Lipid Nanovesicles Research Articles

Lipid Nanovesicles by Microfluidics: Manipulation, Synthesis, and Drug Delivery.

Lipid nanovesicles, including endogenous exosomes and synthetic lipid nanoparticles, have shown great potential in disease diagnostics, drug delivery, and cancer biology. Naturally secreted nanovesicles are promising biomarkers for early detection of cancers in vitro. Synthetic nanovesicles serve as robust drug delivery systems with enhanced tumor targeting in vivo. Microfluidic platforms with features of excellent flow control and rapid mixing are exploited as versatile tools for studying lipid nanovesicles of small sizes and delicate structures. Here, an overview of microfluidics for precise manipulation and synthesis of lipid nanovesicles is provided. The mechanisms of isolation and detection of exosomes in microfluidics, as well as the clinical utility of exosomes for cancer diagnosis, are discussed. Several microfluidic designs for controlled assembly of a variety of lipid nanovesicles are highlighted. Opportunities and outstanding challenges of microfluidics-based investigation of lipid nanovesicles are discussed.

Microfluidic Preparation of Liposomes Using Ethyl Acetate/n-Hexane Solvents as an Alternative to Chloroform

Although liposomes have been used as a nutrient delivery carrier in the pharmaceutical, cosmetic, and food industries, they still suffer from the critical issue caused by the use of halogenated solvents (e.g., chloroform), which may be harmful to humans. Nonhalogenated solvents have been screened as candidate substitutes for chloroform based on their physicochemical properties. However, none of the candidates examined to date could form stable inverted micelles when used alone. Here, to obtain physicochemical properties similar to chloroform, combined mixtures were prepared using various ratios of each candidate. Based on the results of random combination trials with numerous candidates, ethyl acetate: n-hexane = 4 : 1(v/v) was selected as the optimum ratio because it could form stable inverted micelles and a transparent liposome solution without phase separation. The ethyl acetate and n-hexane mixture are a potential substitute for chloroform, which may resolve concerns regarding the toxicity of residual halogenated solvents in lipid nanovesicles.

Peripheral nerve targeting by procaine-conjugated ribavirin-loaded dual drug nanovesicle.

Procaine that is able to reach the peripheral nervous system (PNS) was conjugated as a ligand with lipid nanovesicle and loaded with ribavirin (a broad spectrum antiviral drug incapable of entering the PNS on its own) to target the PNS with a dual-drug effect. Different physicochemical characterizations,γ-scintigraphy and electromyography of the developed nanovesicle were conducted. Marked capability of the optimized radiolabeled formulation to target PNS was observed in rats. Electromyography signals were reduced after treatment with the formulation on conscious rats. The developed nanocarrier can deliver drug successfully at the PNS and reduce excitation of the nerve and thus give a better therapeutic option for treatment of various diseases and disorders of the PNS.

Flexible nano-sized lipid vesicles for the transdermal delivery of colchicine; in vitro/in vivo investigation

Colchicine (CL) is the most effective treatment of acute gout, however, it is associated with side effects in 80% of the patients at therapeutic doses, in addition, it's a water-soluble strong base (pKa ∼12.8) which ionizes at physiological gastrointestinal pH resulting in low oral bioavailability of 44%. This work employed enhancing the bioavailability and reducing the side effects of CL through combining the benefits of the transdermal route together with those of elastic lipid nano-vesicles. Transfersomes (TRs) have been studied as vehicles for transdermal drug delivery, however, poor encapsulation of drugs and drug leaking of the vesicles required complexation of CL with β-cyclodextrin (β-CD) before formulation. The composition of the designed CL-β-CD-TR was studied to balance the flexibility of the vesicles to their entrapment ability. CL-β-CD-TR were characterized for their shape, size, entrapment efficiency, elasticity, release profile, ex vivo skin permeation, pharmacological efficacy, and histopathological effect. Encapsulation efficiency of CL-β-CD complex in the vesicular formulations ranged from 42.3% to 93.8%. Particle size ranged from 70.6 nm to 138.5 nm and zeta potential ranged from 16.1 mV to 23.4 mV. The in vitro release of CL from the selected CL-β-CD-TR formulation (F3) showed a controlled, biphasic profile. Ex vivo study reported the great potential of F3 (CL-β-CD-TR) for skin permeation. In vivo experiment demonstrated that F3 (CL-β-CD-TR) possessed high biological efficacy with reduced skin irritation.

Formulation of Transfersomal Green Tea (Camellia sinensis L. Kuntze) Leaves Extract Cream and In Vitro Penetration Study Using Franz Difussion Cell

Introduction: Green tea is a natural ingredient that contains polyphenolic compounds in the form of catechin derivatives known as epigallocatechin gallate (EGCG). It has an antioxidant activity that is beneficial for body health. However, EGCG has a considerable molecular weight and high hydrophilicity resulting in the difficulty of skin penetration. The aim of this study was to improve EGCG penetration through the skin by using the formation of lipid nanovesicles, called transfersomes, that was formulated into a cream dosage form. Methods: Transfersomes were prepared by thin layer hydration method in three formulations with different concentration of phospholipid and surfactant. The amount of green tea leaves extract used for all formulations was same, (equal to 3% EGCG). All transfersomes formulation was characterized their physical properties, and the most promising formulation was chosen to be formulated into a cream dosage form. A cream contained untreated extract was prepared as a control. The in vitro penetration study of EGCG from both creams was evaluated using Franz diffusion cells with the abdomen skin of Sprague- Dawley rats as a membrane between the compartments. Results and Discussion: The results showed that F1 was the most promising and optimal formulation with a spherical shape, particle size of 80.6 nm, a polydispersity index of 0.214, a zeta potential at -48.2 ± 1.78 mV, and the highest entrapment efficiency of 49.36 ± 4.03%. The cumulative amount of EGCG penetrated from transfersomal and non transfersomal cream were 1003.61 ± 157.93 μg/cm2 and 400.09 ± 47.53 μg/cm2, respectively (P Key words: Cream, Epigallocatechin gallate, Franz diffusion cell, Green tea leaves extract, Transfersome.

Intravenous administration of synthetic platelets (SynthoPlate) in a mouse liver injury model of uncontrolled hemorrhage improves hemostasis.

Clinical resuscitative treatment of traumatic hemorrhage involves transfusion of RBC, platelets and plasma in controlled ratios. However, use of such blood components, especially platelets, present many challenges including availability, portability, contamination risks, and short shelf-life, which limit the use of platelet transfusions outside of large trauma centers such as remote civilian hospitals and austere prehospital settings. This has prompted significant research in platelet substitutes that may resolve the above issues while providing platelet-mimetic hemostatic action. In this framework, we have developed a synthetic platelet surrogate, SynthoPlate, by integrative decoration of platelet function mimetic peptides on a biocompatible lipid nanovesicle platform. We have previously demonstrated hemostatic capability of SynthoPlate in correcting tail-bleeding time in thrombocytopenic mice. Building on this, we hypothesized that SynthoPlate transfusion would decrease bleeding in a murine model of acute hemorrhagic shock. A validated model of uncontrolled intraperitoneal hemorrhage, via liver laceration was used to induce hemorrhagic shock in mice. SynthoPlate, control (unmodified) particles, and normal saline were administered as pretreatment and recue infusions to mice undergoing liver laceration and evaluated for hemostatic benefit by determining differences in blood loss and monitoring real-time hemodynamic data. Pretreatment SynthoPlate transfusion resulted in significant reduction of blood loss following hemorrhage, compared with control particles or normal saline treatment (0.86 ± 0.16 g control particles [CP] vs. 0.84 ± 0.13 g normal saline [NS] vs. 0.68 ± 0.09 g SynthoPlate, p < 0.005). SynthoPlate transfused mice demonstrated improved hemodynamics taking significantly longer to develop post-injury hypotension (168.3 ± 106.6 seconds CP vs. 137 ± 58 seconds NS vs. 546.7 ± 329.8 seconds SynthoPlate, p < 0.05). SynthoPlate infusion following liver laceration, that is, rescue transfusion, also resulted in a significant decrease in blood loss (0.89 ± 0.17 g CP vs. 0.92 ± 0.19 g NS vs. 0.69 ± 0.18 g SynthoPlate, p < 0.05). Transfusion of SynthoPlate particles reduces blood loss in a murine model of liver injury, and SynthoPlates may represent a viable transfusion product for the mitigation of blood loss in acute, severe hemorrhagic shock.

Absence of indicators of hypercoagulability and antiphospholipid syndrome in bxq-350 first in human study.

e14533Background: Saposin C is a lysosomal activator protein that can be combined with a phosphatidylserine lipid to create a nanovesicle that targets cancer cells. Similar lipid nanovesicle prepar...

Combinations of nanovesicles and physical methods for enhanced transdermal delivery of a model hydrophilic drug

Here, we combined lipid nanovesicles (ethosomes, liposomes) as the drug carrier systems with two physical methods (electroporation, sonoporation) to enhance transdermal delivery of a hydrophilic model molecule, calcein. First, using different formulations, ethosomes greatly enhanced calcein permeation by passive diffusion compared to liposomes and calcein in buffer, which is most likely due to a synergism between the ethanol action on the stratum corneum lipids and the penetration of the elastic vesicles. Liposomes permeated poorly through the skin and, as also suggested by other authors, seem to remain confined to the outer layers of the skin. By creating localized effects, liposomes would be better suited to topical dermal delivery than transdermal delivery. Using sonoporation as the physical enhancement method, sonication (5 min) showed improvement over passive diffusion; however, only for the ethosome formulation, and not for solution and liposomes. Similarly, electroporation greatly enhanced delivery of calcein, which was again more pronounced for ethosomes than liposomes and calcein in buffer. Finally, three different transdermal delivery enhancement methods were coupled, using ethosomes as carriers, along with both electroporation and sonoporation, to investigate the potential for synergistic effects. However, these combinations failed to achieve not only synergistic effects, but also additive effects. Nevertheless, combination of the ethosome formulation of calcein with either of electroporation or sonoporation achieves significant enhancement of transdermal molecular delivery being safe for potential clinical use.

Role of Lymphocyte Subsets in the Immune Response to Primary B Cell-Derived Exosomes.

Exosomes are lipid nanovesicles released after fusion of the endosomal limiting membrane with the plasma membrane. In this study, we investigated the requirement for CD4 T cells, B cells, and NK cells to provide help for CD8 T cell-mediated response to B cell-derived exosomes. CTL responses to Ag-loaded exosomes were dependent on host MHC class I, with a critical role for splenic langerin+ CD8α+ dendritic cells (DCs) in exosomal Ag cross-presentation. In addition, there was an absolute dependence on the presence of CD4 T cells, CD8 T cells, and NK cells, where the loss of any one of these subsets led to a complete loss of CTL response. Interestingly, NK cell depletion experiments demonstrated a critical cutoff point for depletion efficacy, with low-level residual NK cells providing sufficient help to allow optimal CD8 T cell proliferative responses to exosomal protein. Despite the potential role for B cells in the response to B cell-derived exosomal proteins, B cell depletion did not alter the exosome-induced CTL response. Similarly, a possible role for the BCR or circulating Ab in mediating CTL responses to B cell-derived exosomes was ruled out using DHLMP2A mice, which lack secreted and membrane-bound Ab, yet harbor marginal zone and follicular B cells. In contrast, CTL responses to DC-derived exosomes were significantly inhibited within Ab-deficient DHLMP2A mice compared with wild-type mice. However, this response was not restored upon serum transfer, implicating a role for the BCR, but not circulating Ab, in DC-derived exosome responses.

Liposomes in Polymersomes: Multicompartment System with Temperature-Triggered Release.

Multicompartmentalization is a key feature of eukaryotic cells, allowing separation and protection of species within the membrane walls. During the last years, several methods have been reported to afford synthetic multicompartment lipidic or polymeric vesicles that mimic biological cells and that allow cascade chemical or enzymatic reactions within their lumen. We hereby report on the preparation and study of liposomes in polymersomes (LiPs) systems. We discuss on the loading and coloading of lipidic nanovesicles made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipentadecanoyl-sn-glycero-3-phosphocholine (diC15-PC), or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) inside the lumen of giant poly(butadiene)-b-poly(ethylene oxide) (PBut-b-PEO) polymersomes. These LiPs systems were characterized by confocal microscopy and UV-visible spectroscopy. We further demonstrate that we can achieve controlled sequential release of dyes from diC15-PC and DPPC liposomes at defined temperatures inside the giant PBut-b-PEO polymersomes. This controlled release could be used as a means to initiate cascade reactions on demand in confined microreactors.

Novel Transdermal Ethosomal Gel Containing Green Tea (Camellia sinensis L. Kuntze) Leaves Extract: Formulation and In vitro Penetration Study

Background: One of the natural products that have many benefits is green tea ( Camellia sinensis L.). It can be used to maintain health because of its high antioxidant activity from epigallocatechin gallate (EGCG). However, EGCG has a low ability to penetrate through the skin due to its hydrophilicity. The aim of this study is to improve the penetration of EGCG through the skin by lipid nanovesicle, namely ethosome. Methods: Ethosomes were made using thin layer hydration method. Ethosomes were formulated with different concentration, equal to 1% (F1), 1.5% (F2), and 2% (F3) of EGCG from green tea leaves extract. They were characterized, then a chosen formula would be formulated into a gel dosage form (ethosomal gel). A gel without ethosome (non-ethosomal gel) was prepared as a control. Both of them were evaluated their penetration enhancement ability using Franz diffusion cells with the abdomen skin of Sprague-Dawley rats. Results and Discussion: Based on the results, F1 had a spherical shape, Dv90 129.00 ± 0.00 nm, polydispersity index 0.05 ± 0.00, zeta potential at -62.6 ± 5.05 mV, and the highest entrapment efficiency of 54.39 ± 0.03 %. The cumulative amount of EGCG penetrated from ethosomal and non-ethosmal gel were 1364.28 ± 56.32 μg/cm 2 and 490.17 ± 2.60 μ g/cm 2 , respectively ( p μ g/cm 2 . hour and 31.09 ± 0.29 μ g/cm 2 . hour, respectively. Conclusion: It can be concluded that ethosomal gel can increase the penetration of EGCG from green tea leaves extract. Key words: EGCG, Epigallocatechin Gallate, Ethosomes, Franz Diffusion Cell, Green Tea Leaves Extract, in-vitro Penetration Test.

Diclofenac Loaded Lipid Nanovesicles Prepared by Double Solvent Displacement for Skin Drug Delivery.

Herein, we detail a promising strategy of nanovesicle preparation based on control of phospholipid self-assembly: the Double Solvent Displacement. A systematic study was conducted and diclofenac as drug model encapsulated. In vitro skin studies were carried out to identify better formulation for dermal/transdermal delivery. This method consists in two solvent displacements. The first one, made in a free water environment, has allowed triggering a phospholipid pre-organization. The second one, based on the diffusion into an aqueous phase has led to liposome formation. Homogeneous liposomes were obtained with a size close to 100nm and a negative zeta potential around -40mV. After incorporation of acid diclofenac, we obtained nanoliposomes with a size between 101±45 and 133±66nm, a zeta potential between 34±2 and 49±3mV, and the encapsulation efficiency (EE%) was between 58±3 and 87±5%. In vitro permeation studies showed that formulation with higher EE% dispayed the higher transdermal passage (18,4% of the applied dose) especially targeting dermis and beyond. Our results suggest that our diclofenac loaded lipid vesicles have significant potential as transdermal skin drug delivery system. Here, we produced cost effective lipid nanovesicles in a merely manner according to a process easily transposable to industrial scale. Graphical Abstract ᅟ.

Chlorosome-Inspired Synthesis of Templated Metallochlorin-Lipid Nanoassemblies for Biomedical Applications.

Chlorosomes are vesicular light-harvesting organelles found in photosynthetic green sulfur bacteria. These organisms thrive in low photon flux environments due to the most efficient light-to-chemical energy conversion, promoted by a protein-less assembly of chlorin pigments. These assemblies possess collective absorption properties and can be adapted for contrast-enhanced bioimaging applications, where maximized light absorption in the near-infrared optical window is desired. Here, we report a strategy for tuning light absorption toward the near-infrared region by engineering a chlorosome-inspired assembly of synthetic metallochlorins in a biocompatible lipid scaffold. In a series of synthesized chlorin analogues, we discovered that lipid conjugation, central coordination of a zinc metal into the chlorin ring, and a 3(1)-methoxy substitution were critical for the formation of dye assemblies in lipid nanovesicles. The substitutions result in a specific optical shift, characterized by a bathochromically shifted (72 nm) Qy absorption band, along with an increase in absorbance and circular dichroism as the ratio of dye-conjugated lipid was increased. These alterations in optical spectra are indicative of the formation of delocalized excitons states across each molecular assembly. This strategy of tuning absorption by mimicking the structures found in photosynthetic organisms may spur new opportunities in the development of biophotonic contrast agents for medical applications.

Chitosan-modified lipid nanovesicles for efficient systemic delivery of l-asparaginase

The goal of this study was to evaluate the enhanced catalytic activity, increased stability, in vitro anti-cancer effects on H446 cells and in vivo bioavailability of novel enzyme delivery nanovesicles (l-asparaginase containing chitosan modified lipid nanovesicles, ACLNs) when administered intravenously. It was the first time for the chitosan-modified lipid nanovesicles to be fabricated to deliver l-asparaginase (ASP, a therapeutic enzyme) efficiently. It was indicated that ACLNs markedly increased the enzymatic activity, improved the temperature/acid-base/proteolytic stabilities and favorably changed the in vivo kinetic characteristics. Moreover, ACLNs exhibited higher anti-lung-cancer activity than free ASP. The possible existence status of ASP in ACLNs and the fluorescence changes of ACLNs reflecting the conformational changes after heat treatment were preliminary explored. ACLNs might be novel promising nanovesicles for effective systemic delivery of therapeutic enzyme ASP.

Stable J-aggregation enabled dual photoacoustic and fluorescence nanoparticles for intraoperative cancer imaging.

J-aggregates display nanoscale optical properties which enable their use in fluorescence and photoacoustic imaging applications. However, control over their optical properties in an in vivo setting is hampered by the conformational lability of the J-aggregate structure in complex biological environments. J-aggregating nanoparticles (JNP) formed by self-assembly of bacteriopheophorbide-lipid (Bchl-lipid) in lipid nanovesicles represents a novel strategy to stabilize J-aggregates for in vivo bioimaging applications. We find that 15 mol% Bchl-lipid embedded within a saturated phospholipid bilayer vesicle was optimal in terms of maximizing Bchl-lipid dye loading, while maintaining a spherical nanoparticle morphology and retaining spectral properties characteristic of J-aggregates. The addition of cholesterol maintains the stability of the J-aggregate absorption band for up to 6 hours in the presence of 90% FBS. In a proof-of-concept experiment, we successfully applied JNPs as a fluorescence contrast agent for real-time intraoperative detection of metastatic lymph nodes in a rabbit head-and-neck cancer model. Lymph node metastasis delineation was further verified by visualizing the JNP within the excised lymph node using photoacoustic imaging. Using JNPs, we demonstrate the possibility of using J-aggregates as fluorescence and photoacoustic contrast agents and may potentially spur the development of other nanomaterials that can stably induce J-aggregation for in vivo cancer bioimaging applications.

Organized Aggregation of Porphyrins in Lipid Bilayers for Third Harmonic Generation Microscopy

AbstractNonlinear optical microscopy has become a powerful tool for high‐resolution imaging of cellular and subcellular composition, morphology, and interactions because of its high spatial resolution, deep penetration, and low photo‐damage to tissue. Developing specific harmonic probes is essential for exploiting nonlinear microscopic imaging for biomedical applications. We report an organized aggregate of porphyrins (OAP) that formed within lipidic nanoparticles showing fingerprint spectroscopic properties, structure‐associated second harmonic generation, and superradiant third harmonic generation. The OAP facilitated harmonic microscopic imaging of living cells with significantly enhanced contrast. The structure‐dependent switch between harmonic (OAP‐intact) and fluorescence (OAP‐disrupted) generation enabled real‐time multi‐modality imaging of the cellular fate of nanoparticles. Robustly produced under various conditions and easily incorporated into pre‐formed lipid nanovesicles, OAP provides a biocompatible nanoplatform for harmonic imaging.

Organized Aggregation of Porphyrins in Lipid Bilayers for Third Harmonic Generation Microscopy.

Nonlinear optical microscopy has become a powerful tool for high-resolution imaging of cellular and subcellular composition, morphology, and interactions because of its high spatial resolution, deep penetration, and low photo-damage to tissue. Developing specific harmonic probes is essential for exploiting nonlinear microscopic imaging for biomedical applications. We report an organized aggregate of porphyrins (OAP) that formed within lipidic nanoparticles showing fingerprint spectroscopic properties, structure-associated second harmonic generation, and superradiant third harmonic generation. The OAP facilitated harmonic microscopic imaging of living cells with significantly enhanced contrast. The structure-dependent switch between harmonic (OAP-intact) and fluorescence (OAP-disrupted) generation enabled real-time multi-modality imaging of the cellular fate of nanoparticles. Robustly produced under various conditions and easily incorporated into pre-formed lipid nanovesicles, OAP provides a biocompatible nanoplatform for harmonic imaging.

Lipidic nanovesicles stabilize suspensions of metal oxide nanoparticles

We have studied the effect of adding lipid nanovesicles (liposomes) on the aggregation of commercial titanium oxide (TiO2), zinc oxide (ZnO), or cerium oxide (CeO2) nanoparticles (NPs) suspensions in Hepes buffer. Liposomes were prepared with pure phospholipids or mixtures of phospholipids and/or cholesterol. Changes in turbidity were recorded as a function of time, either of metal nanoparticles alone, or for a mixture of nanoparticles and lipidic nanovesicles. Lipid nanovesicles markedly decrease the NPs tendency to sediment irrespective of size or lipid compositions, thus keeping the metal oxide NPs in suspension. Cryo-electron microscopy, fluorescence anisotropy of TMA-DPH and general polarization of laurdan failed to reveal any major effect of the NPs on the lipid bilayer structure or phase state of the lipids. The above data may help in developing studies of the interaction of inhaled particles with lung surfactant lipids and alveolar macrophages.

Effective delivery of recombinant proteins to rod photoreceptors via lipid nanovesicles

The potential of liposomes to deliver functional proteins in retinal photoreceptors and modulate their physiological response was investigated by two experimental approaches. First, we treated isolated mouse retinas with liposomes encapsulating either recoverin, an important endogenous protein operating in visual phototransduction, or antibodies against recoverin. We then intravitrally injected in vivo liposomes encapsulating either rhodamin B or recoverin and we investigated the distribution in retina sections by confocal microscopy. The content of liposomes was found to be released in higher amount in the photoreceptor layer than in the other regions of the retina and the functional effects of the release were in line with the current model of phototransduction. Our study sets the basis for quantitative investigations aimed at assessing the potential of intraocular protein delivery via biocompatible nanovesicles, with promising implications for the treatment of retinal diseases affecting the photoreceptor layer.

Content Delivery of Lipidic Nanovesicles in Electropermeabilized Cells.

Lipidic nanovesicles (the so-called liposomes) were among the one of the earliest forms of nanovectors. One of their limits was our lack of knowledge on the delivery pathway of their content to the target cell cytoplasm. In most models, it appears to be linked to endocytotic transfer. Their direct content delivery can be enhanced by electric field pulses applied to a cell liposomes mixture. The optimal form for liposomes was shown to be large unilamellar vesicles (LUV). The present communication describes an optimization to enhance the delivery. When lipidic nanovesicles (LUVs) are electrostatically brought in contact with electropermeabilized cells by a salt bridge, their content is delivered into the cytoplasm of electropermeabilized cells. The PEF parameters are selected to affect specifically the cells leaving the vesicles unaffected. Cell viability is positively affected by the treatment. High-field short pulses are more efficient than low-field long pulses. A homogeneous cytoplasm labeling is observed under digitized videomicroscopy. The process is a content mixing, not an endocytotic pathway. The lipidic composition of the LUV should contain charged lipids (phosphatidylserine), fusion promoting lipids (phosphatidylethanolamine), and cholesterol.