutic agents. Exosomes are extracellular nanometric vesicles that are released by almost all types of cells, which includes cancer cells. These vesicles play a crucial role in tumor immunity. Many in vitro studies have reproduced the aggressive secretion of exosomes following treatment with conventional anticancer drugs. Nevertheless, how chemotherapeutic agents including nanomedicines such as Doxil® affect the in vivo secretion of exosomes is yet to be elucidated. In this study, the effect of intravenous injection of either free doxorubicin (DXR) or liposomal DXR formulation (Doxil®) on exosome secretion was evaluated in BALB/c mice. Exosomes were isolated from serum by using an ExoQuick™ kit. Free DXR treatment markedly increased serum exosome levels in a post-injection time-dependent manner, while Doxil® treatment did not. Exosomal size distribution and marker protein expressions (CD9, CD63, and TSG101) were studied. The physical/biological characteristics of treatment-induced exosomes were comparable to those of control mice. Interestingly, splenectomy significantly suppressed the copious exosomal secretions induced by free DXR. Collectively, our results indicate that conventional anticancer agents induce the secretion of circulating exosomes, presumably via stimulating immune cells of the spleen. As far as we know, this study represents the first report indicating that conventional chemotherapeutics may induce exosome secretion which might, in turn, contribute partly to the antitumor effect of chemotherapeutic agents.

Development, characterization &invivo evaluation of proniosomal based transdermal delivery system of Atenolol

The potential of proniosomes as a transdermal drug delivery system for Atenolol was investigated by encapsulating the drug in various formulations of proniosomal gel composed of various ratios of sorbitan fatty acid esters, cholesterol, lecithin prepared by Coacervation-phase separation method. The objectives of the present study were to define effects on the antihypertension activity and pharmacokinetics of a novel transdermal Proniosomal gel incorporating Atenolol. The formulated systems were characterized in vitro for size, drug entrapment, In vitro and in vivo drug permeation profiles and vesicular stability at different storage conditions. The optimized Atenolol proniosomes (AT8) showed nanometric vesicle size, high entrapment efficiency and marked enhancement in transdermal permeation. The prepared Proniosomal gel showed the relative bioavailability of 365.38 fold increased for AT8 than oral. The maximal concentrations (Cmax), of drug were significantly reduced while the areas under the plasma concentration–time curve (AUC), and mean residence times (MRT), t1/2 were evidently increased and extended, respectively. The results suggest that proniosomes can act as promising carrier which offers an alternative approach for transdermal delivery of Atenolol.

Nanopore Opening at Flat and Nanotip Conical Electrodes during Vesicle Impact Electrochemical Cytometry.

The oxidation of catecholamine at a microelectrode, following its release from individual vesicles, allows interrogation of the content of single nanometer vesicles with vesicle impact electrochemical cytometry (VIEC). Previous to this development, there were no methods available to quantify the chemical load of single vesicles. However, accurate quantification of the content is hampered by uncertainty in the proportion of substituent molecules reaching the electrode surface (collection efficiency). In this work, we use quantitative modeling to calculate this collection efficiency. For all vesicles except those at the very edge of the electrode, modeling shows that ∼100% oxidation efficiency is achieved when employing a 33 μm diameter disk microelectrode for VIEC, independent of the location of the vesicle release pore. We use this to experimentally determine a precise distribution of catecholamine in individual vesicles extracted from PC12 cells. In contrast, we calculate that when a nanotip conical electrode (∼4 μm length, ∼1.5 μm diameter at the base) is employed, as in intracellular VIEC (IVIEC), the current-time response depends strongly on the position of the catecholamine-releasing pore in the vesicle membrane. When vesicle release occurs with the pore opening occurring far from the electrode, lower currents and partial oxidation (∼75%) of the catecholamine are predicted, as compared to higher currents and ∼100% oxidation, when the pore is close to/at the electrode surface. As close agreement is observed between the experimentally measured vesicular content in intracellular and extracted vesicles from the same cell line using nanotip and disk electrodes, respectively, we conclude that pores open at the electrode surface. Not only does this suggest that electroporation of the vesicle membrane is the primary driving force for catecholamine release from vesicles at polarized electrodes, but it also indicates that IVIEC with nanotip electrodes can directly assess vesicular content without correction.

Cover Picture: Zinc Regulates Chemical‐Transmitter Storage in Nanometer Vesicles and Exocytosis Dynamics as Measured by Amperometry (Angew. Chem. Int. Ed. 18/2017)

Cover Picture: Zinc Regulates Chemical‐Transmitter Storage in Nanometer Vesicles and Exocytosis Dynamics as Measured by Amperometry (Angew. Chem. Int. Ed. 18/2017)

Zinc Regulates Chemical‐Transmitter Storage in Nanometer Vesicles and Exocytosis Dynamics as Measured by Amperometry

AbstractWe applied electrochemical techniques with nano‐tip electrodes to show that micromolar concentrations of zinc not only trigger changes in the dynamics of exocytosis, but also vesicle content in a model cell line. The vesicle catecholamine content in PC12 cells is significantly decreased after 100 μm zinc treatment, but, catecholamine release during exocytosis remains nearly the same. This contrasts with the number of molecules stored in the exocytosis vesicles, which decreases, and we find that the amount of catecholamine released from zinc‐treated cells reaches nearly 100 % content expelled. Further investigation shows that zinc slows down exocytotic release. Our results provide the missing link between zinc and the regulation of neurotransmitter release processes, which might be important in memory formation and storage.

Zinc Regulates Chemical-Transmitter Storage in Nanometer Vesicles and Exocytosis Dynamics as Measured by Amperometry.

We applied electrochemical techniques with nano-tip electrodes to show that micromolar concentrations of zinc not only trigger changes in the dynamics of exocytosis, but also vesicle content in a model cell line. The vesicle catecholamine content in PC12 cells is significantly decreased after 100 μm zinc treatment, but, catecholamine release during exocytosis remains nearly the same. This contrasts with the number of molecules stored in the exocytosis vesicles, which decreases, and we find that the amount of catecholamine released from zinc-treated cells reaches nearly 100 % content expelled. Further investigation shows that zinc slows down exocytotic release. Our results provide the missing link between zinc and the regulation of neurotransmitter release processes, which might be important in memory formation and storage.

Formulation by design based risperidone nano soft lipid vesicle as a new strategy for enhanced transdermal drug delivery: In-vitro characterization, and in-vivo appraisal

The present study was designed to formulate and optimize transdermal risperidone soft lipid vesicles. The formulation optimized with phospholipid, safranal and ethanol were incorporated as permeation and absorption enhancers. The optimized risperidone soft lipid vesicle was further evaluated for skin irritation study, in-vivo pharmacokinetic study and locomotor activity. Three factor three level Box–Behnken design (BBD) was used to statistically optimize soft lipid vesicle using safranal (A), ethanol (B)and phospholipid (C) as independent variable, while their effect was observed for vesicle size (Y1), entrapment efficiency (Y2) and flux (Y3). The optimized risperidone soft lipid vesicle (Ris-opt) showed nanometric vesicle size, high entrapment efficiency and marked enhancement in transdermal flux. The extent of absorption from Ris-opt was greater when compared to oral suspension with relative bioavailability of 177%. The histopathological evaluation revealed developed formulation did not showed skin irritation compared to standard irritant. The significant findings presented here encourage further studies with risperidone soft lipid vesicles for treatment of schizophrenia.

Exosome-Based Cancer Therapy: Implication for Targeting Cancer Stem Cells.

Drug resistance, difficulty in specific targeting and self-renewal properties of cancer stem cells (CSCs) all contribute to cancer treatment failure and relapse. CSCs have been suggested as both the seeds of the primary cancer, and the roots of chemo- and radio-therapy resistance. The ability to precisely deliver drugs to target CSCs is an urgent need for cancer therapy, with nanotechnology-based drug delivery system being one of the most promising tools to achieve this in the clinic. Exosomes are cell-derived natural nanometric vesicles that are widely distributed in body fluids and involved in multiple disease processes, including tumorigenesis. Exosome-based nanometric vehicles have a number of advantages: they are non-toxic, non-immunogenic, and can be engineered to have robust delivery capacity and targeting specificity. This enables exosomes as a powerful nanocarrier to deliver anti-cancer drugs and genes for CSC targeting therapy. Here, we will introduce the current explorations of exosome-based delivery system in cancer therapy, with particular focus on several exosomal engineering approaches that have improved their efficiency and specificity for CSC targeting.

Enhancement of 8-methoxypsoralen topical delivery via nanosized niosomal vesicles: Formulation development, in vitro and in vivo evaluation of skin deposition

The aim of the present study is to enhance the skin penetration and deposition of 8-methoxypsoraln (8-MOP) via niosomal vesicles to increase its local efficacy and safety. 8-MOP niosomes were prepared by the thin film hydration method using Span 60 or Span 40 along with cholesterol at five different molar ratios. The obtained vesicles revealed high entrapment efficiencies (83.04–89.90%) with nanometric vesicle diameters (111.1–198.8nm) of monodisperse distribution (PDI=0.145–0.216), zeta potential values <−48.3mV and spherical morphology under transmission electron microscopy. Optimized niosomal formulations depicted a biphasic in vitro release pattern in phosphate buffer (pH 5.5)/ethanol (7:3v/v) and displayed good physical stability after storage for 6 months at room (20–25°C) and refrigeration (4–8°C) temperatures. The two optimized formulations were incorporated in 5% sodium carboxy methylcellulose based hydrogel matrix which showed optimum pH values (7.37–7.39), pseudoplastic with thixotropic rheological behavior and more retarded 8-MOP release, by 23.82 and 14.89%, compared to niosomal vesicles after 24h. In vitro drug permeation and deposition studies, using rat skins, revealed promoted penetration and accumulation of 8-MOP after 8h. The skin penetration was further confirmed in vivo by confocal laser scanning microscopy, after 2h application period using rhodamine-loaded niosomal hydrogels compared to plain rhodamine hydrogel, as a florescence marker. Therefore, enhanced permeation and skin deposition of 8-MOP delivered by niosomes may help in improving the efficacy and safety of long-term treatment with 8-MOP.

Quantitative Chemical Measurements of Vesicular Transmitters with Electrochemical Cytometry.

Electrochemical cytometry adds a new dimension to our ability to study the chemistry and chemical storage of transmitter molecules stored in nanometer vesicles. The approach involves the adsorption and subsequent rupture of vesicles on an electrode surface during which the electroactive contents are quantitatively oxidized (or reduced). The measured current allows us to count the number of molecules in the vesicles using Faraday's law and to correlate this to the amount of molecules released when single exocytosis events take place at communicating cells. The original format for this method involved a capillary electrophoresis separation step to singly address each vesicle, but we have more recently discovered that cellular vesicles tend to adsorb to carbon electrodes and spontaneously as well as stochastically rupture to give mostly single vesicle events. This approach, called impact electrochemical cytometry, even though the impact is perhaps not the important part of this process, has been studied and the vesicle rupture appears to be at the interface between the vesicle and the electrode and is probably driven by electroporation. The pore size and rate of content electrolysis are a function of the pore diameter and the presence of a protein core in the vesicles. In model liposomes with no protein, events appear extremely rapidly as the soft nanoparticles impact the electrode and the contents are oxidized. It appears that the proteins decorating the surface of the vesicle are important in maintaining a gap from the electrode and when this gap is closed electroporation takes place. Models of the event response times suggest the pores formed are small enough so we can carry out these measurements at nanotip electrodes and we have used this to quantify the vesicle content in living cells in a mode we call intracellular impact electrochemical cytometry. The development of electrochemical cytometry allows comparison between vesicle content and vesicular release and we have found that only part of the vesicle content is released in typical exocytotic cases measured by amperometry. This has led to the novel hypothesis that most exocytosis from dense core vesicles is via mechanism where vesicles fuse with the cell membrane, some content is released and then close again to be reloaded and reused. It leaves open the possibility that cells regulate release during individual events. This might be important in learning and memory and be a nonreceptor pharmaceutical target for brain-related disorders. Indeed, the concept of the chemo-brain observed in cisplatin-treated cancer patients appears to be at least in part the result of changing the fraction of transmitter released and we have been able to show this by using the combined amperometric measurement of release and electrochemical cytometry at model cells.

Mass spectrometry imaging of lipids and metabolites: from fruit fly brains to single nanometer vesicles

Alzheimer׳s disease is one of the main causes of dementia in the elderly and its frequency is on the rise worldwide. It is considered the result of complex interactions between genetic and environmental factors, being many of them unknown. Therefore, there is a dire necessity for the identification of novel molecular players for the understanding of this disease. In this data article we determined the protein expression profiles of whole protein extracts from cortex regions of brains from patients with Alzheimer׳s disease in comparison to a normal brain. We identified 721 iTRAQ-labeled polypeptides with more than 95% in confidence. We analyzed all proteins that changed in their expression level and located them in the KEGG metabolic pathways, as well as in the mitochondrial complexes of the electron transport chain and ATP synthase. In addition, we analyzed the over- and sub-expressed polypeptides through IPA software, specifically Core I and Biomarkers I modules. Data in this article is related to the research article “Identification of proteins that are differentially expressed in brains with Alzheimer’s disease using iTRAQ labeling and tandem mass spectrometry” (Minjarez et al., 2016) [1].

The Effect of Hydrophile Topology in RAFT-Mediated Polymerization-Induced Self-Assembly.

Polymerization-induced self-assembly (PISA) was employed to compare the self-assembly of different amphiphilic block copolymers. They were obtained by emulsion polymerization of styrene in water using hydrophilic poly(N-acryloylmorpholine) (PNAM)-based macromolecular RAFT agents with different structures. An average of three poly (ethylene glycol acrylate) (PEGA) units were introduced either at the beginning, statistically, or at the end of a PNAM backbone, resulting in formation of nanometric vesicles and spheres from the two former macroRAFT architectures, and large vesicles from the latter. Compared to the spheres obtained with a pure PNAM macroRAFT agent, composite macroRAFT architectures promoted a dramatic morphological change. The change was induced by the presence of PEGA hydrophilic side-chains close to the hydrophobic polystyrene segment.

The Effect of Hydrophile Topology in RAFT‐Mediated Polymerization‐Induced Self‐Assembly

AbstractPolymerization‐induced self‐assembly (PISA) was employed to compare the self‐assembly of different amphiphilic block copolymers. They were obtained by emulsion polymerization of styrene in water using hydrophilic poly(N‐acryloylmorpholine) (PNAM)‐based macromolecular RAFT agents with different structures. An average of three poly (ethylene glycol acrylate) (PEGA) units were introduced either at the beginning, statistically, or at the end of a PNAM backbone, resulting in formation of nanometric vesicles and spheres from the two former macroRAFT architectures, and large vesicles from the latter. Compared to the spheres obtained with a pure PNAM macroRAFT agent, composite macroRAFT architectures promoted a dramatic morphological change. The change was induced by the presence of PEGA hydrophilic side‐chains close to the hydrophobic polystyrene segment.

Soft biodegradable polymersomes from caprolactone-derived polymers

Polymersomes have promise for advanced theranostic delivery. We report here the design and characterization of a series of block copolymers that assemble into soft, bioresorbable, and non-toxic vesicles. The polymers are based on poly(ethylene glycol)–poly(caprolactone), but the caprolactone (CL) is copolymerized with a second monomer, 1,4,8-trioxaspiro-[4,6]-9-undecanone (TOSUO). Because TOSUO polymers have no crystalline character, copolymerizing TOSUO with CL should reduce the crystallinity of the polymersomes. After synthesizing polymers with different ratios of CL to TOSUO, we found that all copolymers assemble into both micron and nano-metric vesicles. Increasing the TOSUO content of the copolymer reduces the polymer crystalline melting temperature and the area expansion modulus of vesicle membranes. Membranes with partial crystalline structure exhibit hysteresis in the tension versus strain curve during aspiration. Vesicles are not cytotoxic and exhibit first-order release of encapsulated gemcitabine. These materials are promising for the development of deformable, biodegradable polymersomes for biomedical applications.

Acute Inhibition of Selected Membrane-Proximal Mouse T Cell Receptor Signaling by Mitochondrial Antagonists

T cells absorb nanometric membrane vesicles, prepared from plasma membrane of antigen presenting cells, via dual receptor/ligand interactions of T cell receptor (TCR) with cognate peptide/major histocompatibility complex (MHC) plus lymphocyte function-associated antigen 1 (LFA-1) with intercellular adhesion molecule 1. TCR-mediated signaling for LFA-1 activation is also required for the vesicle absorption. Exploiting those findings, we had established a high throughput screening (HTS) platform and screened a library for isolation of small molecules inhibiting the vesicle absorption. Follow-up studies confirmed that treatments (1 hour) with various mitochondrial antagonists, including a class of anti-diabetic drugs (i.e., Metformin and Phenformin), resulted in ubiquitous inhibition of the vesicle absorption without compromising viability of T cells. Further studies revealed that the mitochondrial drug treatments caused impairment of specific membrane-proximal TCR signaling event(s). Thus, activation of Akt and PLC-γ1 and entry of extracellular Ca2+ following TCR stimulation were attenuated while polymerization of monomeric actins upon TCR triggering progressed normally after the treatments. Dynamic F-actin rearrangement concurring with the vesicle absorption was also found to be impaired by the drug treatments, implying that the inhibition by the drug treatments of downstream signaling events (and the vesicle absorption) could result from lack of directional relocation of signaling and cell surface molecules. We also assessed the potential application of mitochondrial antagonists as immune modulators by probing effects of the long-term drug treatments (24 hours) on viability of resting primary T cells and cell cycle progression of antigen-stimulated T cells. This study unveils a novel regulatory mechanism for T cell immunity in response to environmental factors having effects on mitochondrial function.

Recombination of Nanometric Vesicles during Freeze-Drying

Concentrated dispersions of nanometric lipid vesicles (mean diameter 20 nm) in water/maltose solutions have been freeze-dried and then redispersed in water, yielding again dispersions of lipid vesicles. At each stage of the freeze-drying process, the organization of the vesicles in the dispersion and their size distribution were examined through small-angle neutron scattering and gel permeation chromatography. It was found that the osmotic deswelling of the vesicles caused them to recombine into larger vesicles. A single burst of recombination events occurred when the maltose concentration in the aqueous phase rose above 100 g/L. The final vesicle population was monopopulated, with a central diameter about twice as large as that of the original dispersion.