Anil Vangala

A comparative study of cationic liposome and niosome-based adjuvant systems for protein subunit vaccines: characterisation, environmental scanning electron microscopy and immunisation studies in mice.

Vesicular adjuvant systems composing dimethyldioctadecylammonium (DDA) can promote both cell‐mediated and humoral immune responses to the tuberculosis vaccine fusion protein in mice. However, these DDA preparations were found to be physically unstable, forming aggregates under ambient storage conditions. Therefore there is a need to improve the stability of such systems without undermining their potent adjuvanticity. To this end, the effect of incorporating non‐ionic surfactants, such as 1‐monopalmitoyl glycerol (MP), in addition to cholesterol (Chol) and trehalose 6,60‐dibehenate (TDB), on the stability and efficacy of these vaccine delivery systems was investigated. Differential scanning calorimetry revealed a reduction in the phase transition temperature (Tc) of DDA‐based vesicles by ∼12°C when MP and cholesterol (1:1 molar ratio) were incorporated into the DDA system. Transmission electron microscopy (TEM) revealed the addition of MP to DDA vesicles resulted in the formation of multi‐lamellar vesicles. Environmental scanning electron microscopy (ESEM) of MP‐Chol‐DDA‐TDB (16:16:4:0.5 μmol) indicated that incorporation of antigen led to increased stability of the vesicles, perhaps as a result of the antigen embedding within the vesicle bilayers. At 4°C DDA liposomes showed significant vesicle aggregation after 28 days, although addition of MP‐Chol or TDB was shown to inhibit this instability. Alternatively, at 25°C only the MP‐based systems retained their original size. The presence of MP within the vesicle formulation was also shown to promote a sustained release of antigen in‐vitro. The adjuvant activity of various systems was tested in mice against three subunit antigens, including mycobacterial fusion protein Ag85B‐ESAT‐6, and two malarial antigens (Merozoite surface protein 1, MSP1, and the glutamate rich protein, GLURP). The MP‐and DDA‐based systems induced antibody responses at comparable levels whereas the DDA‐based systems induced more powerful cell‐mediated immune responses.

Liposome-mediated DNA immunisation via the subcutaneous route.

Compared to naked DNA immunisation, entrapment of plasmid-based DNA vaccines into liposomes by the dehydration–rehydration method has shown to enhance both humoural and cell-mediated immune responses to encoded antigens administered by a variety of routes. In this paper, we have investigated the application of liposome-entrapped DNA and their cationic lipid composition on such potency after subcutaneous immunisation. Plasmid pI.18Sfi/NP containing the nucleoprotein (NP) gene of A/Sichuan/2/87 (H3N2) influenza virus in the pI.18 expression vector was incorporated by the dehydration–rehydration method into liposomes composed of 16 μmol egg phosphatidylcholine (PC), 8 μmoles dioleoyl phosphatidylethanolamine (DOPE) or cholesterol (Chol) and either the cationic lipid 1,2-diodeoyl-3-(trimethylammonium) propane (DOTAP) or cholesteryl 3̣-N-(dimethyl amino ethyl) carbamate (DC-Chol). This method, entailing mixing of small unilamellar vesicles (SUV) with DNA, followed by dehydration and rehydration, yielded incorporation values of 90–94% of the DNA used. Mixing or rehydration of preformed cationic liposomes with 100 μg plasmid DNA also led to similarly high complexation values (92–94%). In an attempt to establish differences in the nature of DNA association with these various liposome preparations their physico-chemical characteristics were investigated. Studies on vesicle size, zeta potential and gel electrophoresis in the presence of the anion sodium dodecyl sulphate (SDS) indicate that, under the conditions employed, formulation of liposomal DNA by the dehydration–rehydration generated submicron size liposomes incorporating most of the DNA in a manner that prevents DNA displacement through anion competition. The bilayer composition of these dehydration–rehydration vesicles (DRV(DNA)) can also further influence these physico-chemical characteristics with the presence of DOPE within the liposome bilayer resulting in a reduced vesicle zeta potential. Subcutaneous liposome-mediated DNA immunisation employing two DRV(DNA) formulations as well as naked DNA revealed that humoural responses (immunoglobulin total IgG, and subclasses IgG1 and 1gG2a) engendered by the plasmid encoded NP were substantially higher after dosing twice, 28 days apart with 10 μg liposome-entrapped DNA compared to naked DNA. At all time points measured, mice immunised with naked DNA showed no greater immune response compared to the control, non-immunised group. In contrast, as early as day 49, responses were significantly higher in mice injected with DNA entrapped in DRV liposomes containing DOTAP compared to the control group and mice immunised with naked DNA. By day 56, all total IgG responses from mice immunised with both DRV formulations were significantly higher. Comparison between the DRV formulations revealed no significant difference in immune responses elicited except at day 114, where the humoural responses of the group injected with liposomal formulation containing DC-Chol dropped to significantly lower levels that those measured in mice which received the DOTAP formulation. Similar results were found when the IgG1 and IgG2a subclass responses were determined. These results suggest that, not only can DNA be effectively entrapped within liposomes using the DRV method but that such DRV liposomes containing DNA may be a useful system for subcutaneous delivery of DNA vaccines.

Robust spatially resolved pressure measurements using MRI with novel buoyant advection-free preparations of stable microbubbles in polysaccharide gels

MRI of fluids containing lipid coated microbubbles has been shown to be an effective tool for measuring the local fluid pressure. However, the intrinsically buoyant nature of these microbubbles precludes lengthy measurements due to their vertical migration under gravity and pressure-induced coalescence. A novel preparation is presented which is shown to minimize both these effects for at least 25 min. By using a 2% polysaccharide gel base with a small concentration of glycerol and 1,2-distearoyl-sn-glycero-3-phosphocholine coated gas microbubbles, MR measurements are made for pressures between 0.95 and 1.44 bar. The signal drifts due to migration and amalgamation are shown to be minimized for such an experiment whilst yielding very high NMR sensitivities up to 38% signal change per bar.

Environmental Scanning Electron Microscopy Offers Real-Time Morphological Analysis of Liposomes and Niosomes

Liposomes have been imaged using a plethora of techniques. However, few of these methods offer the ability to study these systems in their natural hydrated state without the requirement of drying, staining, and fixation of the vesicles. However, the ability to image a liposome in its hydrated state is the ideal scenario for visualization of these dynamic lipid structures and environmental scanning electron microscopy (ESEM), with its ability to image wet systems without prior sample preparation, offers potential advantages to the above methods. In our studies, we have used ESEM to not only investigate the morphology of liposomes and niosomes but also to dynamically follow the changes in structure of lipid films and liposome suspensions as water condenses on to or evaporates from the sample. In particular, changes in liposome morphology were studied using ESEM in real time to investigate the resistance of liposomes to coalescence during dehydration thereby providing an alternative assay of liposome formulation and stability. Based on this protocol, we have also studied niosome-based systems and cationic liposome/DNA complexes.

Lipoplexes formulation and optimisation: in vitro transfection studies reveal no correlation with in vivo vaccination studies

The aim of these studies was to compare the effect of liposome composition on physico-chemical characteristics and transfection efficacy of cationic liposomes both in vitro and in vivo. Comparison between 4 popularly used cationic lipids, showed 3b-N-(dimethylaminoethyl)carbamate (DC-Chol) to promote the highest transfect levels in cells in vitro with levels being at least 6 times higher than those of 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA). 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and dimethyldioctadecylammonium (DDA) and approximately twice as efficient as dipalmitoyl-trimethylammonium-propane (DPTAP). To establish the role of the helper lipid, DC-Chol liposomes were formulated in combination with either 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) or cholesterol (Chol) (1:1 molar ratio) with and without the addition of phosphatidyl choline. The choice of helper lipid incorporated within the bilayer was found to influence the formation of complexes, their resultant structure and their transfection efficiency in vitro, with SUV-DNA complexes containing optimum levels of DOPE giving higher transfection than those containing cholesterol. The inclusion of PC within the formulation also reduced transfection efficiency in vitro. However, when administered in vivo, SUV-DNA complexes composed of PC:Chol:DC-Chol at a molar ratio of 16:8:4 micromole/ml were the most effective at inducing splenocyte proliferation upon exposure to antigen in comparison to control spleens. These results demonstrate that there is no in vitro/in vivo correlation between the transfection efficacy of these liposome formulations and in vitro transfection in the above cell model cannot be taken as a reliable indicator for in vivo efficacy of DNA vaccines.

Formulation and corneal permeation of ketorolac tromethamine-loaded chitosan nanoparticles

Abstract The aim of this work was to formulate chitosan (CS)-based nanoparticles (NPs) loaded with ketorolac tromethamine (KT) intended for topical ocular delivery. NPs were prepared using ionic gelation method incorporating tri-polyphosphate (TPP) as cross-linker. Following the preparation, the composition of the system was optimized in terms of their particle size, zeta potential, entrapment efficiency (EE) and morphology, as well as performing structural characterization studies using Fourier transform infrared spectroscopy (FT-IR) and differential scanning calorimetry (DSC). The data suggested that the size of the NPs was affected by CS/TPP ratio where the diameter of the NPs ranged from 108.0 ± 2.4 nm to 257.2 ± 18.6 nm. A correlation between drug EE and the corresponding drug concentration added to the formulation was observed, where the EE of the NPs increased with increasing drug concentration, for up to 10 mg/mL. FT-IR and DSC revealed that KT was dispersed within the NPs where the phosphate groups of TPP were associated with the ammonium groups of CS. The in vitro release profile of KT from CS NPs showed significant differences (p < 0.05) compared to KT solution. Furthermore, mucoadhesion studies revealed adhesive properties of the formulated NPs. The KT-loaded NPs were found to be stable when stored at different storage conditions for a period of 3 months. The ex vivo corneal permeation studies performed on excised porcine eye balls confirmed the ability of NPs in retaining the drug on the eye surface for a relatively longer time. These results demonstrate the potential of CS-based NPs for the ocular delivery of KT.

Poloxamer-based thermoresponsive ketorolac tromethamine in situ gel preparations: Design, characterisation, toxicity and transcorneal permeation studies

&NA; This study was aimed at preparing, characterising and evaluating in situ gel formulations based on a blend of two hydrophilic polymers i.e. poloxamer 407 (P407) and poloxamer 188 (P188) for a sustained ocular delivery of ketorolac tromethamine (KT). Drug‐polymer interaction studies were performed using DSC and FT‐IR. The gelation temperature (Tsol‐gel), gelation time, rheological behaviour, mucoadhesive characteristics of these gels, transcorneal permeation and ocular irritation as well as toxicity was investigated. DSC and FT‐IR studies revealed that there may be electrostatic interactions between the drug and the polymers used. P188 modified the Tsol/gel of P407 bringing it close to eye temperature (35 °C) compared with the formulation containing P407 alone. Moreover, gels that comprised P407 and P188 exhibited a pseudoplastic behaviour at different concentrations. Furthermore, mucoadhesion study using mucin discs showed that in situ gel formulations have good mucoadhesive characteristics upon increasing the concentration of P407. When comparing formulations PP11 and PP12, the work of adhesion decreased significantly (P < 0.001) from 377.9 ± 7.79 mN mm to 272.3 ± 6.11 mN mm. In vitro release and ex vivo permeation experiments indicated that the in situ gels were able to prolong and control KT release as only 48% of the KT released within 12 h. In addition, the HET‐CAM and BCOP tests confirmed the non‐irritancy of KT loaded in situ gels, and HET‐CAM test demonstrated the ability of ocular protection against strongly irritant substances. MTT assay on primary corneal epithelial cells revealed that in situ gel formulations loaded with KT showed reasonable and acceptable percent cell viability compared with control samples. Graphical abstract In this study, the thermoresponsive behaviour of poloxamers was employed as a trigger for the formation of in situ gel systems incorporating ketorolac tromethamine (KT) (A). The protective effect of KT loaded in situ gel preparation on 10 day old chorioallantoic membrane (CAM) has been investigated after treating the CAM with a strong irritant, NaOH (B). The BCOP test revealed the corneal opacity and permeability of the prepared in situ gel system (C). The MTT cytotoxicity assay demonstrated that the cell viability when treated with the selected in situ gel preparations was at an acceptable level compared to the control samples (D). Figure. No caption available.

Preparation and Characterization of Gas-filled Liposomes: Can They Improve Oil Recovery?

Although well known for delivering various pharmaceutical agents, liposomes can be prepared to entrap gas rather than aqueous media and have the potential to be used as pressure probes in magnetic resonance imaging (MRI). Using these gas-filled liposomes (GFL) as tracers, MRI imaging of pressure regions of a fluid flowing through a porous medium could be established. This knowledge can be exploited to enhance recovery of oil from the porous rock regions within oil fields. In the preliminary studies, we have optimized the lipid composition of GFL prepared using a simple homogenization technique and investigated key physico-chemical characteristics (size and the physical stability) and their efficacy as pressure probes. In contrast to the liposomes possessing an aqueous core which are prepared at temperatures above their phase transition temperature (Tc), homogenization of the phospholipids such as 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-distearoyl-sn-glycero-3-phosphocoline (DSPC) in aqueous medium below their Tc was found to be crucial in formation of stable GFL. DSPC based preparations yielded a GFL volume of more than five times compared to their DPPC counter part. Although the initial vesicle sizes of both DSPC and DPPC based GFL were about 10 μm, after 7 days storage at 25°C, the vesicle sizes of both formulations significantly (p < 0.05) increased to 28.3 ± 0.3 μm and 12.3 ± 1.0 μm, respectively. When the DPPC preparation was supplemented with cholesterol at a 1:0.5 or 1:1 molar ratio, significantly (p < 0.05) larger vesicles were formed (12–13 μm), however, compared to DPPC only vesicles, both cholesterol supplemented formulations displayed enhanced stability on storage indicating a stabilizing effect of cholesterol on these gas-filled vesicles. In order to induce surface charge on the GFL, DPPC and cholesterol (1: 0.5 molar ratio) liposomes were supplemented with a cationic surfactant, stearylamine, at a molar ratio of 0.25 or 0.125. Interestingly, the ζ potential values remained around neutrality at both stearylamine ratios suggesting the cationic surfactant was not incorporated within the bilayers of the GFL. Microscopic analysis of GFL confirmed the presence of spherical structures with a size distribution between 1–8 μm. This study has identified that DSPC based GFL in aqueous medium dispersed in 2% w/v methyl cellulose although yielded higher vesicle sizes over time were most stable under high pressures exerted in MRI.

Designing nonionic surfactant vesicles for the delivery of antigens for systemic and alternative delivery routes

Bilayer vesicles can be prepared from a range of molecules including nonionic surfactants. Vesicles built from nonionic surfactants are known as nonionic surfactant vesicles or niosomes. Whilst structurally similar to liposomes, the use of nonionic surfactants in a formulation may offer advantages in terms of chemical stability and reduced cost in some cases. In general, the ability of surfactant blends to form vesicles is dependent on their combined critical packing parameter, with cholesterol often being used to support the formation of vesicle constructs. To enhance the potency and delivery of antigens, niosomes can be designed to protect antigens against degradation in harsh in vivo environments, including the oral route, and enhance delivery of antigens to appropriate target sites. Key considerations in the design of niosomal adjuvants include the choice of surfactants, the surface properties of the vesicles, the method of preparation, the cholesterol content and the inclusion of immunostimulatory agents. Manipulation of these attributes allows vesicle constructs to be designed and built that can be used to deliver antigens via a range of delivery routes.

Environmental scanning electron microscope imaging of vesicle systems

The structural characteristics of liposomes have been widely investigated and there is certainly a strong understanding of their morphological characteristics. Imaging of these systems, using techniques such as freeze-fracturing methods, transmission electron microscopy, and cryo-electron imaging, has allowed us to appreciate their bilayer structures and factors which can influence this. However, there are few methods which all us to study these systems in their natural hydrated state; commonly the liposomes are visualized after drying, staining, and/or fixation of the vesicles. Environmental Scanning Electron Microscopy (ESEM) offers the ability to image a liposome in its hydrated state without the need for prior sample preparation. Within our studies we were the first to use ESEM to study liposomes and niosomes and we have been able to dynamically follow the hydration of lipid films and changes in liposome suspensions as water condenses on to, or evaporates from, the sample in real time. This provides insight into the resistance of liposomes to coalescence during dehydration, thereby providing an alternative assay of liposome formulation and stability.