Liposomes are widely investigated for their use as drug delivery systems, where they have to meet strict stability criteria. Hence, it is of common interest to establish appropriate storage conditions to improve the shelf life of liposomes. In general, long-term stability can be achieved by freezing as well as freeze-drying, and different carbohydrates or polyalcohols, such as mannitol or glycerol are considered as cryoprotective agents to inhibit liposomal fusion or degradation during freezing procedures. Here, we determined the impact of different cryoprotectants on physicochemical parameters of sterically stabilized PEGylated liposomes, which become increasingly important for pharmaceutical applications. We investigated particle stability in terms of size, lamellarity and thickness of the lipid bilayer using photon correlation spectroscopy and small angle X-ray scattering. Besides, we evaluated the impact of cryoprotectants on the thermal lipid phase behavior of either frozen/thawn or lyophilised/rehydrated PEGylated liposome formulations by differential scanning calorimetry. Optimal results for the preservation of the average size of the extruded unilamellar liposomes during freezing were achieved using a mixture of glycerol and carbohydrate concentrations of about 1% (w/v), irrespective of the carbohydrate used. We found no significant changes in the bilayer organisation, and the transition behavior of lipids was almost uneffected by freezing. In case of freeze-drying, similar carbohydrate concentrations as used for freezing were sufficient to maintain the size of PEGylated liposomes after reconstitution of the dried lyophilised cakes, but our small angle X-ray scattering data provide strong evidence that the lyophilisation/rehydration process affects lipid membrane reorganisation on a molecular level such that a swelling of the bilayer might occur. These internal structural changes, which are not detected by standard particle size analysis, might well influence drug release profiles and the pharmacological performance of a liposomal drug delivery system.

Radiolabeling of nanoparticles (NPs) has been performed for a variety of reasons, such as for studying pharmacokinetics, for imaging, or for therapy. Here, we describe the in vitro and in vivo evaluation of DTPA-derivatized lipid-based NP (DTPA-NP) radiolabeled with different radiometals, including In-111 and Tc-99m, for single-photon emission computed tomography (SPECT), Ga-68 for positron emission tomography (PET), and Lu-177 for therapeutic applications. PEGylated DTPA-NP with varying DTPA amounts, different composition, and size were radiolabeled with In-111, Lu-177, and Ga-68, using various buffers. Tc-99m-labeling was performed directly and by using the carbonyl aquaion, [99mTc(H2O)(3) (CO)(3)](+). Stability was tested and biodistribution evaluated. High labeling yields (> 90%) were achieved for all radionuclides and different liposomal formulations. Specific activities (SAs) were highest for In-111 (> 4 MBq/mu g liposome), followed by Ga-68 and Lu-177; for 99mTc, high labeling yields and SA were only achieved by using [99mTc(H2O)(3) (CO)(3)](+). Stability toward DTPA/histidine and in serum was high (> 80 % RCP, 24 hours postpreparation). Biodistribution in Lewis rats revealed no significant differences between NP in terms of DTPA loading and particle composition; however, different uptake patterns were found between the radionuclides used. We observed lower retention in blood (< 3.3 % ID/g) and lower liver uptake (< 2.7 % ID/g) for Tc-99m- and 68Ga, compared to In-111-NP (blood, < 4 % ID/g; liver, < 3.6 % ID/g). Imaging potential was shown by both PET magnetic resonance imaging fusion imaging and SPECT imaging. Overall, our study shows that PEGylated DTPA-NP are suitable for radiolabeling studies with a variety of radiometals, thereby achieving high SA suitable for targeting applications.

Inhalation of vasoactive intestinal peptide (VIP) was suggested as promising treatment option of various lung diseases like asthma and pulmonary hypertension. However, the medical use of peptides is limited by their short half-life due to rapid enzymatic degradation in the airways. For that reason, we recently developed unilamellar nano-sized VIP-loaded liposomes (VLL). Now we investigated their applicability for inhalation purposes.After nebulisation by a mouthpiece ventilation inhaler we found the particle size almost unaffected, being in a size range appropriate for bronchiolar deposition; we observed no peptide release due to nebulisation. The VIP release kinetics from VLL were tested by an ex vivo vasorelaxation model. Exposure to target organs revealed an immediate response, which was significantly retarded for VLL as compared to free VIP (p = 0.001). Using vasorelaxation as endpoint, we observed a sustained release and an extended pharmacological effect compared to equimolar free VIP.The liposomes have the potential to improve VIP inhalation therapy by providing a "dispersible peptide depot" in the bronchi. Thereby, the release of VIP from liposomes may be triggered by exposure to cells, i.e. directly by ligand-receptor interactions. (c) 2008 Elsevier B.V. All rights reserved.

Inhalative administration of vasoactive intestinal peptide (VIP) is a promising approach for the treatment of severe lung diseases. However, the clinical use of VIP is limited by the fact that the peptide is prone to rapid degradation mechanisms and proteolytic digestion. Accordingly, VIP exhibits a very short period of activity in the lung. To overcome this problem, we have designed a liposomal drug delivery system for VIP and characterized it in terms of its potential to protect VIP from enzymatic cleavage. The proteolytic conditions of the lung, the target site of aerosolic administered VIP, were mimicked by bronchoalveolar lavage fluid (BALF), a lung surfactant solution, obtained by fiberoptic bronchoscopy. Thus, the stability of VIP was assessed by its resistance to enzymatic degradation in BALF, using a combination of high pressure liquid chromatography with mass spectrometry. We found that free VIP was rapidly digested, whereas liposomal-associated VIP remained intact. By fluorescence spectroscopic techniques using fluorescent-labelled VIP we got strong indications that the tight association of VIP with the lipid membrane is only minimally affected upon incubation with BALF. Loading capacity and stability of EtCy3-VIP loaded liposomes were measured by fluorescence fluctuation spectroscopy. Finally, the protective properties of the liposomes were also expressed in the maintained biological activity of the peptide incubated with BALF.

A polymer-grafted liposomal formulation that has the potential to be developed for aerosolic pulmonary delivery of vasoactive intestinal peptide (VIP), a potent vasodilatory neuropeptide, is described. As VIP is prone to rapid proteolytic degradation in the microenvironment of the lung a proper delivery system is required to increase the half-life and bioavailability of the peptide. Here we investigate structural parameters of unilamellar liposomes composed of palmitoyl-oleoyl-phosphatidylcholine, lyso-stearyl-phosphatidylglycerol and distearyl-phosphatidyl-ethanolamine covalently linked to polyethylene glycol 2000, and report on VIP–lipid interaction mechanisms. We found that the cationic VIP is efficiently entrapped by the negatively charged spherical liposomes and becomes converted to an amphipathic α-helix. By fluorescence spectroscopy using single Trp-modified VIP we could show that VIP is closely associated to the membrane. Our data suggest that the N-terminal random-coiled domain is embedded in the interfacial headgroup region of the phospholipid bilayer. By doing so, neither the bilayer thickness of the lipid membrane nor the mobility of the phospholipid acyl chains are affected as shown by small angle X-ray scattering and electron spin resonance spectroscopy. Finally, in an ex vivo lung arterial model system we found that liposomal-associated VIP is recognized by its receptors to induce vasodilatory effects with comparable high relaxation efficiency as free VIP but with a significantly retarded dilatation kinetics. In conclusion, we have designed and characterized a liposomal formulation that is qualified to entrap biologically active VIP and displays structural features to be considered for delivery of VIP to the lung.