Preparation and characterization of nanometer-sized mechano-responsive liposomes for physically-triggered drug delivery

Cardiovascular diseases remain the leading causes of death worldwide, accounting for 17.7 million deaths every year - 31% of all global deaths. Atherosclerosis is an underlying disease process in blood vessels that leads to the accumulation of cholesterol plaques and a narrowing of the arterial lumen. If the plaque bursts, the components are flushed into the bloodstream, triggering intravascular thrombosis and leading to vascular occlusions. Heart attack or stroke are the most dangerous medical consequences of this. In such an event, time is of utmost importance because the infarcted organ suffers from necrosis without re-establishment of blood perfusion within a few minutes. Currently, emergency treatment provided in the ambulance involves intravenous injection of vasodilators that act systemically to dilate blood vessels and re-establish blood supply. However, this works systemically and even at a low dose lead to peripheral resistance decrease of the vessels and thereby hypotension. This generally inhibits the blood perfusion and thus the drug cannot work optimally at the location of the constricted vessel. Therefore, the development of a smart and effective drug delivery system, capable of releasing the drug locally, is desired. Critically constricted arteries give rise to increased wall shear stress that can be used as a physical trigger to release the therapeutics. Liposomes belong to the most attractive carriers for drug targeting in medical fields. Recently, mechano-responsive liposomes prepared from artificial phospholipids were suggested as nanocontainers for delivery and release of vasodilators at constrictions of arteries. This thesis project gives insight into the physicochemical properties of the mechano-responsive liposomes, determines their thermal stability at physiologically relevant body temperatures, and demonstrates their in vitro immunocompatibility. The preliminary characterization of nanometer-size liposomes is essential for the development of clinically relevant drug delivery systems. The mechano-sensitive liposomes Pad-PC-Pad and Rad-PC-Rad were studied by means of dynamic light scattering and transmission electron microscopy at cryogenic temperatures to determine size distribution and shape. In both cases, the liposomes were found to be around 100 nm in size with a variety of shapes. To prevent liposome aggregation as a consequence of the low zeta potential, a steric stabilization using polyethylene-glycol-grafted phospholipids was applied. To ensure mechano-responsive behavior at body temperature, the liposomes’ structure should be stable at physiological and elevated body temperatures. Therefore, the structural changes of liposomes were evaluated in a temperature range from 22 to 42 °C. Small-angle neutron scattering was used to measure the radius, eccentricity, and bilayer thickness of liposomes. Pad-PC-Pad liposomes already undergo structural changes at 35 °C. Further heating to 42 °C and subsequent cooling to room temperature resulted in a decreased eccentricity by an order of magnitude and a 20% increase of bilayer thickness, indicating the loss of membrane interdigitation. Rad-PC-Rad liposomes, however, show thermal stability up to 42 °C. Thus, Rad-PC-Rad liposomes possess sufficient thermal stability for drug delivery to atherosclerotic human blood vessels. To advance this technology towards clinical applications, the in vitro immunocompatibility of liposomes was investigated. The systemic administration of liposomes may trigger an immediate activation of the immune system, resulting in a hypersensitivity reaction. This reaction is driven by the activation of the complement system, which can stimulate the production of pro-inflammatory cytokines. Experiments demonstrated that both the Pad-PC-Pad and Rad-PC-Rad liposomal formulations exhibited low-to-moderate levels of complement proteins compared to the Food-and-Drug-Administration-approved liposomal drugs such as Doxil® and AmBisome®. Within the restricted number of individuals, one outliner was detected, suggesting that a substantially higher number of donors should be incorporated into future studies

X-ray micro computed tomography for the visualization of an atherosclerotic human coronary artery

Atherosclerosis refers to narrowing or blocking of blood vessels that can lead to a heart attack, chest pain or stroke. Constricted segments of diseased arteries exhibit considerably increased wall shear stress, compared to the healthy ones. One of the possibilities to improve patient's treatment is the application of nano-therapeutic approaches, based on shear stress sensitive nano-containers. In order to tailor the chemical composition and subsequent physical properties of such liposomes, one has to know precisely the morphology of critically stenosed arteries at micrometre resolution. It is often obtained by means of histology, which has the drawback of offering only two-dimensional information. Additionally, it requires the artery to be decalcified before sectioning, which might lead to deformations within the tissue. Micro computed tomography (muCT) enables the three-dimensional (3D) visualization of soft and hard tissues at micrometre level. muCT allows lumen segmentation that is crucial for subsequent flow simulation analysis. In this communication, tomographic images of a human coronary artery before and after decalcification are qualitatively and quantitatively compared. We analyse the cross section of the diseased human coronary artery before and after decalcification, and calculate the lumen area of both samples

Immunocompatibility of Rad-PC-Rad liposomes in vitro, based on human complement activation and cytokine release

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 has preliminary character. 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

Immunological response to nitroglycerin-loaded shear-responsive liposomes in vitro and in vivo

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 20 mg/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

Liposomes - bio-inspired nano-containers for physically triggered targeted drug delivery

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

UNSP 99670O: Histology-validated x-ray tomography for imaging human coronary arteries

Heart disease is the number one cause of death worldwide. To improve therapy and patient outcome, the knowledge of anatomical changes in terms of lumen morphology and tissue composition of constricted arteries is crucial for designing a localized drug delivery to treat atherosclerosis disease. Traditional tissue characterization by histology is a pivotal tool, although it brings disadvantages such as vessel morphology modification during decalcification and slicing. X-ray tomography in absorption and phase contrast modes yields a deep understanding in blood vessel anatomy in healthy and diseased stages: measurements in absorption mode make visible highly absorbing tissue components including cholesterol plaques, whereas phase contrast tomography gains better contrast of the soft tissue components such as vessel walls. Established synchrotron radiation-based micro-CT techniques ensure high performance in terms of 3D visualization of highly absorbing and soft tissues