Aniket Magarkar

Hydrophobin film structure for HFBI and HFBII and mechanism for accelerated film formation.

Hydrophobins represent an important group of proteins from both a biological and nanotechnological standpoint. They are the means through which filamentous fungi affect their environment to promote growth, and their properties at interfaces have resulted in numerous applications. In our study we have combined protein docking, molecular dynamics simulation, and electron cryo-microscopy to gain atomistic level insight into the surface structure of films composed of two class II hydrophobins: HFBI and HFBII produced by Trichoderma reesei. Together our results suggest a unit cell composed of six proteins; however, our computational results suggest P6 symmetry, while our experimental results show P3 symmetry with a unit cell size of 56 Å. Our computational results indicate the possibility of an alternate ordering with a three protein unit cell with P3 symmetry and a smaller unit cell size, and we have used a Monte Carlo simulation of a spin model representing the hydrophobin film to show how this alternate metastable structure may play a role in increasing the rate of surface coverage by hydrophobin films, possibly indicating a mechanism of more general significance to both biology and nanotechnology.

Molecular dynamics simulation of PEGylated bilayer interacting with salt ions: a model of the liposome surface in the bloodstream.

PEGylation is an effective mechanism to prolong the bloodstream lifetime, and thus efficacy, of drug delivery liposomes. The mechanism through which poly(ethylene glycol) (PEG) increases bloodstream lifetime is, however, not completely understood. The interaction with salt ions found in the bloodstream is known to play a role in this. We have used all-atom molecular dynamics simulation to study the effect of PEGylated lipid density, salt concentration, and the interaction with KCl and CaCl(2) salts in addition to NaCl. Increasing the PEGylated lipid concentration in the formulation from 1:18 to 1:9 molar density decreased the extent to which the Cl(-) ions penetrated the PEG layer, thus causing the PEG layer to become effectively positively charged. The interaction of the PEG with the K(+) ions was weaker than for the Na(+) ions, and nonexistent for the Ca(2+) ions. This work expands on our previous work where we studied the gel and liquid crystalline membranes in physiological salt concentration. Our results provide both an explanation for the experimental observation that PEGylation inhibits calcium-induced liposome fusion and further insight into the mechanisms through which PEG may inhibit uptake of the liposome by the reticuloendothilial system (RES).

Analysis of cause of failure of new targeting peptide in PEGylated liposome: Molecular modeling as rational design tool for nanomedicine

Drug nanocarriers are often derivatized with targeting moieties to achieve site specific delivery, however, the results from this approach have, as yet, not reached expectations. We have tested a new phage display based targeting moiety, the activated endothelium targeting peptide (AETP), for its vascular endothelium directed targeting efficiency, when anchored to a PEGylated liposome via maleimide chemistry. Our results have, however, not shown any evidence of improved targeting. We have hypothesized that the failure of the AETP moiety is due to its availability to target receptors being restricted, as a result of steric hindrance due to the PEG polymer, and possibly affinity for bloodstream proteins, particularly human serum albumin (HSA). In this context, molecular modeling was used to contrast the properties of the AETP moiety to those of the RGD targeting peptide, already found to be effective in previous trials. Our molecular dynamics simulation results indicate the AETP moiety is located within the PEG layer, and its hydrophobic nature causes it to be obscured by PEG to a greater extent than the more hydrophilic RGD targeting peptide. Protein-ligand docking results indicated similar affinities for HSA of both the AETP moiety and a PEG fragment, and a significantly lower affinity for the RGD peptide. We know of no means to investigate this experimentally with atomic level resolution, thus our use of computational methods to investigate this can be seen as a new tool for rational design in nanomedicine.

Cholesterol level affects surface charge of lipid membranes in saline solution

Cholesterol is an important component of all biological membranes as well as drug delivery liposomes. We show here that increasing the level of cholesterol in a phospholipid membrane decreases surface charge in the physiological environment. Through molecular dynamics simulation we have shown that increasing the level of cholesterol decreases Na+ ion binding. Complementary experimental ζ – potential measurements have shown a decreased ζ – potential with increasing cholesterol content, indicative of reduced surface charge. Both experiments and simulations have been carried out on both saturated 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and monounsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes. This result is particularly important because membrane surface charge plays an important role in the interactions of biomembranes with peripheral membrane proteins and drug delivery liposomes with the immune system.

Oncolytic adenoviruses coated with MHC-I tumor epitopes increase the antitumor immunity and efficacy against melanoma

ABSTRACT The stimulation of the immune system using oncolytic adenoviruses (OAds) has attracted significant interest and several studies suggested that OAds immunogenicity might be important for their efficacy. Therefore, we developed a versatile and rapid system to adsorb tumor-specific major histocompatibility complex class I (MHC-I) peptides onto the viral surface to drive the immune response toward the tumor epitopes. By studying the model epitope SIINFEKL, we demonstrated that the peptide-coated OAd (PeptiCRAd) retains its infectivity and the cross presentation of the modified-exogenous epitope on MHC-I is not hindered. We then showed that the SIINFEKL-targeting PeptiCRAd achieves a superior antitumor efficacy and increases the percentage of antitumor CD8+ T cells and mature epitope-specific dendritic cells in vivo. PeptiCRAds loaded with clinically relevant tumor epitopes derived from tyrosinase-related protein 2 (TRP-2) and human gp100 could reduce the growth of primary-treated tumors and secondary-untreated melanomas, promoting the expansion of antigen-specific T-cell populations. Finally, we tested PeptiCRAd in humanized mice bearing human melanomas. In this model, a PeptiCRAd targeting the human melanoma-associated antigen A1 (MAGE-A1) and expressing granulocyte and macrophage colony-stimulating factor (GM-CSF) was able to eradicate established tumors and increased the human MAGE-A1-specific CD8+ T cell population. Herein, we show that the immunogenicity of OAds plays a key role in their efficacy and it can be exploited to direct the immune response system toward exogenous tumor epitopes. This versatile and rapid system overcomes the immunodominance of the virus and elicits a tumor-specific immune response, making PeptiCRAd a promising approach for clinical testing.

Design of cholesterol arabinogalactan anchored liposomes for asialoglycoprotein receptor mediated targeting to hepatocellular carcinoma: In silico modeling, in vitro and in vivo evaluation

We have developed active targeting liposomes to deliver anticancer agents to ASGPR which will contribute to effective treatment of hepatocellular carcinoma. Active targeting is achieved through polymeric ligands on the liposome surface. The liposomes were prepared using reverse phase evaporation method and doxorubicin hydrocholoride, a model drug, was loaded using the ammonium sulphate gradient method. Liposomes loaded with DOX were found to have a particle size of 200nm with more than 90% entrapment efficiency. Systems were observed to release the drug in a sustained manner in acidic pH in vitro. Liposomes containing targeting ligands possessed greater and selective toxicity to ASGPR positive HepG2 cell lines due to specific ligand receptor interaction. Bio-distribution studies revealed that liposomes were concentrated in the liver even after 3h of administration, thus providing conclusive evidence of targeting potential for formulated nanosystems. Tumor regression studies indicated greater tumor suppression with targeted liposomes thereby establishing superiority of the liposomal system. In this work, we used a novel methodology to guide the determination of the optimal composition of the targeting liposomes: molecular dynamics (MD) simulation that aided our understanding of the behaviour of the ligand within the bilayer. This can be seen as a demonstration of the utility of this methodology as a rational design tool for active targeting liposome formulation.

Properties of the membrane binding component of catechol-O-methyltransferase revealed by atomistic molecular dynamics simulations.

We used atomistic simulations to study the membrane-bound form of catechol-O-methyltransferase (MB-COMT). In particular we investigated the 26-residue transmembrane α-helical segment of MB-COMT together with the 24-residue fragment that links the transmembrane component to the main protein unit that was not included in our model. In numerous independent simulations we observed the formation of a salt bridge between ARG27 and GLU40. The salt bridge closed the flexible loop that formed in the linker and kept it in the vicinity of the membrane-water interface. All simulations supported this conclusion that the linker has a clear affinity for the interface and preferentially arranges its residues to reside next to the membrane, without a tendency to relocate into the water phase. Furthermore, an extensive analysis of databases for sequences of membrane proteins that have a single transmembrane helical segment brought about an interesting view that the flexible loop observed in our work can be a common structural element in these types of proteins. In the same spirit we close the article by discussing the role of salt bridges in the formation of three-dimensional structures of membrane proteins that exhibit a single transmembrane helix.

Increased Binding of Calcium Ions at Positively Curved Phospholipid Membranes

Calcium ion is the ubiquitous messenger in cells and plays a key role in neuronal signaling and fusion of synaptic vesicles. These vesicles are typically ∼20-50 nm in diameter, and thus their interaction with calcium ions cannot be modeled faithfully with a conventional flat membrane bilayer setup. Within our newly developed molecular dynamics simulations setup, we characterize here interactions of the calcium ion with curved membrane interfaces with atomistic detail. The present molecular dynamics simulations together with time-dependent fluorescence shift experiments suggest that the mode and strength of interaction of calcium ion with a phospholipid bilayer depends on its curvature. Potential of mean force calculations demonstrate that the binding of calcium ion to the positively curved side of the bilayer is significantly stronger compared with that to a flat membrane.

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.

A computational study suggests that replacing PEG with PMOZ may increase exposure of hydrophobic targeting moiety

ABSTRACT In a previous study we showed that the cause of failure of a new, proposed, targeting ligand, the AETP moiety, when attached to a PEGylated liposome, was occlusion by the poly(ethylene glycol) (PEG) layer due to its hydrophobic nature, given that PEG is not entirely hydrophilic. At the time we proposed that possible replacement with a more hydrophilic protective polymer could alleviate this problem. In this study we have used computational molecular dynamics modelling, using a model with all atom resolution, to suggest that a specific alternative protective polymer, poly(2‐methyloxazoline) (PMOZ), would perform exactly this function. Our results show that when PEG is replaced by PMOZ the relative exposure to the solvent of AETP is increased to a level even greater than that we found in previous simulations for the RGD peptide, a targeting moiety that has previously been used successfully in PEGylated liposome based therapies. While the AETP moiety itself is no longer under consideration, the results of this computational study have broader significance: the use of PMOZ as an alternative polymer coating to PEG could be efficacious in the context of more hydrophobic targeting ligands. In addition to PMOZ we studied another polyoxazoline, poly(2‐ethyloxazoline) (PEOZ), that has also been mooted as a possible alternate protective polymer. It was also found that the RDG peptide occlusion was significantly greater for the case of both oxazolines as opposed to PEG and that, unlike PEG, neither oxazoline entered the membrane. As far as we are aware this is the first time that polyoxazolines have been studied using molecular dynamics simulation with all atom resolution.