Liposomes formulated from the 1,3-diamidophospholipid Pad-PC-Pad are shear-responsive and thus promising nano-containers to specifically release a vasodilator at stenotic arteries. The recommended preclinical safety tests for therapeutic liposomes of nanometer size include the in vitro assessment of complement activation and the evaluation of the associated risk of complement activation-related pseudo-allergy (CARPA) in vivo. For this reason, we measured complement activation by Pad-PC-Pad formulations in human and porcine sera, along with the nanopharmaceutical-mediated cardiopulmonary responses in pigs. The evaluated formulations comprised of Pad-PC-Pad liposomes, with and without polyethylene glycol on the surface of the liposomes, and nitroglycerin as a model vasodilator. The nitroglycerin incorporation efficiency ranged from 25% to 50%. In human sera, liposome formulations with 20mg/mL phospholipid gave rise to complement activation, mainly via the alternative pathway, as reflected by the rises in SC5b-9 and Bb protein complex concentrations. Formulations having a factor of ten lower phospholipid content did not result in measurable complement activation. The weak complement activation induced by Pad-PC-Pad liposomal formulations was confirmed by the results obtained by performing an in vivo study in a porcine model, where hemodynamic parameters were monitored continuously. Our study suggests that, compared to FDA-approved liposomal drugs, Pad-PC-Pad exhibits less or similar risks of CARPA.

Cardio-vascular diseases are the main cause of death, emphasizing the need to improve patient treatment and survival. One therapeutic approach is a liposome-based drug carrier system specifically targeting constricted arteries. The recently discovered mechano-sensitive liposomes use hemodynamic shear-stress differences between healthy and constricted blood vessels as trigger for drug release. Liposomes are promising delivery containers but are being recognized as foreign by the immune system. Complement activation as essential factor of the recognition leads to adverse effects. Here, we tested complement activation by liposomes formulated from the artificial phospholipid Pad-PC-Pad in vitro. Surprisingly no complement activation was detected in human sera and porcine plasma. In in vivo experiments with three pigs, neither anaphylactic reactions nor other significant hemodynamic changes were observed even at comparably high liposome doses. The pilot study holds promise for an absence of complement-mediated adverse effects of Pad-PC-Pad liposomes in human.From the Clinical Editor: A lot of research has been done on new treatment for cardiovascular diseases. Liposome-based carrier systems have also shown promises. In this article, the authors studied the potential risks of complement activation by liposomes in in-vivo experiments. The absence of complement activation by Pad-PC-Pad liposomes may indicate its use in humans.

Liposomes of specific artificial phospholipids, such as Pad-PC-Pad and Rad-PC-Rad, are mechanically responsive. They can release encapsulated therapeutics via physical stimuli, as naturally present in blood flow of constricted vessel segments. The question is how these synthetic liposomes change their structure in the medically relevant temperature range from 22 to 42 °C. In the present study, small-angle neutron scattering (SANS) was employed to evaluate the temperatureinduced structural changes of selected artificial liposomes. For Rad-PC-Rad, Pad-Pad-PC, Sur-PC-Sur, and Sad-PC-Sad liposomes, the SANS data have remained constant because the phase transition temperatures are above 42 °C. For Pad-PC-Pad and Pes-PC-Pes liposomes, whose phase transitions are below 42 °C, the q-plots have revealed temperature-dependent structural changes. The average diameter of Pad-PC-Pad liposomes remained almost constant, whereas the eccentricity decreased by an order of magnitude. Related measurements using transmission electron microscopy at cryogenic temperatures, as well as dynamic light scattering before and after the heating cycles, underpin the fact that the non-spherical liposomes flatten out. The SANS data further indicated that, as a consequence of the thermal loop, the mean bilayer thickness increased by 20%, associated with the loss of lipid membrane interdigitation. Therefore, Pad-PC-Pad liposomes are unsuitable for local drug delivery in the atherosclerotic human blood vessel system. In contrast, Rad-PC-Rad liposomes are thermally stable for applications within the human body.

Liposomal drug delivery systems can protect pharmaceutical substances and control their release. Systemic administration of liposomes, however, often activate the innate immune system, resulting in hypersensitivity reactions. These pseudo-allergic reactions can be interpreted as activating the complement system. Complement activation destroys and eliminates foreign substances, either directly through opsonization and the formation of the membrane attack complex (MAC), or by activating leukocytes and initiating inflammatory responses via mediators, such as cytokines. In this study, we investigated the in vitro immune toxicity of the recently synthesized Rad-PC-Rad liposomes, analyzing the liposome-induced complement activation. In five human sera, Rad-PC-Rad liposomes did not induce activation, but in one serum high sensitivity via alternative pathway was detected. Such a behavior in adverse phenomena is characteristic for patient-to-patient variation and, thus, the number of donors should be in the order of hundreds rather than tens, hence the present study based on six donors is preliminary. In order to further prove the suitability of mechano-responsive Rad-PC-Rad liposomes for clinical trials, the production of pro-inflammatory cytokines was examined by human white blood cells. The concentrations of the pro-inflammatory cytokines, IL-6, IL-12p70, TNF-α, and IL-1β, induced by Rad-PC-Rad liposomal formulations, incubated with whole blood samples, were smaller or comparable to saline (negative control). Because of this favorable in vitro hemo-compatibility, in vivo investigations using these mechano-responsive liposomes should be designed.

For natural scientists and engineers, learning from nature has tradition and is often driven by bio-inspired processes and materials. For example, engineers have designed multifunctional materials with hierarchical structures. Lipid bilayers, the principal components of cell membranes, can form vesicles, termed liposomes. Such liposomes are usually recognized as foreign by the immune system of a patient, which makes it challenging to use liposomes as containers for targeted drug delivery. There are, however, promising non-spherical, mechano-sensitive, artificial liposomes about 100 nm in diameter, which were recently identified. These bio-inspired containers offer a wide range of applications. In particular, the targeted release at critically stenosed arteries formed as a result of atherosclerosis significantly reduces the undesired side effects such as a drop of blood pressure. It is well known that FDA-approved liposomal drugs, currently on the market, often induce adverse immune responses. Therefore, to exclude the hypersensitivity of the recently discovered mechano-sensitive liposomes, we have performed in vitro complement activation experiments and related animal studies with pigs. Recently, it has been shown that the drug-free Pad-PC-Pad liposomes surprisingly lack any complement activation. In this study, we demonstrate that nitroglycerin-loaded liposomes with relevant human therapeutic dosage exhibit low complement activation compared to the FDA-approved phospholipid drugs, including Abelcet. Furthermore, the liposomal suspensions applied are stable for a period of more than two months. Consequently, the non-spherical liposomes of nanometer size we have developed are promising containers for physically triggered, targeted drug delivery.