Non-phospholipid Liposomes Research Articles

Development of hyaluronic acid hydrogel containing prednisolone-encapsulated nonphospholipid liposomes for the treatment of rheumatoid arthritis.

Rheumatoid arthritis (RA) requires therapeutic approaches that alleviate symptoms and inhibit the progression of joint damage. Glucocorticoids (GCs) have been a cornerstone of RA treatment, yet their use is often limited by side effects. Recent advancements suggest that liposome-based delivery systems can improve GC biodistribution, minimizing toxicity. This study introduces an innovative tool for RA treatment using prednisone-encapsulated nonphospholipid liposomes (NPLs) in combination with a hyaluronic acid (HA) hydrogel. Our methodology involved incorporating prednisone (PR) with palmitic acid and cholesterol to formulate stable NPLs using a thin-film hydration technique. The synthesized PR-NPLs, characterized by a mean size of 150 nm, demonstrated uniform distribution and higher drug encapsulation in comparison with conventional phospholipid liposomes. In vitro assays revealed that PR-NPL markedly reduced inflammatory responses in macrophages. Additionally, we successfully incorporated PR-NPL into an HA hydrogel, employing a photoinitiated cross-linking process. This novel composite offered modulable PR release, governed by the degree of hydrogel cross-linking. The developed system presents a promising advancement in RA management, especially suited for intraarticular injections. It potentially enables targeted, controlled drug release with a reduced risk of side effects, signifying a significant improvement over existing RA therapies.

Fabrication of Non-phospholipid Liposomal Nanocarrier for Sustained-Release of the Fungicide Cymoxanil

Liposome nanocarriers can be used to solve problems of pesticide instability, rapid degradation and a short period of efficacy. Cymoxanil with antifungal activity requires an ideal drug loading system due to its degradation issues. In this paper, cholesterol and stearylamine were used to prepare non-phospholipid liposomes (sterosomes) as a pesticide nanocarrier, and were characterized with field emission scanning electron microscopy (FE-SEM), X-ray powder diffraction (XRD), Fourier-transform infrared (FT-IR) spectrometer, size distribution, and ζ-potential. The results showed sterosomes were successfully loaded with cymoxanil. The loading efficiency and the drug-to-lipid ratio were 92.6% and 0.0761, respectively. Prolonged drug release was obtained for 3 days, improving the short duration of the drug itself. The addition of cymoxanil-loaded sterosomes in culture medium effectively inhibited the growth of yeast cells, which serve as model fungal targets. Sterosomes as nanocarriers significantly improved the stability and efficacy of cymoxanil, thus introducing practical and economically desirable strategies for the preparation of novel pesticide formulations.

Preparation of photothermal palmitic acid/cholesterol liposomes.

Indocyanine green (ICG) is the only FDA-approved near-infrared dye and it is currently used clinically for diagnostic applications. However, there is significant interest in using ICG for triggered drug delivery applications and heat ablation therapy. Unfortunately, free ICG has a short half-life in vivo and is rapidly cleared from circulation. Liposomes have been frequently used to improve ICG's stability and overall time of effectiveness in vivo, but they have limited stability due to the susceptibility of phospholipids to hydrolysis and oxidation. In this study, nonphospholipid liposomes were used to encapsulate ICG, and the resulting liposomes were characterized for size, encapsulation efficiency, stability, and photothermal response. Using the thin-film hydration method, an ICG encapsulation efficiency of 54% was achieved, and the liposomes were stable for up to 12 weeks, with detectable levels of encapsulated ICG up to week 4. Additionally, ICG-loaded liposomes were capable of rapidly producing a significant photothermal response upon exposure to near-infrared light, and this photothermal response was able to induce changes in the mechanical properties of thermally responsive hydrogels. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1384-1392, 2019.

Design and Characterization of a Therapeutic Non-phospholipid Liposomal Nanocarrier with Osteoinductive Characteristics To Promote Bone Formation.

Sterosomes are recently developed types of non-phospholipid liposomes formed from single-chain amphiphiles and high content of sterols. Although sterosomes presented significantly increased stability compared to conventional phospholipid liposomes, current sterosome biomaterials are not truly bioactive and have no intrinsic therapeutic effects. The purpose of this study was to develop a sterosome formulation with osteoinductive properties by an effective selection of sterol, one of the sterosome components. Oxysterols are oxidized derivatives of cholesterol and are known to stimulate osteogenesis and bone formation. Thus, 20S-hydroxycholesterol (Oxy), one of the most potent oxysterols for bone regeneration, was examined as a promising candidate molecule to form fluid lamellar phases with a single-chain amphiphile, namely, stearylamine (SA). First, the optimal composition was identified by investigating the phase behavior of SA/Oxy mixtures. Next, the capacity of the optimized SA/Oxy sterosomes to promote osteogenic differentiation of bone marrow stromal cells was assessed in vitro in a hydrogel environment. Furthermore, we explored the effects of osteogenic oxysterol sterosomes in vivo with the mouse critical-sized calvarial defect model. Our results showed that SA/Oxy sterosomes induced osteogenic differentiation in vitro and enhanced calvarial healing without delivery of additional therapeutic agents, indicating their intrinsic bone-forming potential. This study suggests a promising non-phospholipid liposomal platform with osteoinductive properties for delivery of small molecular drugs and/or other therapeutic genes for enhanced bone formation.

Delivery of siRNA via cationic Sterosomes to enhance osteogenic differentiation of mesenchymal stem cells

Noggin is a specific antagonist of bone morphogenetic proteins (BMPs) that can prevent the interaction of BMPs with their receptors. RNA interfering molecules have been used to downregulate noggin expression and thereby stimulate BMP signaling and osteogenesis. Cationic liposomes are considered one of the most efficient non-viral systems for gene delivery. In the past decade, non-phospholipid liposomes (Sterosomes) formulated with single-chain amphiphiles and high content of sterols have been developed. In particular, Sterosomes composed of stearylamine (SA) and cholesterol (Chol) display distinct properties compared with traditional phospholipid liposomes, including increased positive surface charges and enhanced particle stability. Herein, we report SA/Chol Sterosome and small interfering RNA (siRNA) complexes that significantly enhanced cellular uptake and gene knockdown efficiencies in adipose derived mesenchymal stem cells with minimal cytotoxicity compared with commercially available lipofectamine 2000. Furthermore, we confirmed osteogenic efficacy of these Sterosomes loaded with noggin siRNA in in vitro two- and three-dimensional settings as well as in a mouse calvarial defect model. The delivery of siRNA via novel SA/Chol Sterosomes presents a powerful method for efficient gene knockdown. These distinct nanoparticles may present a promising alternative approach for gene delivery.

Nonphospholipid fluid liposomes with switchable photocontrolled release.

We created novel nonphospholipid photosensitive liposomes from a mixture of a monoacylated azobenzene amphiphile (AzoC10N(+)) and cholesterol sulfate (Schol). This system belongs to the family of sterol-enriched nonphospholipid liposomes that were shown to form stable large unilamellar vesicles (LUVs) with enhanced impermeability. Fluid bilayers were successfully prepared from AzoC10N(+)/Schol (25/75 molar ratio) mixtures, and LUVs could be derived at room temperature using standard extrusion methods. The isomerization process of the bilayer-inserted AzoC10N(+) was characterized. Leakage from these liposomes could be induced by the photoconversion of AzoC10N(+) from its trans form to its cis form. This photocontrolled release from fluid liposomes contrasts with the case of phospholipid-based azo-containing liposomes, which are generally required to be in the gel phase to be photosensitive. It is proposed that the very high degree of conformational order of the monoalkylated amphiphile and the tight packing of the hydrophobic core of the AzoC10N(+)/Schol liposomes make them responsive to the presence of the bulky cis azo isomer. Interestingly, the liposome impermeability could be fully restored by the photoisomerization of the cis form back to the trans form, providing a sharp on-and-off control of payload release. In addition, these nonphospholipid liposomes display a very limited passive release. Therefore, it is shown that AzoC10N(+)/Schol LUVs can be used as nanocontainers, whose content can be released by light in a controlled and switchable manner.

Impact of interfacial cholesterol-anchored polyethylene glycol on sterol-rich non-phospholipid liposomes

HypothesisLiposomes made of single-chain amphiphiles and a large amount of sterols display several advantages including a limited permeability. In the present paper, we examine the possibility to prepare such non-phospholipid liposomes with interfacial polyethylene glycol (PEG) in order to improve their circulation in the blood stream. Cholesterol (Chol) was chosen as the PEG anchor. ExperimentsThe phase behavior of mixtures of palmitic acid (PA) and cholesterol including various proportions of PEGylated cholesterol (PEG-Chol) was characterized. In conditions leading to the formation of fluid bilayers, properties of the resulting liposomes were assessed. FindingsUp to 20mol% of PEGylated cholesterol could be introduced without significant perturbations in fluid bilayers made of PA and cholesterol. With 10mol% PEG-Chol, PA/Chol/PEG-Chol liposomes showed a very limited permeability to calcein and doxorubicin. Doxorubicin could be actively loaded in PA/Chol/PEG-Chol liposomes with a high drug loading efficiency and a high drug to lipid ratio. Pharmaco-kinetic experiments in rats indicated that interfacial PEG reduced the clearance of PA/Chol liposomes compared to the naked ones. However the lifetime of these non-phospholipid liposomes in the blood circulation was considerably shorter than that observed for control PEGylated phospholipid liposomes, a phenomenon associated with the negative interfacial charge of the PA/Chol/PEG-Chol liposomes.

Interactions between non-phospholipid liposomes containing cetylpyridinium chloride and biofilms of Streptococcus mutans: modulation of the adhesion and of the biodistribution

Cetylpyridinium chloride (CPC) is a surfactant that binds strongly to bacteria and bacterial biofilms. In this study, fluorescence-based techniques were used to determine the penetration and adhesion of CPC when it was introduced in liposomes. In spite of a reduced adhesion as compared to pure CPC micelles, CPC-containing liposomes adhered significantly to the biofilms of Streptococcus mutans. In contrast, no binding was observed for liposomes that were composed of phosphatidylcholine-cholesterol. The influence of the charge of the liposome on its adhesion to biofilms was studied using cholesterol (Chol) and cholesterol sulfate (Schol). In spite of similar binding to the biofilms, positively charged CPC/Chol liposomes were located mainly in the core of the biofilm microcolonies, whereas the negatively charged CPC/Schol liposomes were mainly concentrated at their periphery. This effect may be attributed to the different availability of the CPC head group. In summary, this work demonstrates the high potential for tailoring drug nanovectors by modulating sterol selection in order to selectively target and bind biofilms.

Interaction of pH-sensitive non-phospholipid liposomes with cellular mimetic membranes

Surfactant nanocarriers have received considerable attention in the last several years as interesting alternative to classic liposomes. Different pH-sensitive vesicular colloidal carriers based on Tween 20 derivatives, obtained after functionalization of the head groups of the surfactant with natural, or simply modified, amino acids, were proposed as drug nanocarriers. Dynamic light scattering, Small Angle X-ray Scattering, Trasmission Electron Microscopy and fluorescence studies were used for the physico-chemical characterization of vesicles and mean size, size distribution, zeta potential, vesicle morphology and bilayer properties were evaluated. The pH-sensitivity and the stability of formulations, in absence and in presence of foetal bovine serum, were also evaluated. Moreover, the contact between surfactant vesicles and liposomes designed to model the cellular membrane was investigated by fluorescence studies to preliminary explore the potential interaction between vesicle and cell membranes. Experimental findings showed that physico-chemical and technological features of pH-sensitive vesicles were influenced by the composition of the carriers. Furthermore, proposed carriers are able to interact with mimetic cell membrane and it is reasonable to attribute the observed differences in interaction to the architectural/structural properties of Tween 20 derivatives. The findings reported in this investigation showed that a deep and extensive physico-chemical characterization of the carrier is a fundamental step, according to the evidence that the knowledge of nanocarrier properties is necessary to translate its potentiality to in vitro/in vivo applications.

Non-phospholipid liposomes with high sterol content display a very limited permeability

We demonstrate that it is possible to form non-phospholipid fluid bilayers in aqueous milieu with a mixture of palmitic acid (PA), cholesterol (Chol), and cholesterol sulfate (Schol) in a molar proportion of 30/28/42. These self-assemblies are shown to be bilayers in the liquid ordered phase. They are stable between pH 5 and 9. Over this pH range, the protonation/deprotonation of PA carboxylic group is observed but this change does not appear to alter the stability of these bilayers, a behavior contrasting with that observed for binary mixtures of PA/Chol, and PA/Schol. The multilamellar dispersions formed spontaneously from the PA/Chol/Schol mixture could be successfully extruded to form Large Unilamellar Vesicles (LUVs). These LUVs show interesting permeability properties, linked with their high sterol content. These non-phospholipid liposomes can sustain a pH gradient (pHinternal 8/pHexternal 6) 100 times longer than LUVs made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and cholesterol, with a molar ratio of 60/40. Moreover, the non-phospholipid LUVs are shown to protect ascorbic acid from an oxidizing environment (1 mM iron(III)). Once entrapped in liposomes, ascorbic acid displays a degradation rate similar to that obtained in the absence of iron(III). These results show the possibility to form novel nanocontainers from a mixture of a monoalkylated amphiphile and sterols, with a good pH stability and showing interesting permeability properties.

Formation of Fluid Lamellar Phase and Large Unilamellar Vesicles with Octadecyl Methyl Sulfoxide/Cholesterol Mixtures

Systems composed of a monoalkylated amphiphile and a sterol have been shown to form stable liquid-ordered (lo) lamellar phases; these include negatively charged mixtures of unprotonated palmitic acid/cholesterol (Chol) or cholesterol sulfate (Schol) and mixtures of positively charged cetylpyridinium chloride/Schol. Large unilamellar vesicles (LUVs) could be formed by these systems, using conventional extrusion methods. The passive permeability of these LUVs was drastically limited, a phenomenon associated with the high sterol content. In the present paper, we showed that octadecyl methyl sulfoxide (OMSO), a neutral monoalkylated amphiphile, can form, in the presence of cholesterol, LUVs that are stable at room temperature. Differential scanning calorimetry, infrared spectroscopy, and nuclear magnetic resonance spectroscopy of deuterium were used to characterize the phase behavior of OMSO/Chol mixtures. A temperature-composition diagram summarizing the behavior of the OMSO/Chol system is proposed; it includes a eutectic with an OMSO/Chol molar ratio of 5/5. It is found that the fluid phase observed at temperature higher than 43 degrees C is metastable at room temperature, and this situation allows extruding these mixtures to form stable LUVs at room temperature. This distinct behavior is associated with the strong H-bond capability of the sulfoxide group. The properties associated with this neutral formulation expand the potential of these non-phospholipid liposomes for applications in several areas such as drug delivery.

Development of Non-Phospholipid Liposomes Containing a High Cholesterol Concentration

We present a novel formulation of non-phospholipid liposomes formed from cholesterol and palmitic acid. Despite the fact that these two lipidic species do not form individually fluid bilayers, we show that once mixed together, fluid bilayers can be obtained and, moreover, these can be extruded using classical extrusion processes to form liposomes. The chemical analysis indicates that these liposomes contain 70 mol % cholesterol, a content that is considerably higher that the saturation limit generally reported for phospholipid bilayers. These cholesterol-rich liposomes, formed with molecules that have low toxicity in vivo, display an improved impermeability relative to that of traditional phospholipid liposomes. In addition, because of the presence of palmitic acid, the stability of the liposomes is pH-dependent, and it is possible to trigger the release of encapsulated materials by pH stimuli.

Liposomal Formulations of Inflammatory Bowel Disease Drugs: Local versus Systemic Drug Delivery in a Rat Model

Based on adherence to intestinal mucosa, intralumenally administered liposomal formulations of 5-aminosalicylate (5-ASA) and 6-mercaptopurine (6-MP) were studied for their potential to enhance local drug delivery to intestinal tissue for the treatment of inflammatory bowel disease. 5-ASA was encapsulated in standard phospholipid liposomes while 6-MP required encapsulation in nonphospholipid liposomes to obtain equivalent drug loading. Encapsulation efficiency was measured by size-exclusion chromatography/high-performance liquid chromatogtaphy (HPLC). Liposomal formulations or solution of the drugs were injected into unligated jejunum to compare pharmacokinetics and into ligated loops of rat ileum and colon to evaluate local delivery. Dextran sulfate and acetic acid induced colitis were used as models of lower intestinal inflammation. Plasma, tissue and luminal drug and metabolite levels were measured by liquid scintillation counting or HPLC. Encapsulation efficiency of 6-MP was dependent on lipid content and composition. While liposomal encapsulation significantly reduced systemic absorption of 5-ASA this was not the case for 6-MP. Liposomal adherence to intestinal tissue resulted in increased tissue levels for 5-ASA; however, 6-MP local tissue levels were not improved compared to solution drug. Nonphospholipid liposomes optimize encapsulation of 6-MP. While liposomal formulations show potential for local drug delivery to diseased bowel, drug physicochemical properties, absorption, and metabolic profiles dictate tissue-targeting potential. Liposomes reduce systemic availability from paracellular absorption of hydrophilic 5-ASA, but fail to improve local tissue delivery of 6-MP, a molecule absorbed by passive membrane permeation that undergoes extensive first- pass metabolism.

An assessment by fluorescence spectroscopy of the stability of polyanions/positively charged liposome systems in the presence of polycations

The interactions between cationic non-phospholipid liposomes and pyrene-labeled hydrophobically modified sodium poly-2-(acrylamido)-2-methylpropane sulfonates (HM-PAMPS) have been examined by fluorescence spectroscopy and dynamic light scattering. Evidence from changes in the relative intensity of pyrene excimer and monomer emissions indicates that the polyanions bind to cationic liposomes and that, for systems in aqueous 0.2M NaCl, the binding is only mildly affected by the charge density of the liposomes. The reversibility of the polyanion binding to the liposomes was assessed by addition of the polycation (N-isopropylacrylamide)–N-dimethylethyl(2-acrylamidoethyl)ammonium bromide copolymer (PNIPAM–DMEAB). Based on the level of pyrene fluorescence recovery upon addition of the polycation to complexes between PAMPS and liposomes containing a quencher of fluorescence in their bilayer, we demonstrate reversibility of the binding of sodium poly-2-[acrylamido-2-methylsulfonate–N-(1-pyrenylmethyl)]acrylamide (PAMPS–Py1). In contrast, under the same conditions, a HM-PAMPS sample carrying along its backbone 5mol% of n-octadecyl groups is not desorbed from the surface of the liposome.

Contribution of Hydrogen Bonding to the Association of Liposomes and an Anionic Hydrophobically Modified Poly(N-isopropylacrylamide)

The interactions between a random copolymer of N-isopropylacrylamide (NIPAM), N-[4-(1-pyrenyl)-butyl]-N-n-octadecylacrylamide, and N-glycylacrylamide (Gly) (PNIPAM-Py-Gly) with a series of vesicles have been examined by fluorescence spectroscopy, centrifugation assays, and capillary electrophoresis. The copolymer exhibits pH-sensitivity due to the carboxylic acid group of the glycine residues (15 mol %), thermosensitivity due to the NIPAM residues (84 mol %), and amphiphilic characteristics due to the pyrenyl octadecyl moieties (1 mol %). In the presence of salts, complex formation was detected between the copolymer and cationic or anionic nonphospholipid liposomes based on the nonionic surfactant n-octadecyldiethylene oxide ((EO) 2 C 18 H 37 ) and cationic phospholipid liposomes. Weak interaction was detected between the copolymer and neutral phospholipid liposomes under the same conditions. The effects of the charge density of the liposomes and the lipid concentration on the copolymer/liposome interactions were examined as a function of pH and temperature. The binding of the copolymer to the liposomes is shown to be controlled primarily through hydrogen bond formation between the hydroxyl groups of the (EO) 2 C 18 H 37 and the amide groups of the NIPAM residues, through electrostatic interactions between the charged surfactants of the bilayer and the glycine residues, and, a lesser extent, by hydrophobic forces.

Non-phospholipid fusogenic liposomes

We have demonstrated the capacity of non-phospholipid liposomes composed primarily of dioxyethylene acyl ethers and cholesterol to fuse with membranes composed primarily of phospholipid. Phase-contrast microscopy, freeze-fracture electron microscopy and a macromolecular probe indicate that these non-phospholipid liposomes can fuse with the plasma membranes of erythrocytes and fibroblasts. Furthermore, fluorescence probe experiments have demonstrated fusion between phosphatidylcholine liposomes and non-phospholipid liposomes. Mixing of internal contents was shown by a terbium/dipicolinate assay. Mixing of membrane lipid components was demonstrated by measuring (i) fluorescence resonance energy transfer between N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine and N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine, after phosphatidylcholine liposomes were mixed with non-phospholipid liposomes, and (ii) reduced concentration quenching of rhodaminephosphatidylethanolamine and octadecylrhodamine incorporated into phosphatidylcholine liposomes after mixing with the non-phospholipid liposomes. The degree of apparent fusion reported by the different probe techniques ranged from 25% to 64%.

Adjuvant properties of non-phospholipid liposomes (Novasomes ®) in experimental animals for human vaccine antigens

Non-phospholipid liposomes composed of dioxyethylene cetyl ether, cholesterol and oleic acid were evaluated as adjuvants with human vaccine antigens, tetanus toxoid (TT) and diphtheria toxoid (DT), in mice and rabbits. Antigens encapsulated in or mixed with liposomes elicited antitoxin levels similar to those elicited by antigens given with Freund's adjuvant or adsorbed onto aluminum phosphate. All liposomal antigen preparations, antigen given with Freund's adjuvant or adsorbed onto aluminum phosphate, elicited significantly higher IgG antibodies and antitoxin levels than soluble antigens in mice after a single injection and in rabbits after each of three injections. TT encapsulated in liposomes elicited sustained anti-TT IgG antibody levels in mice after a single injection as compared to TT mixed with liposomes. TT mixed with or encapsulated within liposomes containing monophosphoryl lipid Alsqualene or squalene alone, as well as aluminum phosphate adsorbed TT elicited greater primary responses in mice than TT mixed with or encapsulated within plain liposomes. Liposomal TT preparations produced a slightly higher anamnestic response in mice than aluminum phosphate adsorbed TT. Subclass analysis of anti-TT antibodies showed that the majority of the antibodies belong to IgG1 subclass. Liposomal TT preparations, particularly those with encapsulated monophosphoryl lipid Alsqualene or squalene alone, consistently elicited higher levels of anti-TT IgG2a and IgG2b than aluminum phosphate adsorbed or soluble TT. None of the preparations elicited IgG3 or IgM antibodies. It appears that non-phospholipid liposomes are as potent adjuvants as the currently employed adjuvant for human vaccines (aluminum phosphate) or a benchmark adjuvant for experimental immunology (Freund's adjuvant), and may be able to modulate the immune response towards the Th1 type.

Encapsulation of hemoglobin in non-phospholipid vesicles.

The efficiency of encapsulating hemoglobin in non-phospholipid liposomes by rapidly mixing hemoglobin with lipids heated above their solid-liquid phase transition temperature was examined. Human hemoglobin was mixed at 55-60 degrees C with a lipid solution containing polyoxyethylene-2 cetyl ether and cholesterol (molar ratio, 3:1) at 60-65 degrees C. Repeated mixing was carried out through a high-shear orifice, followed by rapid cooling and additional mixing. Lipid vesicles were heterogeneous in size, with diameters from approximately 300 nm to 10 microns. The non-encapsulated aqueous phase was removed by centrifugation, and total hemoglobin was determined spectrophotometrically. Encapsulation efficiency was calculated as the percentage of hemoglobin associated with the liposome phase (i.e., encapsulated) as a function of hemoglobin concentration and the aqueous:lipid hydration ratio. Hemoglobin concentrations were varied from 1 to 10 nM (in heme). Aqueous:lipid ratios of 8:1 and 4:1 were tested. Percent encapsulation varied from 13-30%, with the greatest efficiency, i.e., 30%, at a 4:1 hydration ratio of hemoglobin:lipid at 5.6 mM hemoglobin.