Molecular dynamics simulations of drug delivery liposomes and their interactions with bloodstream elements

Drug delivery is a vital issue in pharmaceutical research; once a drug candidate molecule is identified, it must be delivered to the target area of the body where it can take effect. In addition, non-specific distribution of drug molecules to areas other than the drug target must be decreased to avoid unwanted side effects. To achieve this, nanotechnological drug delivery systems can be used. Nanotechnological drug delivery systems come in a wide variety of forms, including liposomes, dendrimers, nanoparticles, and polymeric micelles. Of these, our research is focused on drug delivery liposomes. Drug delivery liposomes are composed of a membrane that forms a closed spherical sack, with a diameter of approximately 100 nm that can contain drug molecules. The criteria for effectiveness of these drug delivery liposomes (DDLs) are structural stability, its lifetime in the bloodstream, the release rate of the encapsulated content and site specific targeting. Cholesterol is one of the crucial lipid components of the DDL known to increase its stability. They also can have a protective polymer coating such as polyethylene glycol (PEG) that protects the DDL from the body s defense mechanisms. Also the DDL can posses targeting moieties, able to direct the PEGylated liposomes to the specific target. In this study we have investigated surface structure of the DDL and its interactions with elements of the blood stream. While it is difficult to determine an accurate picture of the DDL surface and its interactions with ions and bloodstream proteins with atomistic resolution by experiments alone, computational molecular modelling techniques can provide insights into it. Hence, we have used computational modelling and molecular dynamics simulations to understand the role of each component of the DDL in its structure. The three of the five reported studies in this thesis (I, II, III) are focused on how surface charge plays an important role in the liposome, how it is affected by various components of the DDLs, and how the specific interactions of DDLs and ions present in the blood stream influence it. The chapter IV deals with understanding the properties by systematically varying components such as cholesterol and PEG. Also we have produced the first ever model of the first FDA approved drug delivery liposome (DOXIL ®) at atomistic resolution details. The last study (V) deals with the application of molecular dynamics in targeted drug delivery research. In this study we could identify the reason for failure of specific novel targeting peptide (AETP), which is used to functionalize the DDL, by identifying its interactions with the protective PEG polymer.Tämä väitöskirja käsittelee lääkeaineiden kantajina toimivien liposomien eli lipidikalvoista muodostuvien rakkuloiden tutkimusta laskennallisin menetelmin. Tutkimme liposomien ominaisuuksia ja pintarakenteita sekä vuorovaikutusta verenkierron yhdisteiden kanssa. Käytimme molekyylimallinnusmenetelmiä yhdessä kokeellisten tutkimusten kanssa saadaksemme kattavamman kuvan liposomeista lääkeaineiden kantajina. Tuntemalla nämä rakenteet ja niiden vuorovaikutukset atomien tasolla, on mahdollista kehittää edistyksellisiä lääkeainekantajia esimerkiksi syövän hoitoon

Determinants of Orexin Receptor Binding and Activation—A Molecular Dynamics Study

We assess the stability of two previously suggested binding modes for the neuropeptide orexin-A in the OX2 receptor through extensive molecular dynamics simulations. As the activation determinants of the receptor remain unknown, we simulated an unliganded receptor and two small-molecular ligands, the antagonist suvorexant and the agonist Nag26 for comparison. Each system was simulated in pure POPC membrane as well as in the 25% cholesterol–POPC membrane. In total, we carried out 36 μs of simulations. Through this set of simulations, we report a stable binding mode for the C-terminus of orexin-A. In addition, we suggest interactions that would promote orexin receptor activation, as well as others that would stabilize the inactive state.Peer reviewe

Mechanistic Insight into How PEGylation Reduces the Efficacy of pH-Sensitive Liposomes from Molecular Dynamics Simulations

Liposome-based drug delivery systems composed of DOPE stabilized with cholesteryl hemisuccinate (CHMS) have been proposed as a drug delivery mechanism with pH-triggered release as the anionic form (CHSa) is protonated (CHS) at reduced pH; PEGylation is known to decrease this pH sensitivity. In this manuscript, we set out to use molecular dynamics (MD) simulations with a model with all-atom resolution to provide insight into why incorporation of poly(ethyleneglycol) (PEG) into DOPE–CHMS liposomes reduces their pH sensitivity; we also address two additional questions: (1) How CHSa stabilizes DOPE bilayers into a lamellar conformation at a physiological pH of 7.4? and (2) how the change from CHSa to CHS at acidic pH triggers the destabilization of DOPE bilayers? We found that (A) CHSa stabilizes the DOPE lipid membrane by increasing the hydrophilicity of the bilayer surface, (B) when CHSa changes to CHS by pH reduction, DOPE bilayers are destabilized due to a reduction in bilayer hydrophilicity and a reduction in the area per lipid, and (C) PEG stabilizes DOPE bilayers into the lamellar phase, thus reducing the pH sensitivity of the liposomes by increasing the area per lipid through penetration into the bilayer, which is our main focus.Peer reviewe

Membrane Binding of Recoverin : From Mechanistic Understanding to Biological Functionality

Recoverin is a neuronal calcium sensor involved in vision adaptation that reversibly associates with cellular membranes via its calcium-activated myristoyl switch. While experimental evidence shows that the myristoyl group significantly enhances membrane affinity of this protein, molecular details of the binding process are still under debate. Here, we present results of extensive molecular dynamics simulations of recoverin in the proximity of a phospholipid bilayer. We capture multiple events of spontaneous membrane insertion of the myristoyl moiety and confirm its critical role in the membrane binding. Moreover, we observe that the binding strongly depends on the conformation of the N-terminal domain. We propose that a suitable conformation of the N-terminal domain can be stabilized by the disordered C-terminal segment or by binding of the target enzyme, i.e., rhodopsin kinase. Finally, we find that the presence of negatively charged lipids in the bilayer stabilizes a physiologically functional orientation of the membrane-bound recoverin.Peer reviewe

642 oncolytic vaccines with modified tumor epitopes for cancer immunotherapy

Oncolytic adenoviruses (OAds) are capable of killing tumor cells while activating the immune system due to their immunogenicity. Hance, they are an excellent platform for oncolytic vaccine. We previously demonstrated that the injection of peptide-coated conditionally replicating adenoviruses (PeptiCRAd) is capable of reducing the growth of established aggressive melanomas (murine B16).Oncolytic vaccines, like PeptiCRAds, often rely on inducing an immune response against specific tumor antigens. However, many tumor antigens are also self-antigens, hence the peripheral tolerance might impair the activity of tumor-reactive T-cells. Therefore, mutated epitopes represent an optimal tool to break the tolerance, exploiting the cross-reactivity of T-cells. To this end we developed an Epitope Discovery and Improvement System (EDIS) framework to study native epitopes and predict, in silico, mutated forms suitable for cancer therapy. The novel aspect of EDIS is the ability to interrogate different prediction servers, integrate the different results and validate these by molecular dynamics simulations.We started by studying the model epitope SIINFEKL. According to our in silico predictions, two mutated variants were suggested to be more immunogenic than the native SIINFEKL peptide. To test whether this prediction would reflect in enhanced vaccine effect we studied the immune response against these peptides in B16-OVA bearing mice. Mice were challenged with B16-OVA and treated with three different PeptiCRAds coated with SIINFEKL and the two predicted derivates. By ELISPOT assay we assessed the anti-peptide response and demonstrated that the two mutated forms were in fact more effective in reducing the growth of established B16OVA tumors.Finally, we studied the native epitope SVYDFFVWL from the tyrosinase-related protein 2 (TRP2), a melanoma antigen in clinical evaluation. By using the EDIS framework we selected two mutated variants that show increased MHC-I binding affinity and we tested them by treating aggressive B16F10 tumors. As expected, treatment with the native TRP2 reduced the growth of the tumors compared to the controls. Suprisingly, one of the two analogues improved significantly the survival of mice and reduced the growth of their tumors compared to the group treated with the native TRP2 epitope. In conclusion, we demonstrated that the integration of different in silico methods increases the accuracy when predicting mutated epitopes for cancer immunotherapy

Influence of doxorubicin on model cell membrane properties : insights from in vitro and in silico studies

Despite doxorubicin being commonly used in chemotherapy there still remain significant holes in our knowledge regarding its delivery efficacy and an observed resistance mechanism that is postulated to involve the cell membrane. One possible mechanism is the efflux by protein P-gp, which is found predominantly in cholesterol enriched domains. Thereby, a hypothesis for the vulnerability of doxorubicin to efflux through P-gp is its enhanced affinity for the ordered cholesterol rich regions of the plasma membrane. Thus, we have studied doxorubicin's interaction with model membranes in a cholesterol rich, ordered environment and in liquid-disordered cholesterol poor environment. We have combined three separate experimental protocols: UV-Vis spectrophotometry, fluorescence quenching and steady-state anisotropy and computational molecular dynamics modeling. Our results show that the presence of cholesterol induces a change in membrane structure and doesn't impair doxorubicin's membrane partitioning, but reduces drug's influence on membrane fluidity without directly interacting with it. It is thus possible that the resistance mechanism that lowers the efficacy of doxorubicin, results from an increased density in membrane regions where the efflux proteins are present. This work represents a successful approach, combining experimental and computational studies of membrane based systems to unveil the behavior of drugs and candidate drug molecules.Peer reviewe

Cholesterol level affects surface charge of lipid membranes in saline solution

Peer reviewe

Two cations, two mechanisms : interactions of sodium and calcium with zwitterionic lipid membranes

Adsorption of metal cations onto a cellular membrane changes its properties, such as interactions with charged moieties or the propensity for membrane fusion. It is, however, unclear whether cells can regulate ion adsorption and the related functions via locally adjusting their membrane composition. We employed fluorescence techniques and computer simulations to determine how the presence of cholesterol-a key molecule inducing membrane heterogeneity-affects the adsorption of sodium and calcium onto zwitterionic phosphatidylcholine bilayers. We found that the transient adsorption of sodium is dependent on the number of phosphatidylcholine head groups, while the strong surface binding of calcium is determined by the available surface area of the membrane. Cholesterol thus does not affect sodium adsorption and only plays an indirect role in modulating the adsorption of calcium by increasing the total surface area of the membrane. These observations also indicate how lateral lipid heterogeneity can regulate various ion-induced processes including adsorption of peripheral proteins, nanoparticles, and other molecules onto membranes.Peer reviewe