J. Andrew MacKay

Imaging and drug delivery using theranostic nanoparticles

Nanoparticle technologies are significantly impacting the development of both therapeutic and diagnostic agents. At the intersection between treatment and diagnosis, interest has grown in combining both paradigms into clinically effective formulations. This concept, recently coined as theranostics, is highly relevant to agents that target molecular biomarkers of disease and is expected to contribute to personalized medicine. Here we review state-of-the-art nanoparticles from a therapeutic and a diagnostic perspective and discuss challenges in bringing these fields together. Major classes of nanoparticles include, drug conjugates and complexes, dendrimers, vesicles, micelles, core-shell particles, microbubbles, and carbon nanotubes. Most of these formulations have been described as carriers of either drugs or contrast agents. To observe these formulations and their interactions with disease, a variety of contrast agents have been used, including optically active small molecules, metals and metal oxides, ultrasonic contrast agents, and radionuclides. The opportunity to rapidly assess and adjust treatment to the needs of the individual offers potential advantages that will spur the development of theranostic agents.

Self-assembling chimeric polypeptide–doxorubicin conjugate nanoparticles that abolish tumours after a single injection

New strategies to self-assemble biocompatible materials into nanoscale, drug-loaded packages with improved therapeutic efficacy are needed for nanomedicine. To address this need, we developed artificial recombinant chimeric polypeptides (CPs) that spontaneously self-assemble into sub-100 nm size, near monodisperse nanoparticles upon conjugation of diverse hydrophobic molecules, including chemotherapeutics. These CPs consist of a biodegradable polypeptide that is attached to a short Cys-rich segment. Covalent modification of the Cys residues with a structurally diverse set of hydrophobic small molecules, including chemotherapeutics leads to spontaneous formation of nanoparticles over a range of CP compositions and molecular weights. When used to deliver chemotherapeutics to a murine cancer model, CP nanoparticles have a four-fold higher maximum tolerated dose than free drug, and induce nearly complete tumor regression after a single dose. This simple strategy can promote co-assembly of drugs, imaging agents, and targeting moieties into multifunctional nanomedicines.

Structural analyses reveal phosphatidyl inositols as ligands for the NR5 orphan receptors SF-1 and LRH-1

Vertebrate members of the nuclear receptor NR5A subfamily, which includes steroidogenic factor 1 (SF-1) and liver receptor homolog 1 (LRH-1), regulate crucial aspects of development, endocrine homeostasis, and metabolism. Mouse LRH-1 is believed to be a ligand-independent transcription factor with a large and empty hydrophobic pocket. Here we present structural and biochemical data for three other NR5A members-mouse and human SF-1 and human LRH-1-which reveal that these receptors bind phosphatidyl inositol second messengers and that ligand binding is required for maximal activity. Evolutionary analysis of structure-function relationships across the SF-1/LRH-1 subfamily indicates that ligand binding is the ancestral state of NR5A receptors and was uniquely diminished or altered in the rodent LRH-1 lineage. We propose that phospholipids regulate gene expression by directly binding to NR5A nuclear receptors.

Distribution in brain of liposomes after convection enhanced delivery; modulation by particle charge, particle diameter, and presence of steric coating

We have investigated the role of diameter, charge, and steric shielding on the brain distribution of liposomes infused by convection enhanced delivery (CED) using both radiolabeled and fluorescent-labeled particles. Liposomes of 40 and 80-nm diameter traveled the same distance but penetrated significantly less than a 10-kDa dextran; whereas 200-nm-diameter liposomes penetrated less than 80 nm liposomes. A neutral liposome shielded by polyethylene glycol (PEG; 2 kDa; 10% by mole) penetrated significantly farther than an unshielded liposome. Even when shielded with PEG, positive surface charge (10% by mole) significantly reduced the penetration radius compared to a neutral or negative charged liposome (10% by mole). A mathematical CED model including a term for liposome cell binding was applied to analyze the radius of particle penetration. Neutral liposomes had a binding constant of k=0.0010+/-0.0002 min-1, whereas for positive charged liposomes k increased 50-fold. The binding constant was independently verified using a degradable lipid radiolabel that eliminated from the brain with a 9.9+/-2.0 h half-life, equivalent to the calculated elimination constant k=0.0012+/-0.0002 min-1. During CED, liposomes accumulated in a subpopulation of perivascular cells within the brain. A non-degradable lipid radiolabel showed that lipid components remained within these perivascular brain cells for at least 2 days. To reduce this uptake, 100-fold molar excess of non-labeled liposomes were co-infused with labeled liposomes, which significantly increased liposome penetration. These studies suggest that optimization of therapeutic CED using particles such as drug-loaded liposomes, polymeric nanoparticles, non-viral DNA complexes, and viruses will require a strategy to overcome particle binding and clearance by cells within the CNS.

Fabrication of Elastin-Like Polypeptide Nanoparticles for Drug Delivery by Electrospraying

The development of environmentally responsive drug carriers requires new methods for assembling stimuli-responsive nanoparticulates. This communication describes a novel application of electrospray to construct bioresponsive peptide-based particulates, which can encapsulate drugs. These particles are composed from genetically engineered elastin-like polypeptides (ELPs), a biodegradable, biocompatible, and bioresponsive polymer. To generate nanoparticles (300-400 nm in diameter), ELPs and drugs are codissolved in organic solvent, accelerated across a voltage gradient, dried by evaporation during transit, and collected from a target surface. These findings indicate that particle diameter, polydispersity, and morphology are strong functions of the solvent concentration, spraying voltage, and polymer molecular weight. Surprisingly, the loading of drug at 20 w/w% did not influence particle morphology; furthermore, drug release from these particles correlated with the pH-dependent solubility of the parent ELPs. These studies suggest that electrospray is an efficient and flexible method for generating stimuli-responsive drug particles.

Low-pH-sensitive poly(ethylene glycol) (PEG)-stabilized plasmid nanolipoparticles: effects of PEG chain length, lipid composition and assembly conditions on gene delivery.

We have studied the effects of the poly(ethylene glycol) (PEG) chain length and acyl chain composition on the pH‐sensitivity of acid‐labile PEG‐diorthoester (POD) lipids. The optimal conditions are described for preparation of DNA plasmid encapsulated POD nanolipoparticles (NLPs) which mediate high gene delivery activity in vitro with moderate cytotoxicity.

In situ growth of a stoichiometric PEG-like conjugate at a protein's N-terminus with significantly improved pharmacokinetics

The challenge in the synthesis of protein-polymer conjugates for biological applications is to synthesize a stoichiometric (typically 1:1) conjugate of the protein with a monodisperse polymer, with good retention of protein activity, significantly improved pharmacokinetics and increased bioavailability, and hence improved in vivo efficacy. Here we demonstrate, using myoglobin as an example, a general route to grow a PEG-like polymer, poly(oligo(ethylene glycol) methyl ether methacrylate) [poly(OEGMA)], with low polydispersity and high yield, solely from the N-terminus of the protein by in situ atom transfer radical polymerization (ATRP) under aqueous conditions, to yield a site-specific (N-terminal) and stoichiometric conjugate (1:1). Notably, the myoglobin-poly(OEGMA) conjugate [hydrodynamic radius (Rh): 13 nm] showed a 41-fold increase in its blood exposure compared to the protein (Rh: 1.7 nm) after IV administration to mice, thereby demonstrating that comb polymers that present short oligo(ethylene glycol) side chains are a class of PEG-like polymers that can significantly improve the pharmacological properties of proteins. We believe that this approach to the synthesis of N-terminal protein conjugates of poly(OEGMA) may be applicable to a large subset of protein and peptide drugs, and thereby provide a general methodology for improvement of their pharmacological profiles.

Recursive Directional Ligation by Plasmid Reconstruction Allows Rapid and Seamless Cloning of Oligomeric Genes

This paper reports a new strategy, recursive directional ligation by plasmid reconstruction (PRe-RDL), to rapidly clone highly repetitive polypeptides of any sequence and specified length over a large range of molecular weights. In a single cycle of PRe-RDL, two halves of a parent plasmid, each containing a copy of an oligomer, are ligated together, thereby dimerizing the oligomer and reconstituting a functional plasmid. This process is carried out recursively to assemble an oligomeric gene with the desired number of repeats. PRe-RDL has several unique features that stem from the use of type IIs restriction endonucleases: first, PRe-RDL is a seamless cloning method that leaves no extraneous nucleotides at the ligation junction. Because it uses type IIs endonucleases to ligate the two halves of the plasmid, PRe-RDL also addresses the major limitation of RDL in that it abolishes any restriction on the gene sequence that can be oligomerized. The reconstitution of a functional plasmid only upon successful ligation in PRe-RDL also addresses two other limitations of RDL: the significant background from self-ligation of the vector observed in RDL, and the decreased efficiency of ligation due to nonproductive circularization of the insert. PRe-RDL can also be used to assemble genes that encode different sequences in a predetermined order to encode block copolymers or append leader and trailer peptide sequences to the oligomerized gene.

Mechanism of pH-triggered collapse of phosphatidylethanolamine liposomes stabilized by an ortho ester polyethyleneglycol lipid

The mechanism of pH-triggered destabilization of liposomes composed of a polyethyleneglycol-orthoester-distearoylglycerol lipid (POD) and phosphatidyl ethanolamine (PE) has been studied using an ANTS/DPX leakage and a lipid-mixing assay. We developed a kinetic model that relates POD hydrolysis to liposome collapse. This minimum-surface-shielding model describes the kinetics of the pH-triggered release of POD/PE liposomes. In the model, when acid-catalyzed hydrolysis lowers the mole percentage of POD on the liposome surface to a critical level, intervesicular lipid mixing is initiated, resulting in a burst of contents release. Two phases of content leakage are observed: a lag phase and a burst phase. During the lag phase, less than 20% of liposomal contents are released and the leakage begins to accelerate when approaching to the transition point. During the burst phase, the leakage rate is dependent on interbilayer contact. The burst phase occurs when the surface density of the PEG lipid is 2.3 +/- 0.6 mol%, regardless of the pH. Vesicles containing 4 mol% of a pH-insensitive PEG-lipid conjugate and 10% POD did not leak contents or collapse at any pH. These data are consistent with the stalk theory to describe the lamellar-to-inverted hexagonal phase transition and set a lower bound of approximately 16 PE lipids on the external monolayer as the contact site required for lipid mixing between two bilayers.

Environmentally responsive peptides as anticancer drug carriers.

The tumor microenvironment provides multiple cues that may be exploited to improve the efficacy of established chemotherapeutics; furthermore, polypeptides are uniquely situated to capitalize on these signals. Peptides provide: 1) a rich repertoire of biologically specific interactions to draw upon; 2) environmentally responsive phase behaviors, which may be tuned to respond to signatures of disease; 3) opportunities to direct self-assembly; 4) control over routes of biodegradation; 5) the option to seamlessly combine functionalities into a single polymer via a one-step biosynthesis. As development of cancer-targeted nanocarriers expands, peptides provide a unique source of functional units that may target disease. This review explores potential microenvironmental physiology indicative of tumors and peptides that have demonstrated an ability to target and deliver to these signals.