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Papers by Prashant Agrawal

Research paper thumbnail of De novo design of conformationally flexible transmembrane peptides driving membrane fusion

Proceedings of the National Academy of Sciences, 2004

Fusion of biological membranes is mediated by distinct integral membrane proteins, e.g., soluble ... more Fusion of biological membranes is mediated by distinct integral membrane proteins, e.g., soluble N -ethylmaleimide-sensitive factor attachment protein receptors and viral fusion proteins. Previous work has indicated that the transmembrane segments (TMSs) of such integral membrane proteins play an important role in fusion. Furthermore, peptide mimics of the transmembrane part can drive the fusion of liposomes, and evidence had been obtained that fusogenicity depends on their conformational flexibility. To test this hypothesis, we present a series of unnatural TMSs that were designed de novo based on the structural properties of hydrophobic residues. We find that the fusogenicity of these peptides depends on the ratio of α-helix-promoting Leu and β-sheet-promoting Val residues and is enhanced by helix-destabilizing Pro and Gly residues within their hydrophobic cores. The ability of these peptides to refold from an α-helical state to a β-sheet conformation and backwards was determined ...

Research paper thumbnail of A high relaxivity Gd(III)DOTA-DSPE-based liposomal contrast agent for magnetic resonance imaging

European Journal of Pharmaceutics and Biopharmaceutics, 2009

The field of molecular imaging aims to visualize and quantify (patho)physiological processes at t... more The field of molecular imaging aims to visualize and quantify (patho)physiological processes at the cellular and molecular level. Sensitive and site-targeted contrast agents are employed to visualize molecular constituents of processes of interest. The principal aim of this study was to develop a magnetic resonance imaging (MRI) detectable liposome with high relaxivity and stability. To this end, Gd(III)DOTA-DSPE was synthesized and incorporated in a liposomal formulation. The resulting liposomes were extensively characterized in vitro in terms of contrast agent efficiency and structural properties. The liposomes were shown to have a high longitudinal relaxivity, which is crucial for the detection of low concentration molecular markers in molecular imaging studies. We also demonstrated that Gd(III)DOTA-DSPE exhibits no detectable transmetallation upon incubation with Zn(II). This is important as it significantly contributes to the biocompatibility of the contrast agent. The present liposome preparation will serve as versatile and well characterized platform for molecular imaging and targeted drug delivery studies.

Research paper thumbnail of Solid State NMR Investigation of the Interaction between Biomimetic Lipid Bilayers and de novo Designed Fusogenic Peptides

ChemBioChem, 2007

Regulated fusion of biological membranes can be induced by membrane-associated peptides with low-... more Regulated fusion of biological membranes can be induced by membrane-associated peptides with low-complexity sequences. [1] Such peptides can drive fusion depending on their structural flexibility, but how the peptide changes the lipid phase is not known. Here we study the interaction of synthetic peptides with a biomimetic lipid mixture by using 31 P solid-state NMR spectroscopy. [2, 3] We investigated two peptides: one previously shown to be rigid and virtually nonfusogenic, L16 (K 3 WL 16 K 3), and one that is flexible and highly fusogenic, LV16G8P9 (K 3 WLVLVLVLGPVLVLVLVK 3). [1] A mixture of brain phosphatidylethanolamine (PE), brain phosphatidylserine (PS) and egg phosphatidylcholine (PC) at a 3:1:1 ratio was used to mimic biological membrane composition. [4] Here we show how the de novo designed peptides altered the phase behaviour of biomimetic lipid bilayers at a peptide to lipid ratio of 1:100. While L16 forms a rather stable a helix, LV16G8P9 can readily refold from a helix to b sheet and vice versa, by changing solvent polarity. [1] Samples were prepared by dissolving 1-palmitoyl-2-oleoylsn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-[phospho-l-serine] (DOPS) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE; Avanti Polar Lipids, AL, USA) in chloroform. Peptide dissolved in TFE was added at a peptide/ lipid molar ratio of 0.01, as in previous fusion studies. [1] In the case of POPC lipid-bilayer studies, the peptide was dissolved in TFE, and was then mixed with POPC in chloroform at the

Research paper thumbnail of 13C and 15N NMR evidence for peripheral intercalation of uniformly labeled fusogenic peptides incorporated in a biomimetic membrane

Biochimica et Biophysica Acta (BBA) - Biomembranes, 2007

Membrane fusion requires drastic and transient changes of bilayer curvature and here we have stud... more Membrane fusion requires drastic and transient changes of bilayer curvature and here we have studied the interaction of three de novo designed synthetic hydrophobic peptides with a biomimetic three-lipid mixture by solid state NMR. An experimental approach is presented for screening of peptide-lipid interactions and their aggregation, and their embedding in a biomimetic membrane system using established proton-decoupled 13 C, 15 N and proton spin diffusion heteronuclear 1 H− 13 C correlation NMR methods at high magnetic field. Experiments are presented for a set of denovo designed fusion peptides in interaction with their lipid environment. The data provide additional support for the transmembrane model for the least fusogenic peptide, L16, while the peripheral intercalation model is preferred for the fusogenic peptides LV16 and LV16G8P9. This contributes to converging evidence that peripheral intercalation is both necessary and sufficient to trigger the fusion process for a lipid mixture close to a critical point for phase separation across the bilayer.

Research paper thumbnail of Chitosan-based systems for molecular imaging

Advanced Drug Delivery Reviews, 2010

Imaging techniques Imaging agents MRI CT Ultrasound Optical imaging Molecular imaging enables the... more Imaging techniques Imaging agents MRI CT Ultrasound Optical imaging Molecular imaging enables the non-invasive assessment of biological and biochemical processes in living subjects. Such technologies therefore have the potential to enhance our understanding of disease and drug activity during preclinical and clinical drug development. Molecular imaging allows a repetitive and noninvasive study of the same living subject using identical or alternative biological imaging assays at different time points, thus harnessing the statistical power of longitudinal studies, and reducing the number of animals required and cost. Chitosan is a hydrophilic and non-antigenic biopolymer and has a low toxicity toward mammalian cells. Hence, it has great potential as a biomaterial because of its excellent biocompatibility. Conjugated to additional materials, chitosan composites result in a new class of biomaterials that possess mechanical, physicochemical and functional properties, which have potential for use in advanced biomedical imaging applications. The present review will discuss the strengths, limitations and challenges of molecular imaging as well as applications of chitosan nanoparticles in the field of molecular imaging.

Research paper thumbnail of Surface modification of PLGA nanospheres with Gd-DTPA and Gd-DOTA for high-relaxivity MRI contrast agents

Biomaterials, 2010

The preparation of particulate contrast agents for magnetic resonance imaging (MRI) based on biod... more The preparation of particulate contrast agents for magnetic resonance imaging (MRI) based on biodegradable poly(D,L-lactide-co-glycolide) (PLGA) nanocarriers is reported. By spacer-aided covalent surfacegrafting of the prominent chelating ligands diethylenetriaminepentaacetic acid (DTPA) and 1,4,7, 10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), respectively, up to 236 mg gadolinium per mg PLGA can be immobilized in a stable manner. Due to the localisation at the particle surface, water protons may effectively interact with the gadolinium chelates and the modified particles exhibit high proton relaxivities as confirmed by T1 relaxivities of up to 17.5 mM À1 s À1 (25 C, 1.41 T) in case of Gd-DOTAfunctionalized carriers and also supported by NMRD profiles. The obtained values compare favorably with marketed low-molecular weight contrast agents and thus suggest suitability for in vivo use.

Research paper thumbnail of De novo design of conformationally flexible transmembrane peptides driving membrane fusion

Proceedings of the National Academy of Sciences, 2004

Fusion of biological membranes is mediated by distinct integral membrane proteins, e.g., soluble ... more Fusion of biological membranes is mediated by distinct integral membrane proteins, e.g., soluble N -ethylmaleimide-sensitive factor attachment protein receptors and viral fusion proteins. Previous work has indicated that the transmembrane segments (TMSs) of such integral membrane proteins play an important role in fusion. Furthermore, peptide mimics of the transmembrane part can drive the fusion of liposomes, and evidence had been obtained that fusogenicity depends on their conformational flexibility. To test this hypothesis, we present a series of unnatural TMSs that were designed de novo based on the structural properties of hydrophobic residues. We find that the fusogenicity of these peptides depends on the ratio of α-helix-promoting Leu and β-sheet-promoting Val residues and is enhanced by helix-destabilizing Pro and Gly residues within their hydrophobic cores. The ability of these peptides to refold from an α-helical state to a β-sheet conformation and backwards was determined ...

Research paper thumbnail of A high relaxivity Gd(III)DOTA-DSPE-based liposomal contrast agent for magnetic resonance imaging

European Journal of Pharmaceutics and Biopharmaceutics, 2009

The field of molecular imaging aims to visualize and quantify (patho)physiological processes at t... more The field of molecular imaging aims to visualize and quantify (patho)physiological processes at the cellular and molecular level. Sensitive and site-targeted contrast agents are employed to visualize molecular constituents of processes of interest. The principal aim of this study was to develop a magnetic resonance imaging (MRI) detectable liposome with high relaxivity and stability. To this end, Gd(III)DOTA-DSPE was synthesized and incorporated in a liposomal formulation. The resulting liposomes were extensively characterized in vitro in terms of contrast agent efficiency and structural properties. The liposomes were shown to have a high longitudinal relaxivity, which is crucial for the detection of low concentration molecular markers in molecular imaging studies. We also demonstrated that Gd(III)DOTA-DSPE exhibits no detectable transmetallation upon incubation with Zn(II). This is important as it significantly contributes to the biocompatibility of the contrast agent. The present liposome preparation will serve as versatile and well characterized platform for molecular imaging and targeted drug delivery studies.

Research paper thumbnail of Solid State NMR Investigation of the Interaction between Biomimetic Lipid Bilayers and de novo Designed Fusogenic Peptides

ChemBioChem, 2007

Regulated fusion of biological membranes can be induced by membrane-associated peptides with low-... more Regulated fusion of biological membranes can be induced by membrane-associated peptides with low-complexity sequences. [1] Such peptides can drive fusion depending on their structural flexibility, but how the peptide changes the lipid phase is not known. Here we study the interaction of synthetic peptides with a biomimetic lipid mixture by using 31 P solid-state NMR spectroscopy. [2, 3] We investigated two peptides: one previously shown to be rigid and virtually nonfusogenic, L16 (K 3 WL 16 K 3), and one that is flexible and highly fusogenic, LV16G8P9 (K 3 WLVLVLVLGPVLVLVLVK 3). [1] A mixture of brain phosphatidylethanolamine (PE), brain phosphatidylserine (PS) and egg phosphatidylcholine (PC) at a 3:1:1 ratio was used to mimic biological membrane composition. [4] Here we show how the de novo designed peptides altered the phase behaviour of biomimetic lipid bilayers at a peptide to lipid ratio of 1:100. While L16 forms a rather stable a helix, LV16G8P9 can readily refold from a helix to b sheet and vice versa, by changing solvent polarity. [1] Samples were prepared by dissolving 1-palmitoyl-2-oleoylsn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-[phospho-l-serine] (DOPS) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE; Avanti Polar Lipids, AL, USA) in chloroform. Peptide dissolved in TFE was added at a peptide/ lipid molar ratio of 0.01, as in previous fusion studies. [1] In the case of POPC lipid-bilayer studies, the peptide was dissolved in TFE, and was then mixed with POPC in chloroform at the

Research paper thumbnail of 13C and 15N NMR evidence for peripheral intercalation of uniformly labeled fusogenic peptides incorporated in a biomimetic membrane

Biochimica et Biophysica Acta (BBA) - Biomembranes, 2007

Membrane fusion requires drastic and transient changes of bilayer curvature and here we have stud... more Membrane fusion requires drastic and transient changes of bilayer curvature and here we have studied the interaction of three de novo designed synthetic hydrophobic peptides with a biomimetic three-lipid mixture by solid state NMR. An experimental approach is presented for screening of peptide-lipid interactions and their aggregation, and their embedding in a biomimetic membrane system using established proton-decoupled 13 C, 15 N and proton spin diffusion heteronuclear 1 H− 13 C correlation NMR methods at high magnetic field. Experiments are presented for a set of denovo designed fusion peptides in interaction with their lipid environment. The data provide additional support for the transmembrane model for the least fusogenic peptide, L16, while the peripheral intercalation model is preferred for the fusogenic peptides LV16 and LV16G8P9. This contributes to converging evidence that peripheral intercalation is both necessary and sufficient to trigger the fusion process for a lipid mixture close to a critical point for phase separation across the bilayer.

Research paper thumbnail of Chitosan-based systems for molecular imaging

Advanced Drug Delivery Reviews, 2010

Imaging techniques Imaging agents MRI CT Ultrasound Optical imaging Molecular imaging enables the... more Imaging techniques Imaging agents MRI CT Ultrasound Optical imaging Molecular imaging enables the non-invasive assessment of biological and biochemical processes in living subjects. Such technologies therefore have the potential to enhance our understanding of disease and drug activity during preclinical and clinical drug development. Molecular imaging allows a repetitive and noninvasive study of the same living subject using identical or alternative biological imaging assays at different time points, thus harnessing the statistical power of longitudinal studies, and reducing the number of animals required and cost. Chitosan is a hydrophilic and non-antigenic biopolymer and has a low toxicity toward mammalian cells. Hence, it has great potential as a biomaterial because of its excellent biocompatibility. Conjugated to additional materials, chitosan composites result in a new class of biomaterials that possess mechanical, physicochemical and functional properties, which have potential for use in advanced biomedical imaging applications. The present review will discuss the strengths, limitations and challenges of molecular imaging as well as applications of chitosan nanoparticles in the field of molecular imaging.

Research paper thumbnail of Surface modification of PLGA nanospheres with Gd-DTPA and Gd-DOTA for high-relaxivity MRI contrast agents

Biomaterials, 2010

The preparation of particulate contrast agents for magnetic resonance imaging (MRI) based on biod... more The preparation of particulate contrast agents for magnetic resonance imaging (MRI) based on biodegradable poly(D,L-lactide-co-glycolide) (PLGA) nanocarriers is reported. By spacer-aided covalent surfacegrafting of the prominent chelating ligands diethylenetriaminepentaacetic acid (DTPA) and 1,4,7, 10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), respectively, up to 236 mg gadolinium per mg PLGA can be immobilized in a stable manner. Due to the localisation at the particle surface, water protons may effectively interact with the gadolinium chelates and the modified particles exhibit high proton relaxivities as confirmed by T1 relaxivities of up to 17.5 mM À1 s À1 (25 C, 1.41 T) in case of Gd-DOTAfunctionalized carriers and also supported by NMRD profiles. The obtained values compare favorably with marketed low-molecular weight contrast agents and thus suggest suitability for in vivo use.