Search

Camurus’ FluidCrystal® injection depot is a lipid liquid crystal (LLC) phase formation-based method, comprising of glycerol dioleate (GDO) and soy phosphatidylcholine (SPC), together with minute quantities of N-methyl-2-pyrrolidone solvent (NMP). The present study aimed to develop a method for LLC using sorbitan monooleate (LLC-SMO) instead of GDO to prepare a one-month sustained-release formulation of naltrexone (NTX) that is applied for the treatment of autism and treating alcohol dependence. The optical characteristics of the LLC were assessed by polarizing light microscopy (PLM) to reveal the presence of lamellar, hexagonal, and cubic mesophases. Furthermore, in vitro release of NTX and NMP, degradation, pharmacokinetics, and histopathology of LLC-GDO and LLC-SMO in rats were evaluated and compared to those of Vivitrol®. The PLM images revealed that the structure of LLC-SMO is hexagonal, similar to LLC-GDO. The in vitro release of NTX and its pharmacokinetic results in rats indicted that the LLC-SMO system is more uniform than LLC-GDO and Vivitrol® during 35 days. Histopathological results of LLC-GDO and LLC-SMO confirmed the biocompatibility of our LLC delivery systems. Taken together these data demonstrate that the LLC-SMO-based method, was efficient enough to sustain the release of NTX in vitro and in vivo, confirming the biocompatible nature of this delivery system.

This study explored the transition of lamellar-type liquid crystal (LLC) to biocompatible oil-in-water nanoemulsions able to modify benznidazole (BNZ) release and target the drug to cells infected with the T. cruzi parasite. Three cosolvents (2methylpyrrolidone [NMP], polyethylene glycol [POL], and propylene glycol [PRO] were tested to induce the transition of anisotropic LLC systems to isotropic nanoemulsions. Mixtures of soy phosphatidylcholine with sodium oleate stabilized the dispersions of medium chain triglyceride in water. Rheological measurements, polarized microscopy, and small angle X-ray scattering demonstrated that there is a phase transition from LLC to desired nanoemulsions. These small and narrow droplet-sized nanocarriers exhibited some advantages and promising features, such as the enhanced BNZ aqueous solubility and slow drug release rate. In vitro cell biocompatibility of formulations was assessed in the Vero E6 and SiHa cell lines. Drug-loaded nanoemulsions inhibited the epimastigote growth of the T. cruzi parasite (IC50 0.208 ± 0.052 μg mL−1) and reduced its infective life form trypomastigote (IC50 0.392 ± 0.107 μg mL−1). The oil-in-water nanoemulsions were demonstrated as promising biocompatible liquid drug delivery systems capable of improving the BNZ trypanocidal activity for the treatment of Chagas disease.

Microfluidic enables precise control over the continuous mixing of fluid phases at the micrometre scale, aiming to optimize the processing parameters and to facilitate scale-up feasibility. The optimization of parameters to obtain monodispersed drug-loaded liposomes however is challenging. In this work, two phosphatidylcholines (PC) differing in acyl chain length were selected, and used to control the release of the chemotherapeutic agent doxorubicin hydrochloride, an effective drug used to treat breast cancer. Microfluidics was used to rapidly screen manufacturing parameters and PC formulations to obtain monodispersed unilamellar liposomal formulations with a reproducible size (i.e. 200 nm). Cholesterol was included in all liposomal formulations; some formulations also contained DMPC(1,2-dimyristoyl-sn-glycero-3-phosphocholine) and/or DSPC(1,2-distearoyl-sn-glycero-3-phosphocholine). Systematic variations in microfluidics total flow rate (TFR) settings were performed, while keeping a constant flow rate ratio (FRR). A total of six PC-based liposomes were fabricated using the optimal manufacturing parameters (TFR 500 μL/min, FRR 0.1) for the production of reproducible, stable liposome formulations with a narrow size distribution. Liposomes actively encapsulating doxorubicin exhibited high encapsulation efficiencies (80%) for most of the six formulations, and sustained drug release profiles in vitro over 48 h. Drug release profiles varied as a function of the DMPC/DSPC mol content in the lipid bilayer, with DMPC-based liposomes exhibiting a sustained release of doxorubicin when compared to DSPC liposomes. The PC-based liposomes, with a slower release of doxorubicin, were tested in vitro, as to investigate their cytotoxic activity against three human breast cancer cell lines: the non-metastatic ER+/PR + MCF7 cells, the triple-negative aggressive MDA-MB 231 cells, and the metastatic HER2-overexpressing/PR + BT474 cells. Similar cytotoxicity levels to that of free doxorubicin were reported for DMPC

Ethosomes are widely applied as the carriers for the transdermal delivery of hydrophobic and hydrophilic drugs. Herein, curcumin-loaded ethosomes (CE) with different phospholipid composition were formulated and thoroughly compared. A significant interaction between the unsaturated phosphatidylcholine (PC) and saturated hydrogenated phosphatidylcholine (HPC) was found by molecular simulation and differential scanning calorimetry (DSC), which led to the reduction of PC peroxidation with the presence of HPC. Subsequently, the composite phospholipid ethosomes containing curcumin were prepared for the first time to evaluate their properties in comparison with the conventional ethosomes composed of PC (CE-P) or HPC (CE-H). CE with PC/HPC ratio of 1:1 (CE-P1H1) with the best vesicle stability and flexibility significantly decreased the uptake by HaCaT cells compared to CE-H and free curcumin, indicating reduced skin cell toxicity. Compared with free curcumin, CE-P1H1 had the highest transdermal efficiency (p 0.001), followed by CE-P (p 0.05), partly due to the fact that CE-P1H1 could disturb lipid domain of stratum corneum (SC). Moreover, CE-P1H1 was found to promote curcumin for deep penetration of the skin via the hair follicles route. Our study has shown that using composite phospholipid ethosomes as lipid vesicular carriers could enhance transdermal penetration of drugs and increase in the vesicle stability.

Phospholipid nanoparticles (PNs) encapsulating vitamin C and E derivatives, 3-O-cetyl ascorbic acid (CA) and tocopherol acetate (TA), respectively, were examined using the film rehydration and extrusion method. PN formulations (TA-Cassome) were prepared by mixing CA, soya phosphatidylcholine (Soya PC), sodium cholate, and TA at a molar ratio of 20/80/5/6. Glycerol (GL) or diglycerol (DG) were also added to improve the skin accumulation of CA and TA. Three TA-Cassome formulations were evaluated using a dynamic light scattering (DLS), NMR, TEM, skin accumulation test for CA and TA, and small-angle X-ray diffraction (SAXD) analysis. TA-Cassome formulations (150 nm) were stable for two weeks and they encapsulated 1.8 mg/mL of TA. TEM and SAXD analysis revealed that the nanoparticles formed a spherical multilayer structure. 1H and 31P NMR indicated that GL and DG enhanced the proton mobility of choline groups of soya PC molecules located on the membrane surface of TA-Cassome. Accumulation of CA and TA in the dermis was increased by adding GL and DG. SAXD analysis revealed that GL and DG promoted the formation of new lamellar structures on the stratum corneum, which contributed to improving the skin accumulation of CA and TA.

In this study, we report pH-responsive liposomes consisting of hydrogenated soy phosphatidylcholine (HSPC) as a lipid, hyaluronic acid (HA) grafted with functional 3-diethylaminopropyl (DEAP) groups (hereafter denoted as HA-g-DEAP) as a pH-responsive polymer, and docetaxel (DTX) as an antitumor drug. DTX-loaded HSPC liposomes were prepared via a conventional liposome manufacturing procedure and then were decorated with HA-g-DEAP (HA-g-DEAP0.15, HA-g-DEAP0.25, and HA-g-DEAP0.40, according to the molar conjugate ratio of DEAP to HA) in an aqueous solution (pH 7.4), by sonication. The liposomes with HA-g-DEAP0.40 allowed the efficient release of the encapsulated DTX content when the pH of the solution decreased to 6.5 (i.e., endosomal pH), owing to the acidic pH-induced protonation of the DEAP anchored to the vesicular lipid bilayers. These hyaluronated liposomes were effective at entering the human colon carcinoma HCT-116 cells with a CD44 receptor overexpression. In an in vitro tumor cell cytotoxicity test, the DTX-loaded liposomes caused a significant increase in HCT-116 tumor cell death, revealing their pharmaceutical potential in tumor therapy.

The electron paramagnetic resonance (EPR) spin labeling methodology was used to analyze the interactions of phosphatidylcholine (PC) liposomal formulations that are commonly used as transepidermal drug delivery systems with stratum corneum (SC) membranes. The lipid dynamics of five liposome formulations were evaluated to study the influences of sorbitan monooleate (Span80), cholesterol, and cholesterol with the charged lipids 2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DSPG) on the molecular dynamics of PC vesicles. The EPR spectra of 5-doxyl-stearic acid (5-DSA) showed that the addition of Span80 to the liposomes increased the lipid fluidity, whereas cholesterol had the opposite effect, and the combination of charged lipids and cholesterol did not additionally influence the lipid bilayer dynamics. Fatty acid spin-labeled SC membranes were treated with the liposome formulations, leading to migration of the spin label to the molecular environment of the formulation and the presence of two spectral components representing distinct mobility states. In terms of molecular dynamics, these environments correspond to the lipid domains of the untreated SC and the liposome, indicating a poor interaction between the liposome and SC membranes. However, the contact was sufficient to allow a pronounced exchange of the spin-labeled fatty acid. Our data suggest that flexible liposomes may access the inner intercellular membranes of the SC and facilitate mutual lipid exchange without losing their relative liposomal integrity.

The work aimed to evaluate the feasibility to design hyaluronic acid (HA) decorated flexible liposomes to enhance the skin penetration of nifedipine. Egg phosphatidylcholine (e-PC) based transfersomes (Tween 80) and transethosomes (ethanol) were prepared. HA was reacted with 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (HA-DPPE) and two molar ratios (0.5 and 3%) of conjugate with respect to e-PC were tested. The presence of HA significantly increased the packing order of the bilayer (as verified by differential scanning calorimetry), reducing both the encapsulation efficiency and the flexibility of the decorated liposomes in a dose-dependent manner. In fact, at the highest HA content the constant of deformability (K, N/mm) increased and the carriers remained on the skin surface after topical application. The stiffening effect of HA was counterbalanced by the addition of ethanol as fluidizing agent that allowed to maintain the highest HA concentration, meanwhile reducing the K value of the vesicles. HA-transethosomes allowed a suitable nifedipine permeation (J ∼ 30 ng/cm2/h) and significantly improved the drug penetration, favouring the formation of a drug depot in the epidermis. These data suggest the potentialities of HA-transethosomes as drug delivery systems intended for the treatment of cutaneous pathologies and underline the importance of studying the effect of surface functionalization on carrier deformability to rationalize the design of such systems.

Bupivacaine is the most employed local anesthetic in surgical procedures, worldwide. Its systemic toxicity has directed the synthesis of the less toxic, S(−) enantiomer. This work describes a formulation of ionic gradient liposomes (IGL) containing S75BVC, an enantiomeric excess mixture of 75% S(−) and 25% R(+) bupivacaine. IGL prepared with 250 mM (NH4)2SO4 in the inner aqueous core of phosphatidylcholine and cholesterol (3:2 mol%) vesicles plus 0.5% S75BVC showed average sizes of 312.5 ± 4.5 nm, low polydispersity (PDI

Several biologically relevant phospholipids are considered as potential excipients for IV administration liposome’s formulation of AMB (Biopharmaceututics Classification System Class IV). On the basis of in vivo bioavaibility studies, DMPC and DMPG were ranked as the first potent encapsulation enhancers for this model drug, especially if one expects to target DMPG rich systems as pulmonary surfactant. Subsequently, dispersions (multilayers) of DMPC, DMPG or in binary systems with various molar ratios were prepared with or without AMB (molar ratios AMB/lipid) and further investigated using the 1 H-, 31 P-NMR methods. It was found that equimolar preparations of DMPG/DMPG exhibited both a good encapsulation of AMB, while also probably able to target pulmonary surfactant. Besides DMPG did not exhibit the same solubilization properties. Conversely, no targeting by DMPC dispersion alone was expected, even if a good solubilization was obtained.

Phosphatidylcholines (PCs) have been widely used in pharmaceutical research. Unfortunately, our understanding of how PCs influence the in vivo lipolysis process of drug delivery systems is still limited. In this study, PCs with fatty acid chains of varying lengths and saturability were used as emulsifiers to prepare curcumin-loaded nanoemulsions (Cur-NEs). The differences in particle size as well as drug and free fatty acid release during the lipolysis process were studied in a simulated blood environment. Furthermore, the pharmacokinetics and antitumor efficacy of Cur-NEs were evaluated in mice. The prepared 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)-stabilized Cur-NEs showed similar particle size and stability during storage but exhibited different lipolysis behaviors in vitro and in vivo. Due to the gel state of DPPC in the physiological environment, DPPC-stabilized Cur-NEs had low binding affinity with proteins and maintained their integrity in plasma, leading to sustained drug release, prolonged circulation time and enhanced antitumor efficacy in 4T1 tumor-bearing mice. In contrast, DOPC and DSPC-stabilized Cur-NEs were prone to coalescence in the plasma, resulting in rapid drug release and elimination from circulation. Our findings demonstrated that proper use of PCs is beneficial for obtaining desired transport behavior and drug therapeutic effects, providing guiding principles for rational design of nanodelivery systems.

Inflammation plays a key role in dry eye disease (DED) affecting millions of people worldwide. Non-steroidal anti-inflammatory drugs (NSAIDs) can be used topically to act on the inflammatory component of DED, but their limited aqueous solubility raises formulation issues. The aim of this study was development and optimization of functional cationic nanoemulsions (NEs) for DED treatment, as a formulation approach to circumvent solubility problems, prolong drug residence at the ocular surface and stabilize the tear film. Ibuprofen was employed as the model NSAID, chitosan as the cationic agent, and lecithin as the anionic surfactant enabling chitosan incorporation. Moreover, lecithin is a mixture of phospholipids including phosphatidylcholine and phosphatidylethanolamine, two constituents of the natural tear film important for its stability. NEs were characterized in terms of droplet size, polydispersity index, zeta-potential, pH, viscosity, osmolarity, surface tension, entrapment efficiency, stability, sterilizability and in vitro release. NEs mucoadhesive properties were tested rheologically after mixing with mucin dispersion. Biocompatibility was assessed employing 3D HCE-T cell-based model and ex vivo model using porcine corneas. The results of our study pointed out the NE formulation with 0.05 % (w/w) chitosan as the lead formulation with physicochemical properties adequate for ophthalmic application, mucoadhesive character and excellent biocompatibility. This is the peer-reviewed version of the manuscript: Bisera Jurišić Dukovski et.al., Functional ibuprofen-loaded cationic nanoemulsion: Development and optimization for dry eye disease treatment, International Journal of Pharmaceutics, 2020, 576, 118979, DOI: [https://doi.org/10.1016/j.ijpharm.2019.118979] The published version: [https://cer.ihtm.bg.ac.rs/handle/123456789/4012]

Despite extensive investigations of lamellar liquid crystals for dermal application, the effects of these systems at the cellular level are still not well elucidated. The key aim of this study was to determine the elasticity and morphological features of keratinocytes after exposure to a lamellar liquid crystal system (LLCS) using atomic force microscopy (AFM) as the method of choice. Prior to AFM assessment, a cell proliferation test and light plus fluorescence imaging were applied to determine the sub-toxic concentration of LLCS. According to the AFM results, slightly altered morphology was observed in the case of fixed keratinocytes, while an intact morphology was visualized on live cells. From the quantitative study, decreased Young’s moduli were determined for fixed cells (i.e., 8.6 vs. 15.2 MPa and 1.3 vs. 2.9 MPa for ethanol or PFA-fixed LLCS-treated vs. control cells, respectively) and live cells (i.e., ranging from 0.6 to 2.8 for LLCS-treated vs. 1.1–4.5 MPa for untreated cells), clearly demonstrating increased cell elasticity. This is related to improved membrane fluidity as a consequence of interactions between the acyl chains of cell membrane phosphatidylcholine and those of LLCS. What seems to be of major importance is that the study confirms the potential clinical relevance of such systems in treatment of aged skin with characteristically more rigid epithelial cells.

Drug-loaded thermosensitive liposomes are investigated as drug delivery systems in combination with local mild hyperthermia therapy due to their capacity to release their cargo at a specific temperature range (40–42 °C). Additional benefit can be achieved by the development of such systems that combine two different anticancer drugs, have cell penetration properties and, when heated, release their drug payload in a controlled fashion. To this end, liposomes were developed incorporating at low concentration (5 mol%) a number of monoalkylether phosphatidylcholine lipids, encompassing the platelet activating factor, PAF, and its analogues that induce thermoresponsiveness and have anticancer biological activity. These thermoresponsive liposomes were efficiently (>90%) loaded with doxorubicin (DOX), and their thermal properties, stability and drug release were investigated both at 37 ◦C and at elevated temperatures. In vitro studies of the most advantageous liposomal formulation containing the methylated PAF derivative (methyl-PAF, edelfosine), an established antitumor agent, were performed on human prostate cancer cell lines. This system exhibits controlled release of DOX at 40–42 °C, enhanced cell uptake due to the presence of methyl-PAF, and improved cell viability inhibition due to the combined action of both medications.

Aiming to simultaneous target of methotrexate (MTX), as folate antagonist, and conferone (CON) in various cancer cells, the newly lipid/polymer hybrid nanoparticle containing an albumin targeted succinylchitosan shell and lipoid bilayer core composed of hydrogenated soy phosphatidylcholine and cholesterol was synthesized. The covalently conjugating albumin to the external surface of chitosan was accomplished using N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride and N- hydroxyl succinimide as an activating carboxylic group, and nanoliposomes were fabricated via thin film hydration-sonication method. The molecular structure of MTX@CON-targeted lipid/polymer hybrid nanoparticle (MTX@CON-TLPN) were characterized using FTIR spectroscopy, 1H NMR, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and dynamic light scattering (DLS). The newly nanoparticle with high encapsulation efficiency (85.12%, and 78.4%), acceptable loading capacity (9.8% and 4.6% for MTX and CON) and the stimuli responsiveness drug release behavior in simulated physiologic tumor tissue condition (pH 5.4, 40 °C) was successfully synthetized in the spherical shape with mean average size of approximately 290 nm and ζ-potential of +21 mv. The enhanced efficiency of the targeted nanoparticle was further confirmed using MTT endpoints, cell cycle modulation, apoptosis assessment, and cellular internalization assessments. Collectively, these findings establish the utility of our newly prepared nanoparticle for simultaneous delivery of multiple anti-cancer drugs.

The aim of this study was to develop and evaluate novel polyglycerol fatty acid ester (PGFE)-based nanoparticles (NPs) for the dermal delivery of tocopherol acetate (TA). TA-loaded PGFE-based NPs (PGFE-NPs) were prepared by mixing PGFE, soya phosphatidylcholine, dimyristoylphosphatidylglycerol, and TA with film using the film rehydration and extrusion method. The prepared formulations were analyzed by dynamic light scattering, small-angle X-ray diffraction and polarization microscopy. An in vitro skin accumulation test was performed with TA under occlusive and non-occlusive applications, using Yucatan micropig skin. The size range of the TA-loaded liposome and PGFE-NPs was 107–128 nm, and they were encapsulated in 1.6–2.3 mg/mL TA. All PGFE-NP formulations were negatively charged and stable for 2 weeks. Under occlusive applications, all formulations induced small amounts of TA accumulation in the epidermis but not in the dermis. However, under non-occlusive applications, some of PGFE-NP formulations enhanced TA accumulation in the epidermis. Furthermore, only the polyglycerol 4-laurate (PG4L)-based formulation induced dermal TA accumulation with the change in the formulation from a vesicular to bilayer stacked structure following water evaporation under non-occlusive applications. These results indicated that the novel TA-loaded PG4L formulation enabled the dermal delivery of TA in non-occlusive applications.

Although liquid crystal (LC) systems have been studied before, their utility in drug delivery applications has not been explored in depth. This study examined the development of a 1-month sustained release formulation of leuprolide acetate using an in situ-forming LC matrix. The phase progression upon water absorption was tested through construction of ternary phase diagrams of phosphatidylcholine, sorbitan monooleate, and tocopherol acetate (TA) at increasing water content. Small angle X-ray scattering revealed the presence of lamellar and hexagonal mesophases. The physicochemical characteristics and in vitro drug release were evaluated as a function of the ternary component ratio and its resultant phase behavior. Formulations with increased water uptake capacity displayed greater drug release and enhanced erodability. Removal of TA resulted in increased water uptake capacity and drug release, where 8% (w/w) TA was determined as the critical concentration threshold for divergence of release profiles. In conclusion, characterization of the resultant HII mesophase region provided information of the impact the individual components have on the physicochemical properties and potential drug release mechanisms. This high mitigating impact of TA on drug release indicates the use of TA as a tailoring agent, broadening the therapeutic applications of this LC system.

Intestinal fluids contain several constituents with affinity for cyclodextrins that have the potential of displacing drugs from the cyclodextrin cavity by competition. In this study, the solubilizing capacity of 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) for itraconazole was studied in presence of selected bile salts and phosphatidylcholine. Despite the fact that these competing agents significantly lowered the solubility of itraconazole in presence of cyclodextrins, the addition of concentrated solutions of these bile constituents to a solution containing itraconazole solubilized by HP-β-CD did not result in precipitation, even at bile salt and phospholipid concentrations where itraconazole precipitation would be anticipated based on solubility studies. This phenomenon was further investigated in more dynamic conditions via in vitro transfer studies, mimicking the gastrointestinal transfer of HP-β-CD solutions saturated with itraconazole. Intestinal supersaturation upon transfer was observed for all conditions tested and a concentration dependent impact of bile salts and phospholipids on the precipitation behavior of itraconazole was demonstrated: high concentrations of bile salts and phospholipids generated the highest degrees of supersaturation shortly after the transfer step but also resulted in stronger itraconazole precipitation at later time points. These findings demonstrate the possible impact of the variable intestinal fluid composition on the behavior of cyclodextrin containing formulations. publisher: Elsevier articletitle: Displacement of itraconazole from cyclodextrin complexes in biorelevant media: In vitro evaluation of supersaturation and precipitation behavior journaltitle: International Journal of Pharmaceutics articlelink: http://dx.doi.org/10.1016/j.ijpharm.2016.07.063 content_type: article copyright: © 2016 Elsevier B.V. All rights reserved. ispartof: International Journal of Pharmaceutics vol:511 issue:1 pages:680-687 ispartof: location:Netherlands status: published

Drug inhalation provides localized drug therapy for respiratory diseases. However, the therapeutic efficacy of inhaled drugs is limited by rapid clearance from the lungs. Small hydrophilic compounds have short half-lives to systemic absorption. We developed a liposomal formulation as a sustained-release strategy for pulmonary delivery of procaterol hydrochloride (PRO), a short-acting pulmonary β2-agonist for asthma treatment. After PRO-loaded liposomes were prepared using a pH gradient (remote loading) method, 100-nm liposomes improved residence times of PRO in the lungs. PRO encapsulation efficiency and release profiles were examined by screening several liposomal formulations of lipid, cholesterol, and inner phase. Although PRO loading was not achieved using the conventional hydration method, PRO encapsulation efficiency was >60% using the pH gradient method. PRO release from liposomes was sustained for several hours depending on liposomal composition. The liposomal formulation effects on the PRO behavior in rat lungs were evaluated following pulmonary administration in vivo. Sustained PRO release was achieved using simplified egg phosphatidylcholine (EPC)/cholesterol (8/1) liposome in vitro, and greater PRO remnants were observed in rat lungs following pulmonary administration. Extended pharmacological PRO effects were observed for 120min in a histamine-induced bronchoconstriction guinea pig model. We indicated the simplified EPC/cholesterol liposome potential as a controlled-release PRO carrier for pulmonary administration.

The objective of this study was to develop proliposomal formulation and self micro-emulsifying drug delivery system (SMEDDS) for a poorly bioavailable drug, nisoldipine and to compare their in vivo pharmacokinetics. Proliposomes were prepared by thin film hydration method using different lipids such as Soy phosphatidylcholine (SPC), Hydrogenated Soy phosphatidylcholine (HSPC), Dimyristoylphosphatidylcholine (DMPC) and Dimyristoyl phosphatidylglycerol sodium (DMPG), Distearyl phosphatidylcholine (DSPC), and Cholesterol in various ratios. SMEDDS formulations were prepared using varying concentrations of Capmul MCM, Labrasol, Cremophor EL and Tween 80. Both proliposomes and SMEDDS were evaluated for particle size, zeta potential, in vitro drug release, in vitro permeability and in vivo pharmacokinetics. In vitro drug release was carried out in purified water using USP type II dissolution apparatus. In vitro drug permeation was studied using parallel artificial membrane permeation assay (PAMPA) and everted rat intestinal perfusion techniques. In vivo pharmacokinetic studies were conducted in male Sprague-Dawley rats. Among the different formulations, proliposomes with drug:DMPC:cholesterol in the ratio of 1:2:0.5 and SMEDDS with Capmul MCM (13.04% w/w), Labrasol (36.96% w/w), Cremophor EL (34.78% w/w) and Tween 80 (15.22% w/w) demonstrated the desired particle size and zeta potential. Enhanced drug release was observed with proliposomes and SMEDDS compared to pure nisoldipine in purified water after 1h. Nisoldipine permeability across PAMPA and everted rat intestinal perfusion models was significantly higher with proliposomes and SMEDDS. Following single oral administration of proliposomes and SMEDDS, a relative bioavailability of 301.11% and 239.87% respectively, was achieved compared to pure nisoldipine suspension.

The determination of the permeability of drug candidates across the blood-brain barrier (BBB) is a fundamental step during drug discovery programs. The parallel artificial membrane permeability assay (PAMPA) is a high throughput screening tool applied to evaluate the passive permeability and adapted to predict BBB penetration. Herein, a new PAMPA model was developed using an in-house brain lipid extract capable of discriminating BBB permeable from non-permeable compounds. The apparent permeability (Papp) of 18 reference molecules and 10 test compounds was assessed and compared with phosphatidylcholine and commercial porcine polar brain lipid (PBL). The physicochemical selectivity of the in-house brain lipid extract was demonstrated by correlating Papp values with physicochemical properties and its predictive capacity estimated by establishing in vitro-in vivo correlations. The strong correlations achieved between 2% (w/v) in-house lipid extract and PBL for reference (r(2)=0.77) and test compounds (r(2)=0.94) support an equivalent discriminatory capacity and validate the presented model. Moreover, PAMPA studies performed with PBL and in-house lipid extract exhibited a higher correlation with the in vivo parameter logBB (r(2)=0.76 and r(2)=0.72, respectively) than phosphatidylcholine (r(2)=0.51). Overall, the applied lipid extraction process was reproducible, economical and provided lipid extracts that can be used to reliably assess BBB permeation.

Hydroxyethyl bearing gemini surfactants, alkanediyl-α,ω-bis(N-hexadecyl-N-2-hydroxyethyl-N-methylammonium bromide), 16-s-16(OH), were used to augment phosphatidylcholine based liposomes to achieve higher stability and enhanced cellular uptake and penetration. The developed liposomes were loaded with rhodamine B, doxorubicin hydrochloride, pralidoxime chloride to investigate release properties, cytotoxicity in vitro, as well as ability to cross the blood-brain barrier. At molar ratio of 35:1 (lipid:surfactant) the formulation was found to be of low toxicity, stable for two months, and able to deliver rhodamine B beyond the blood-brain barrier in rats. In vivo, pharmacokinetics of free and formulated 2-PAM in plasma and brain were evaluated, liposomal 2-PAM was found to reactivate 27% of brain acetylcholinesterase, which is, to our knowledge, the first example of such high degree of reactivation after intravenous administration of liposomal drug.

The purpose of this study was to develop an injectable submicron emulsion of eugenol (Eug-SE) and to investigate its antagonism on epilepsy. The formulation was optimized using a complete randomized design, comprising 5% (w/v) eugenol, 5% (w/v) soybean oil, 1.2% (w/v) egg phosphatidylcholine, 0.3% (w/v) poloxamer 188, and 0.03% (w/v) sodium oleate. The prepared Eug-SE was comprehensively evaluated in terms of its pharmaceutical characteristics, physicochemical stability, injection safety, antioxidant activity in vitro, and anti-epileptic effect in vivo. The mean particle size of Eug-SE was 176.1 ± 10.3 nm, the ζ-potential was −40.2 ± 1.8 mV, and the drug content was (95.3 ± 0.4) %. Moreover, the Eug-SE displayed excellent stability and improved safety compared to the eugenol solution. The Eug-SE (20 μg/mL) produced a significant neuroprotective effect against H2O2-induced oxidative damage in PC12 cells, which was attributed to the decrease of cellular reactive oxygen species level and mitochondrial damage. Besides, the in vivo test indicated that Eug-SE exerted an anti-epileptic effect in the PTZ treated mice. These results suggested that Eug-SE was a suitable dosage form of eugenol for injection, and displayed great therapeutic potential for neurological disease in the future.

To face the challenges of oral delivery of peptide and protein (P/P) drugs, self-emulsifying drug delivery systems (SEDDSs) containing monoacyl phosphatidylcholine (MAPC), Labrasol (LAB) and medium-chain (MC) monoglycerides as permeation enhancers (PEs) were evaluated for their effect on intestinal absorption of insulin. In this study, insulin was complexed with phosphatidylcholine (SPC) to form an insulin-SPC complex (ins-SPC) with increased lipophilicity. The following three SEDDSs: MCT(MAPC) (MC triglycerides and MAPC included), MCT(RH40) (MC triglycerides and Kolliphor® RH40 included) and LCT(MAPC) (long-chain triglycerides and MAPC included) were loading with ins-SPC (4% or 8% w/w of SPC). Three SEDDSs generated emulsions with droplet sizes between 50 and 470 nm and with zeta potentials between −5 to −25 mV in a simulated intestinal medium. Mucus-secreting Caco-2/HT29-MTX-E12 co-culture and Caco-2 monolayers were used as in vitro cell transport models to investigate insulin permeability. In comparison to insulin HBSS solution, MCT(MAPC) significantly increased the insulin permeability across co-culture and Caco-2 monolayers (2.0–2.5 × 10–7 cm/s). In an intra-jejunal (i.j.) instillation model in rats, MCT(RH40) significantly decreased the rat blood glucose after 0.5 h by 17.0 ± 2.5% and for MCT(MAPC), it was 23.6 ± 10.6%. Furthermore, a lipase inhibitor orlistat was incorporated into MCT(MAPC) to evaluate the effect of lipid digestion on insulin absorption. Results indicated that the incorporation of orlistat did not significantly alter the in vivo insulin absorption. Overall, the SEDDS MCT(MAPC) composed of natural PEs (MAPC and MC glycerides) and synthetic PE (LAB) significantly increased the intestinal absorption of insulin upon i.j. instillation. Although it is not possible to conclude if a single PE is dominating the intestinal absorption of insulin, MCT(MAPC) seems to have the potential for oral insulin delivery.

The lipid membrane composition defines the physical and pharmacological characteristics of liposomal drugs, and it can be tailored to meet the desired drug delivery needs. The current study is aimed to provide a sharper understanding of the lipid composition effect on doxorubicin (DOX) delivery kinetics, using cholesterol and phosphatidylcholine lipids (PCs) with different acyl chains in liposomal DOX formulations. The PCs were distearoyl (DSPC), dipalmitoyl (DPPC), dimyristoyl (DMPC) and egg-derived PC (EPC), either alone or in combination with cholesterol. Several characteristics were monitored, including DOX loading capacity of liposomes, DOX release in phosphate buffered saline (PBS), PBS/human plasma including buffy coat and human blood, cell uptake, as well as in vivo distribution and therapeutic effects in BALB/c mice bearing C26 colon carcinoma. Addition of cholesterol to liposomal formulation enhanced the particle size stability of the liposomes and the DOX-to-lipid ratio. EPC-liposomes and EPC/Cholesterol-liposomes showed few distinctive features. Overall, cholesterol decreased DOX release from the liposomes, and longer saturated fatty acyl chains in PC decreased DOX release and side-effects and increased the anti-tumor effects of liposomal DOX.

The study aimed to fabricate innovative drug-phospholipid complexes termed “sonocomplexes” adopting ultrasound irradiation to increase the liposolubility and to enhance the intestinal absorption of rosuvastatin as a model drug for BCS class III active pharmaceutical ingredients (APIs). A 22 full factorial design was fashioned to investigate the influence of phosphatidylcholine content in the phospholipid (∼30 and 60%) and molar ratio of phospholipid to rosuvastatin (1:1 and 2:1) on physicochemical properties of sonocomplexes. In comparison to pure drug, sonocomplexes showed a minimum of about 2 folds and a maximum of about 15 folds increase in lipophilicity (expressed in terms of partition coefficient, P). Results of molecular docking, dynamic simulations, Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC) confirmed the strong interactions between rosuvastatin and the phospholipid via hydrogen bonding interaction, van der Waals forces and hydrophobic interaction. The complexation efficiency reached around 99% and transmission electron microscopy (TEM) of the aqueous dispersion of the optimal sonocomplex showed spherical nanosized vesicles. The optimal sonocomplex showed significantly superior Caco-2 cells permeability and markedly better oral bioavailability compared to the pure drug. In summary, sonocomplexes can be considered as effective approach for enhancing the liposolubility and consequently the intestinal permeability of BCS class III drugs.

The mixture of polyethyleneimine (PEI) and cinnamic acid (CA) in HEPES buffer (pH 7.0) exhibited an upper critical solution temperature in the temperature range of 20-50 °C. CA would be electrostatically conjugated with PEI and the PEI-CA conjugate is thought to act as a thermo-sensitive polymer. On the optical microscope image of PEI/CA mixture, microparticles were found at 25 °C, disappeared when heated to 50 °C, and formed again upon cooling to 25 °C. PEI-CA conjugate was immobilized on the surface of egg phosphatidylcholine (EPC) liposome by adding PEI to the suspension of liposome incorporating CA. The size and the zeta potential of the liposome markedly increased by cooling the liposomal suspension from 50 °C to 20 °C. This could be ascribed to the cooling-induced self-assembling property of PEI-CA conjugate. The release profile of Rhodamine B base from liposome incorporating CA with PEI was investigated while the liposome suspension of 50 °C was exposed to the release medium of 20 °C, 30 °C, 40 °C and 50 °C. The release degree was higher at a lower temperature. When exposed to a lower temperature (20 °C, 30 °C, 40 °C), PEI-CA could be self-assembled and change its configuration on the surface of liposome, promoting the release from the liposome.

The overexpression of secretory phospholipase A 2 (sPLA 2) in tumors has opened new avenues for enzyme-triggered active unloading of liposomal antitumor drug carriers selectively at the target tumor. However, the effects of the liposome composition, drug encapsulation, and tumor microenvironment on the activity of sPLA 2 are still not well understood. We carried out a physico-chemical study to characterize the sPLA 2-assisted breakdown of liposomes using dye-release assays in the context of drug delivery and under physiologically relevant conditions. The influence of temperature, lipid concentration, enzyme concentration, and drug loading on the hydrolysis of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, T m = 42°C) liposomes with snake venom sPLA 2 was investigated. The sensitivity of human sPLA 2 to the liposome composition was checked using binary lipid mixtures of phosphatidylcholine (PC) and phosphatidylglycerol (PG) phospholipids with C14 and C16 acyl chains. Increasing temperature (36-41°C) was found to mainly shorten the enzyme lag-time, whereas the effect on lipid hydrolysis rate was modest. The enzyme lag-time was also found to be inversely dependent on the lipid-to-enzyme ratio. Drug encapsulation can alter the hydrolysis profile of the carrier liposomes. The activity of human sPLA 2 was highly sensitive to the phospholipid acyl-chain length and negative surface charge density of the liposomes. We believe our work will prove useful for the optimization of sPLA 2-susceptible liposomal formulations as well as will provide a solid ground for predicting the hydrolysis profile of the liposomes in vivo at the target site.

There is a growing interest in drug–phospholipid complexes and similar formulations that are mostly solid dispersions with high drug load. This study aims to explore the feasibility of such phospholipid-based solid dispersions as well as to characterize them. A particular aim was to compare monoacyl phosphatidylcholine (PC) with the diacyl excipient. The solid dispersions were manufactured using a solvent evaporation technique and characterized by means of differential scanning calorimetry and X-ray diffractometry. Density functional theory was used to calculate molecular frontier orbitals of the different compounds. Finally, the dissolution properties were analyzed in a flow-through cell by means of UV imaging. It was found that the ability to form solid dispersions with the phospholipids containing amorphous or solubilized drug (at equimolar ratio with the lipid) was dependent on the drug’s frontier orbital energy, the enthalpy of fusion, as well as the log P value. In a subsequent dissolution study, UV imaging revealed pronounced surface swelling of the solid dispersions. Only the monoacyl PC was found to substantially enhance in vitro dissolution compared to pure drug. The gained understanding will support a future development of solid drug dispersions using phospholipids as matrix components.

In this work, phospholipid vesicle-based nanoformulations were developed to deliver antioxidant resveratrol (RSV) to the skin. Penetration enhancer-containing vesicles (PEVs) were prepared adding Oramix™ CG110 or Lauroglycol™ FCC to phosphatidylcholine to favor RSV diffusion through the skin, which was investigated using Franz cells. Vesicles were approximately 100 nm in size, negatively charged and fairly round in shape, as shown via transmission electron microscopy. Nuclear magnetic resonance studies were performed to investigate the RSV/vesicle interactions at the molecular scale, which revealed that RSV was deeply embedded in the bilayer, as shown by the restricted mobility of the drug. Moreover, PEVs improved drug local accumulation 1.7- to 2.1-fold, as compared to the control liposomes. Confocal imaging displayed broadened intercellular spaces in the viable epidermis of PEVs treated skin and high degree of hydration, which are presumably due to the occlusive film formed on the skin surface by the vesicles. These phenomena may be responsible for the higher RSV accumulation achieved when administering PEVs, as compared to control liposomes. Finally, the toxicity of the vesicular formulations was evaluated in vitro against 3T3 fibroblasts, showing no alteration on cell viability after 24h incubation with RSV loaded vesicles. The results from this study suggest that the proposed formulations may be a potential therapeutic alternative to treat skin disorders associated with oxidative stress.

Polymeric nanoparticles are widely investigated as drug delivery systems for oral administration. However, the hydrophobic nature of many polymers hampers effective loading of the particles with hydrophilic macromolecules such as insulin. Thus, the aim of this work was to improve the loading of insulin into poly(lactic-co-glycolic) acid (PLGA) nanoparticles by pre-assembly with amphiphilic lipids. Insulin was complexed with soybean phosphatidylcholine or sodium caprate by self-assembly and subsequently loaded into PLGA nanoparticles by using the double emulsion-solvent evaporation technique. The nanoparticles were characterized in terms of size, zeta potential, insulin encapsulation efficiency and loading capacity. Upon pre-assembly with lipids, there was an increased distribution of insulin into the organic phase of the emulsion, eventually resulting in significantly enhanced encapsulation efficiencies (90% as compared to 24% in the absence of lipids). Importantly, the insulin loading capacity was increased up to 20% by using the lipid-insulin complexes. The results further showed that a main fraction of the lipid was incorporated into the nanoparticles and remained associated to the polymer during release studies in buffers, whereas insulin was released in a non-complexed form as a burst of approximately 80% of the loaded insulin. In conclusion, the protein load in PLGA nanoparticles can be significantly increased by employing self-assembled protein-lipid complexes.

Microfluidics has recently emerged as a new method of manufacturing liposomes, which allows reproducible mixing in miliseconds on the nanoliter scale. Here we investigated the feasibility of a microfluidic flow focusing setup built from commercially available fittings to encapsulate phages into liposomes. Two types of Pseudomonas phages, PEV2 (Podovirus, ~65 nm) and PEV40 (Myovirus, ~220 nm), were used as model phages. A mixture of soy phosphatidylcholine and cholesterol at a ratio of 4:1 dissolved in absolute ethanol with a total solid content of 17.5 mg/mL was injected through the center inlet channel of a cross mixer. Phage suspensions were injected into the cross mixer from the two side channels intersecting with the center channel. The total flow rate (TFR) varied 160 – 320 µL/min and the organic/aqueous flow rate ratio (FRR) varied 1:3 to 2:3. The size of liposomes and the encapsulation efficiency both increased with increasing FRR and slightly decreased with increasing TFR. Due to the different size of the two studied phages, the size of liposomes encapsulating PEV2 were smaller (135 – 218 nm) than those encapsulating the Myovirus PEV40 (261 – 448 nm). Highest encapsulation efficiency of PEV2 (59%) and PEV40 (50%) was achieved at a TFR of 160 µL/ml and a FRR of 2:3. Generally, the encapsulation efficiency was slightly higher than that obtained from the conventional thin film hydration followed by extrusion method. In summary, the proposed microfluidic technique was capable of encapsulating phages of different size into liposomes with reasonable encapsulation efficiency and minimal titer reduction. This work was financially supported by the Australian Research Council (Discovery Project DP150103953). SSY Leung is a research fellow supported by the University of Sydney. WJ Britton is funded by the National Health and Medical Research Council Centre of Research Excellence in Tuberculosis Control (APP1043225). The authors wish to acknowledge the technical support of Australian Microscopy & Microanalysis Research Facility at the Australian Centre for Microscopy and Microanalysis, The University of Sydney.

A liquid crystalline (LC) system, composed of phosphatidylcholine, sorbitan monoleate, and tocopherol acetate, was investigated to understand the in vivo transformation after subcutaneous injection, coupled with the physicochemical and pharmacokinetic properties of the formulation. The rat model was utilized to monitor a pseudo-time course transformation from a precursor LC formulation to the LC matrix, coupled with the blood concentration profiles of the formulations containing leuprolide acetate. Three formulations that result in the HII phase, demonstrating dissimilar in vitro release profiles, were used. The formulation showing the highest AUC, Cmax and Tmax, also displayed the greatest release rate in vitro, the lowest viscosity (LC matrix), and an earlier transformation (LC precursor to matrix) in vivo. A potential link between viscosity, phase transformation, and drug release properties of a liquid crystalline system is described.

The potential of two derivatives of vitamin E, i.e. α-tocopheryl phosphate (TP) and di-α-tocopheryl phosphate (T2P), for the protection and delivery of therapeutics is being investigated in our laboratory and some promising results have been obtained so far. Novel nanocarrier systems containing TP, T2P and different lipids/phospholipids (phosphatidylcholine, stearyl amine, Phospholipon 90H, Phospholipon 100H) with and without cholesterol were prepared using Mozafari method. The preparation method did not require utilization of potentially toxic solvents, detergents or employment of harsh treatments such as sonication or high-shear force homogenization. Tocosomes, containing 5-fluorouracil (as a model drug), were shown to possess narrow size distribution, acceptable encapsulation efficiency and long-term stability (more than two years).

Although microemulsions are one of the most promising dermal carrier systems, their clinical use is limited due to their skin irritation potential. Therefore, microemulsions based on naturally derived monoacyl phosphatidylcholine (MAPL) were developed. The influence of the water, oil and surfactant content on dermal delivery of flufenamic acid was systematically investigated for the first time. A water-rich microemulsion led to significantly higher in vitro skin penetration of flufenamic acid compared to other microemulsions. The superiority of the water-rich microemulsion over a marketed flufenamic acid containing formulation was additionally confirmed. Differences in drug delivery could be explained by alterations of the microemulsions after application. Evaporation of isopropanol led to crystal-like structures of MAPL on the skin surface from the surfactant- or oleic acid-rich microemulsions. In contrast, the formation of this additional barrier was hindered in case of the water-rich microemulsion. The skin penetration of MAPL was additionally analyzed by combined ATR-FTIR and tape stripping experiments, where MAPL itself penetrated only into the initial layers of the stratum corneum, independent of the microemulsion composition. Since a surfactant must penetrate the skin to cause irritation, MAPL can be presumed as a skin-friendly emulsifier with the ability to stabilize pharmaceutically acceptable microemulsions.

Quil A is the purified saponin fraction extracted from the bark of Quillaja saponaria Molina. Besides its utilisation as a surfactant, it is commonly used in a pseudo-ternary system with cholesterol and phospholipid to form colloidal structures known as ISCOMs (immunostimulating complexes). Their appropriateness as immune stimulating drug carriers has been widely demonstrated, albeit the evaluation of physico-chemical properties of the ISCOM matrix still draws a heterogeneous picture. The aim of our study was to elucidate the effects of Quil A on liposomal phosphatidylcholine/cholesterol dispersions as this interaction is regarded as the major step for the formation of the ISCOM matrix. Transmission electron microscopy was applied to observe structural changes of liposomal dispersions upon addition of Quil A. A formation of ISCOM matrices readily out of the liposomal membrane was proven. The entrapment efficiency (EE) of Arsenazo III as well as differential thermal analysis (DSC) also demonstrated an interaction between the components above a critical concentration of Quil A. To further clarify the effects of interaction, Langmuir trough experiments of insoluble monolayers of both cholesterol and PC and their interaction with Quil A were performed. Measurable effects even below the critical concentration of Quil A (derived from DSC and EE) were shown. Cholesterol had a major impact on the formation and stabilisation of the ISCOM matrix.

Light- and temperature-responsive liposomes were prepared by immobilizing cinnamoyl Pluronic F127 (CP F127) on the surface of egg phosphatidylcholine liposomes. CP F127 was prepared by a condensation reaction, and the molar ratio of cinnamoyl group to Pluronic F127 was calculated to be 1:1.4 on (1)H NMR spectrum. The cinnamoyl group of CP F127 was readily dimerized under the irradiation of a UV light (254 nm, 6 W). CP F127 decreased the absolute value of the zeta potential of liposome possibly because it can shift the hydrodynamic plane away from the liposome surface. The size of liposome decorated with CP F127, measured on a dynamic light scattering machine and observed on a TEM, was larger than that of bare liposome. The liposome bearing CP F127 seemed to fuse and aggregate each other. The liposome released calcein, a fluorescence dye, in response to a UV irradiation, possibly because the photo-dimerization of cinnamoyl group perturbs the liposomal membrane. Moreover, the liposome released the dye in response to a temperature change, possible due to the phase transition of Pluronic F127 layer on the liposomal surface or the hydrophobic interaction of the polymer with liposomal membrane.

Conventional quantitation of drug content in the liposome formulation involves the breakdown of bulk liposomes, which ignores details on the distribution of the active pharmaceutical ingredient (API) and excipients in liposomes of different sizes. The objective of this study is to develop an analytical method which can separate the liposomes into different sizes and obtain information of the drug and excipient distribution in the different sized liposomes. We developed an asymmetric flow field-flow fractionation (AF4) method for size-based separation of AmBisome, an amphotericin B liposomal formulation, and a high-performance liquid chromatography ultraviolet-visible and charged aerosol detection (HPLC-UV-CAD) method for simultaneous quantitation of the API (Amphotericin B) and the lipid excipients [1,2-Distearoyl-sn-glycero-3-phosphoglycerol (DSPG), hydrogenated soy phosphatidylcholine (HSPC), and cholesterol]. The measured drug content in the bulk liposome formulation was consistent with the drug product labeling. Liposomes were separated using AF4 into eleven size fractions and the liposomes particles sizes of each fraction were measured with nanoparticle tracking analysis. The drug to total lipid ratios in fractionated liposomes increased from 0.1 to 0.45 when the liposome size increased from 75 nm to 124 nm, while the lipid composition remained constant throughout the fractioned size range (cholesterol:DSPG, 0.7 and HSPC:DSPG, 0.3). These study results suggest that, for liposomal formulations of Amphotericin B in liposomes, the drug to lipid ratio increases with the size of the liposomes. This new analytical method provided a more in-depth characterization of liposomes, i.e., determining drug and excipient distributions in different sizes of liposomes, in a more efficient manner with more specific size-based composition information.