Vesicular Phospholipid Gels Research Articles

Vesicular phospholipid gel-based depot formulations for pharmaceutical proteins: Development and in vitro evaluation

The object of this study was to evaluate the potential of vesicular phospholipid gels (VPGs) as an alternative delivery system for therapeutic proteins. Therefore, the model protein erythropoietin (EPO) was incorporated into various VPG formulations by dual asymmetric centrifugation. In order to characterize and to quantify the incorporated protein, different extraction protocols were investigated. Among several detergents and organic solvents an extraction method applying chloroform was found most suitable. SDS-PAGE analysis of EPO extracted from VPGs revealed excellent protein stability which was maintained in the VPGs' matrix after an incorporation process with dual asymmetric centrifuge. Irrespective of the investigated formulation all VPGs delivered the protein over prolonged periods of time at close to linear kinetics without any burst effect. Both the lipid content and the lipid charge had a strong influence on the release behavior. For instance formulations based on 300 mg/g lipid delivered 83% EPO after 280 h while gels based on 550 mg/g lipid liberated 64% within 400 h. From the parallelism of release and erosion kinetics found for all formulations it was concluded that erosion rather than diffusion is the dominant release controlling mechanism for these macromolecule-loaded VPGs. For the first time the present study presents VPGs as promising alternative depot systems for protein drugs.

Dual asymmetric centrifugation (DAC)—A new technique for liposome preparation

This is the first report on the use of a “dual asymmetric centrifuge (DAC)” for preparing liposomes. DAC differs from conventional centrifugation by an additional rotation of the sample around its own vertical axis: While the conventional centrifugation constantly pushes the sample material outwards, this additional rotation constantly forces the sample material towards the center of the centrifuge. This unique combination of two contra rotating movements results in shear forces and thus, in efficient homogenization. We demonstrated that it is possible to prepare liposomes by DAC, by homogenizing a rather concentrated blend of hydrogenated phosphatidylcholine and cholesterol (55:45 mol%) and 0.9% NaCl-solution, which results in a viscous vesicular phospholipid gel (VPG). The resulting VPG can subsequently be diluted to a conventional liposome dispersion. Since DAC is intended to make sterile preparations of liposomes, or to entrap toxic/radioactive compounds, the process was performed within a sealed vial. It could be shown that the DAC speed, the lipid concentration, the homogenization time and the addition of a mixing aid (glass beads) are all critical for the size of the liposomes. Optimized conditions resulted in liposomes of 60 ± 5 nm and a trapping efficacy of 56 ± 3.3% for the model compound calcein.

Vesicular Phospholipid Gels: A Technology Platform

Vesicular phospholipid gels (VPGs) represent semi-solid phospholipid dispersions. Their morphology is truly vesicular with aqueous compartments both within the core of the vesicles and in-between the vesicles. VPGs are suited to carry both hydrophilic, amphiphilic and lipophilic drugs. Their drug load is stable since there is no concentration gradient between the vesicles' core and the surrounding water phase. VPGs are suited to release drugs in a controlled manner, and thus may serve as depot implants. When blended with excess aqueous medium VPGs are easily converted into small-sized liposome (SUV) dispersions showing high encapsulation efficiencies for all kinds of drugs. VPG-formulations with various cytostatic drugs have been tested successfully in human xenografts. Obviously, the vesicles protect the drugs from premature metabolic inactivation and/or elimination and guide them to solid tumors with enhanced vascular permeability (passive targeting).Furthermore, when mounted on a filter support, VPGs represent a tight diffusion barrier suitable for screening of oral drug permeability, as demonstrated by a set of 21 drug compounds. Permeability values were shown to fit well with human absorption in vivo, indicating that the model is suited for rapid screening of passive transport properties of new chemical entities.

Development and in vitro evaluation of a liposome based implant formulation for the decapeptide cetrorelix

Semisolid phospholipid dispersions of vesicular morphology, so-called vesicular phospholipid gels (VPGs), were prepared by high-pressure homogenisation and tested in vitro for their suitability as implantable sustained release system for the decapeptide cetrorelix, a potent LH-RH antagonist. The VPGs contained 300–500 mg/g egg phosphatidylcholine (E80) and 0.5–10 mg/g cetrorelix acetate (CXA). The in vitro release experiments showed a wide variability of the system in release, ranging from complete release within less than 24 h (0.5 mg/g CXA; 400 mg/g E80) to a predicted 80% sustained release over 3 months (8.6 mg/g CXA; 280 mg/g E80). Erosion of the phospholipid matrix, i.e. release of phospholipid vesicles was found to be the main release mechanism, following zero order or first order kinetics depending on the composition of the VPG. CXA-concentration dependent drug–drug or drug–lipid interactions are assumed to be responsible for the change in release kinetics and the decrease of CXA release at high concentrations of the peptide. Multivariate analysis revealed that both lipid concentration and peptide concentration and also the interactions between the two factors are significant factors for the release rate of the peptide. In summary: based on the presented in vitro release data sustained release of therapeutically relevant CXA doses over up to 6 weeks appears feasible. VPGs are thus considered as a promising new approach for the sustained release of peptide hormones.

Effect of β-sitosterol on the characteristics of vesicular gels containing chlorhexidine

Previous studies confirm that β-sitosterol is very effective in altering the molecular packing of soybean lecithin bilayers even more than the cholesterol. The primary aim of the present study was to evaluate the influence of the β-sitosterol portion in the lipid bilayer on the physical–chemical characteristics of the prepared gel systems, and its influence on the consequent drug release from the liposomes obtained from vesicular phospholipid gels (VPG-s) by redispersion. VPG-s were prepared of different molar ratios of lecithin:sterol components (10:90–35:65 mol%). The mixture was hydrated with the aqueous solution of chlorhexidin digluconate in order to achieve 30% (w/w) final concentration of the lipid mixtures and 4% (w/w) concentration of the drug in each homogenized VPG sample. To characterize the obtained VPG systems optical microscopic examinations using polarized light, differential scanning calorimetry (DSC), photon correlation spectroscopy (PCS), and dynamic surface tension measurements were carried out. Vertical type diffusion cell was applied to determine the amount of released chlorhexidine digluconate. As a result of the surface tension-decreasing effect of β-sitosterol, the membrane deformability and the dispersity of the system increased. The increased dispersity and fluidity significantly increased the extent of released chlorhexidine from the vesicles.

5-Fluorouracil in vesicular phospholipid gels for anticancer treatment: entrapment and release properties

Vesicular phospholipid gels (VPG), i.e. highly concentrated liposomal dispersions, are suitable for entrapping substances such as anticancer drugs with particular high encapsulation efficiencies (EE). We prepared different formulations of VPG with 30% (w/w) lipid containing 5-fluorouracil (5-FU) by high pressure homogenization and analysed their EE and drug release. Using mixtures of hydrogenated soy phosphatidylcholine and cholesterol with molar ratios ranging from 55/45 to 75/25, a decreasing amount of cholesterol correlated with an increasing EE, which is probably due to a reduced amount of smaller vesicles and number of lamellae. Using a 5-FU solution of pH 8.6 for VPG preparation, an EE of approximately 40% was found after redispersion of the gel to a liposomal dispersion and separation of free drug from liposomal drug by size exclusion chromatography. The reduced EE for preparations with lower pH values was attributed to a fast initial drug release due to the increased drug lipophilicity below the p K a value of 8. After redispersion of a VPG of pH 8.0, an initially faster release of about a third of the entrapped drug was found during the first 20 min, followed by stable entrapment over many hours. The rapid initial release may be due to the portion of liposomes smaller than 40 nm in diameter, determined by photon correlation spectroscopy. Cryo electron microscopic pictures show a lentil-like shape of these small liposomes. The membrane defects on the edges are probably the reason for the very high initial drug release rate. The half-life time of the release of 5-FU from intact FU-VPG at both pH 7.4 and 8.0 was found to be in the order of 4–5 h and the kinetics are typical for matrix-controlled drug diffusion. The in vitro data of 5-FU loaded VPG suggest their applicability as implants with controlled release properties or, after redispersion, as intravenously injected liposomal formulations.

Pharmacokinetics and antitumor activity of vincristine entrapped in vesicular phospholipid gels.

In vivo antitumoral activity, pharmacokinetics (PK) and biodistribution of a new liposomal formulation of vincristine (VCR-Lip) were compared to VCR in aqueous solution (VCR-Conv). VCR was entrapped into a vesicular phospholipid gel (VPG) consisting of densely packed liposomes. Redispersed VCR-containing VPG (VCR-Lip) consisted of 54% liposomally entrapped and 46% free VCR. In vivo efficacy of VCR-Lip versus VCR-Conv was tested using the s.c. growing human small cell lung carcinoma LXFS 650 and the human mammary carcinoma MX1. PK and biodistribution were evaluated using radiolabeled drug and lipid in LXFS 650 tumor-bearing mice. VCR-Lip at a dose of 1.0 mg/kg (dose near the maximum tolerated dose) led to partial remissions in the MX1 tumor xenograft model (T/C=3.9%). VCR-Conv at an equitoxic dose of 0.6 mg/kg produced only a tumor growth inhibition (T/C=7.0%). In LXFS 650 tumor-bearing mice, VCR-Lip was highly active at doses of 0.75 (T/C=0.7%) and 1.0 (T/C=0.0%) mg/kg, and complete tumor regressions were observed. In contrast, equitoxic doses of VCR-Conv (0.6 mg/kg) resulted only in less pronounced tumor remissions (T/C=4.1%). The PK study revealed that VCR-Lip achieved an over 10-fold higher plasma AUC (22.6 microg x h/ml) than VCR-Conv (2.16 microg x h/ml). Moreover, tumor drug levels were 2.3-fold higher when VCR was injected as VCR-Lip in comparison to VCR-Conv. In some cases, however, VCR-Lip as well as blank VPG appeared to be toxic. We conclude that VCR-Lip is an effective VCR delivery system with superior antitumor activity compared to VCR-Conv. The enhanced efficacy can be explained by sustained release and passive tumor targeting.

Change in pharmacokinetic and pharmacodynamic behavior of gemcitabine in human tumor xenografts upon entrapment in vesicular phospholipid gels

The in vivo pharmacokinetics (PK), biodistribution and antitumor activity of a new liposomal formulation of gemcitabine (GemLip) were compared to the conventional (clinical) formulation of gemcitabine (GemConv). Gemcitabine was entrapped in a vesicular phospholipid gel (VPG) consisting of densely packed liposomes. Redispersed VPG containing GemLip consisted of 33% liposomally entrapped and 67% free gemcitabine. The in vivo efficacies of GemLip and GemConv were compared using the subcutaneously growing human soft tissue sarcoma SXF 1301 and the orthotopically growing human bladder cancer BXF 1299T. PK and biodistribution were evaluated using radiolabeled drug and lipid in SXF 1301 tumor-bearing nude mice. GemLip was highly active in SXF 1301 at a gemcitabine dose of 6-9 mg/kg (days 1, 8 and 15; dose near the MTD). In the 6-mg/kg groups, complete tumor remissions were observed in seven of eight mice. Equimolar doses of GemConv resulted in only moderate tumor growth inhibition. Even at equitoxic doses (360 mg/kg given on days 1, 8 and 15, or 120 mg/kg on days 1, 5 and 8) GemConv was less active than GemLip. Furthermore, GemLip was active in the orthotopically growing BXF 1299T bladder cancer model at 6 mg/kg and prevented distant organ metastasis. In the PK study, GemLip achieved a 35-fold higher plasma AUC (1680 mg x h/ml) than GemConv (47.6 mg x h/ml). The serum half-lives were 0.15 h for free gemcitabine and 13.3 h for liposomal gemcitabine (6 mg/kg each i.v.). Moreover, gemcitabine levels in tumors were fourfold higher following injection of GemLip than following injection of GemConv. GemLip is a highly effective gemcitabine delivery system which results in superior gemcitabine pharmacodynamics and PK than GemConv. The enhanced in vivo efficacy might be explained by sustained release and passive tumor targeting.

MORPHOLOGICAL AND THERMAL PROPERTIES OF VESICULAR PHOSPHOLIPID GELS STUDIED BY DSC, RHEOMETRY AND ELECTRON MICROSCOPY

Semisolid phospholipid preparations have been well known for several years and are still investigated as drug carrier systems, e.g. for potential cancer therapy. They may be applied parenterally as semisolid vesicular phospholipid gels suitable as implants for sustained drug release or as liposomal preparations after redisperging the stable storage form. Due to enhanced stability, mixtures of hydrated phospholipids and cholesterol are more suitable than natural unsaturated phospholipids. In order to describe characteristics of vesicular phospholipid gels, only a few techniques may be useful. Especially the structure of the semisolid preparation is not yet completely understood. We tried to get some more information about these systems by using a combination of freeze-fracture electron microscopy, differential scanning calorimetry and rheometry to elucidate, on the one hand, the inner structure or homogeneity and, on the other, the thermotropic phase transition of the three-dimensional lipid network and the temperature dependency of the fluidity/viscosity of the samples. Using freeze-fracture electron microscopy we found coexisting phospholipid domains of lamellar sheets and vesicular structures. With the help of differential scanning calorimetry the reasons for the different phase behaviour were elucidated. Rheometric measurements show increased intermediate viscosity at the thermotropic phase transition of the lipid bilayers, possibly induced by interacting membrane defects.

Steam sterilisation of vesicular phospholipid gels

Vesicular phospholipid gels (VPGs), highly concentrated phospholipid dispersions of semisolid consistency and vesicular morphology are under investigation as potential implantable depots for sustained release of drugs and as intermediates for subsequent dilution into ‘conventional’ liposome dispersions. It was investigated here if VPGs can be steam sterilised. VPGs prepared from 400 mg/g egg-phosphatidylcholine by high-pressure homogenisation retained their vesicular structure but showed a slight increase in vesicle size (freeze-fracture electron microscopy). However, autoclaving slowed down both, the in vitro release of the hydrophilic marker carboxyfluorescein and vesicles from VPGs. This was assumed to be due to bigger vesicle sizes and corresponding increase in packing density of the vesicular matrix. Upon dilution into a liposome dispersion both negative staining electron microscopy and dynamic laser light scattering analysis confirmed a distinct increase in liposome size, mainly due to fusion of small (20 nm) vesicles with unfavourable curvature. This was consistent with the observed increase in encapsulation efficiency of carboxyfluorescein. Phospholipid hydrolysis during autoclaving was negligible with lysophosphatidylcholine formation of less than 2% (thin layer chromatography). Despite significant change of their morphological and functional properties during autoclaving VPGs retained their main characteristics, such as vesicular structure, sustained release and dilutability to liposome dispersions, and are, therefore, considered as autoclavable.

Erosion and controlled release properties of semisolid vesicular phospholipid dispersions

Phosphatidylcholine, when dispersed in aqueous medium in high concentrations (300 mg/g and above) by high pressure homogenisation, forms semisolid pastes of vesicular morphology. The multivesicular matrix can accommodate hydrophilic compounds in the aqueous compartments. The disintegration and release of hydrophilic marker of these dispersions were studied in vitro by using a flow-through cell. Slow release of the marker over periods of hours up to days took place via two different mechanisms: (1) erosion of the matrix with release of marker-filled liposomes and marker which had been trapped in between the liposomes and (2) diffusion of marker through the membranes. Whereas for lipid concentrations up to 300 mg/g almost spontaneous disintegration occurred, more concentrated pastes (350–400 mg/g) showed zero-order erosion kinetics for 4–6 h. Erosion was rate limiting for overall release. For 450 and 500 mg/g lipid dispersions, the release of free marker followed square root of time kinetics for 13 or 21 h, respectively, which is typical for matrix-controlled diffusion. Underlying the diffusion was a slow zero-order erosion of the matrix. The results are important for the future development of vesicular phospholipid gels as sustained release therapeutic system, e.g. as implantable depot.