Mark Kastantin

Targeting of albumin-embedded paclitaxel nanoparticles to tumors.

We have used tumor-homing peptides to target abraxane, a clinically approved paclitaxel-albumin nanoparticle, to tumors in mice. The targeting was accomplished with two peptides, CREKA and LyP-1 (CGNKRTRGC). Fluorescein (FAM)-labeled CREKA-abraxane, when injected intravenously into mice bearing MDA-MB-435 human cancer xenografts, accumulated in tumor blood vessels, forming aggregates that contained red blood cells and fibrin. FAM-LyP-1-abraxane co-localized with extravascular islands expressing its receptor, p32. Self-assembled mixed micelles carrying the homing peptide and the label on different subunits accumulated in the same areas of tumors as LyP-1-abraxane, showing that Lyp-1 can deliver intact nanoparticles into extravascular sites. Untargeted, FAM-abraxane was detected in the form of a faint meshwork in tumor interstitium. LyP-1-abraxane produced a statistically highly significant inhibition of tumor growth compared with untargeted abraxane. These results show that nanoparticles can be effectively targeted into extravascular tumor tissue and that targeting can enhance the activity of a therapeutic nanoparticle.

Targeting atherosclerosis by using modular, multifunctional micelles

Subtle clotting that occurs on the luminal surface of atherosclerotic plaques presents a novel target for nanoparticle-based diagnostics and therapeutics. We have developed modular multifunctional micelles that contain a targeting element, a fluorophore, and, when desired, a drug component in the same particle. Targeting atherosclerotic plaques in ApoE-null mice fed a high-fat diet was accomplished with the pentapeptide cysteine-arginine-glutamic acid-lysine-alanine, which binds to clotted plasma proteins. The fluorescent micelles bind to the entire surface of the plaque, and notably, concentrate at the shoulders of the plaque, a location that is prone to rupture. We also show that the targeted micelles deliver an increased concentration of the anticoagulant drug hirulog to the plaque compared with untargeted micelles.

Effect of the Lipid Chain Melting Transition on the Stability of DSPE-PEG(2000) Micelles

Micellar nanoparticles are showing promise as carriers of diagnostic and therapeutic biofunctionality, leading to increased interest in their properties and behavior, particularly their size, shape, and stability. This work investigates the physical chemistry of micelles formed from DSPE-PEG(2000) monomers as it pertains to these properties. A melting transition in the lipid core of spheroidal DSPE-PEG(2000) micelles is observed as an endothermic peak at 12.8 degrees C upon heating in differential scanning calorimetry thermograms. Bulky PEG(2000) head groups prevent regular crystalline packing of lipids in both the low-temperature glassy and high-temperature fluid phases, as evidenced by wide-angle X-ray scattering. Equilibrium micelle geometry is spheroidal above and below the transition temperature, indicating that the entropic penalty to force the PEG brush into flat geometry is greater than the enthalpic benefit to the glassy core to pack in an extended configuration. Increased micelle stability is seen in the glassy phase with monomer desorption rates significantly lower than in the fluid phase. Activation energies for monomer desorption are 156+/-6.7 and 79+/-5.0 kJ/mol for the glassy and fluid phases, respectively. The observation of a glass transition that increases micelle stability but does not perturb micelle geometry is useful for the design of more effective biofunctional micelles.

Single-molecule resolution of interfacial fibrinogen behavior: effects of oligomer populations and surface chemistry.

Through the use of single-molecule total internal reflection fluorescence microscopy, the dynamic behavior of fibrinogen was observed at the interface between aqueous solution and various solid surfaces. Multiple populations of objects were observed, as characterized by surface residence times, interfacial diffusion, and fluorescence intensity. On all surfaces, populations exhibited direct links between surface residence time, rate of diffusion, and fluorescence intensity. In particular, longer-lived populations diffused more slowly and exhibited greater fluorescence intensity, leading to the conclusion that the objects represented fibrinogen monomers and discrete oligomer populations (dimers, trimers, etc.), and that these oligomer populations play an important role in the protein-surface interaction because of their long surface residence times. Two or three diffusive modes were observed for most populations, indicating that protein aggregates have multiple mechanisms for interaction with solid substrates. In addition, the fastest diffusive mode is believed to represent a hopping mode that often precedes desorption events. Surprisingly, a monolayer of 5000 Da poly(ethylene glycol) (PEG5000) increased surface residence time and slowed diffusion of fibrinogen relative to bare fused silica or hydrophobically modified fused silica, suggesting that the mechanism of PEG resistance to protein adhesion is more sophisticated than the simple repulsion of individual proteins.

Thermodynamic and Kinetic Stability of DSPE-PEG(2000) Micelles in the Presence of Bovine Serum Albumin

This work investigated the stability of DSPE-PEG(2000) micelles in the presence of bovine serum albumin (BSA). DSPE-PEG(2000) was found to exist in equilibrium among monomeric, micellar, and BSA-bound states, and this equilibrium shifted toward the BSA-bound state when the temperature increased from 20 to 37 °C. The micellar state is thermodynamically unstable at both temperatures when the concentration of BSA approaches that of DSPE-PEG(2000), and micelle breakup occurs with a first-order time constant of 130 ± 9 min at 20 °C and 7.8 ± 1.6 min at 37 °C. Thus, previous targeting experiments that demonstrate synergistic effects in multiply functionalized DSPE-PEG(2000) micelles are likely due to targeting that occurs on a timescale faster than that of micelle breakup. Micelle breakup was limited by diffusion at 20 °C whereas at 37 °C monomer desorption from the micelle was the rate-limiting step. These findings give clear guidance concerning the lifetimes of micelles that may be used as diagnostic and therapeutic nanoparticles.

A bottom-up approach to understanding protein layer formation at solid-liquid interfaces.

A common goal across different fields (e.g. separations, biosensors, biomaterials, pharmaceuticals) is to understand how protein behavior at solid-liquid interfaces is affected by environmental conditions. Temperature, pH, ionic strength, and the chemical and physical properties of the solid surface, among many factors, can control microscopic protein dynamics (e.g. adsorption, desorption, diffusion, aggregation) that contribute to macroscopic properties like time-dependent total protein surface coverage and protein structure. These relationships are typically studied through a top-down approach in which macroscopic observations are explained using analytical models that are based upon reasonable, but not universally true, simplifying assumptions about microscopic protein dynamics. Conclusions connecting microscopic dynamics to environmental factors can be heavily biased by potentially incorrect assumptions. In contrast, more complicated models avoid several of the common assumptions but require many parameters that have overlapping effects on predictions of macroscopic, average protein properties. Consequently, these models are poorly suited for the top-down approach. Because the sophistication incorporated into these models may ultimately prove essential to understanding interfacial protein behavior, this article proposes a bottom-up approach in which direct observations of microscopic protein dynamics specify parameters in complicated models, which then generate macroscopic predictions to compare with experiment. In this framework, single-molecule tracking has proven capable of making direct measurements of microscopic protein dynamics, but must be complemented by modeling to combine and extrapolate many independent microscopic observations to the macro-scale. The bottom-up approach is expected to better connect environmental factors to macroscopic protein behavior, thereby guiding rational choices that promote desirable protein behaviors.

DNA Hairpin Stabilization on a Hydrophobic Surface

DNA hybridization in the vicinity of surfaces is a fundamental process for self-assembled nanoarrays, nanocrystal superlattices, and biosensors. It is widely recognized that solid surfaces alter molecular forces governing hybridization relative to a bulk solution, and these effects can either favor or disfavor the hybridized state depending on the specific sequence and surface. Results presented here provide new insights into the dynamics of DNA hairpin-coil conformational transitions in the vicinity of hydrophilic oligo(ethylene glycol) (OEG) and hydrophobic trimethylsilane (TMS) surfaces. Single-molecule methods are used to observe the forward and reverse hybridization hairpin-coil transition of adsorbed species while simultaneously measuring molecular surface diffusion in order to gain insight into surface interactions with individual DNA bases. At least 35 000 individual molecular trajectories are observed on each type of surface. It is found that unfolding slows and the folding rate increases on TMS relative to OEG, despite stronger attractions between TMS and unpaired nucleobases. These rate differences lead to near-complete hairpin formation on hydrophobic TMS and significant unfolding on hydrophilic OEG, resulting in the surprising conclusion that hydrophobic surface coatings are preferable for nanotechnology applications that rely on DNA hybridization near surfaces.

Apparent Activation Energies Associated with Protein Dynamics on Hydrophobic and Hydrophilic Surfaces

With the use of single-molecule total internal reflection fluorescence microscopy (TIRFM), the dynamics of bovine serum albumin (BSA) and human fibrinogen (Fg) at low concentrations were observed at the solid-aqueous interface as a function of temperature on hydrophobic trimethylsilane (TMS) and hydrophilic fused silica (FS) surfaces. Multiple dynamic modes and populations were observed and characterized by their surface residence times and squared-displacement distributions (surface diffusion). Characteristic desorption and diffusion rates for each population/mode were generally found to increase with temperature, and apparent activation energies were determined from Arrhenius analyses. The apparent activation energies of desorption and diffusion were typically higher on FS than on TMS surfaces, suggesting that protein desorption and mobility were hindered on hydrophilic surfaces due to favorable protein-surface and solvent-surface interactions. The diffusion of BSA on TMS appeared to be activationless for several populations, whereas diffusion on FS always exhibited an apparent activation energy. All activation energies were small in absolute terms (generally only a few kBT), suggesting that most adsorbed protein molecules are weakly bound and move and desorb readily under ambient conditions.

Linker Chemistry Determines Secondary Structure of p5314−29 in Peptide Amphiphile Micelles

Biofunctional micelles formed via self-assembly of synthetic peptide-lipid conjugates are a class of promising biomaterials with applications in drug delivery and tissue engineering. The micelle building block, termed peptide amphiphile, consists of a lipid-like chain covalently linked through a spacer to a peptide headgroup. Self-assembly results in formation of a hydrophobic core surrounded by a dense shell with multiple, functional peptides. We report here on the effect that different linkers between a palmitic tail and a bioactive peptide (p5314-29) have on headgroup secondary structure. Peptide p5314-29 may act as an inhibitor of the interaction between tumor suppressor p53 and human double minute-2 (hDM2) proteins by binding hDM2 in a partially helical form, leading to the release of p53 and the induction of apoptosis in certain tumors. Circular dichroism and fluorescence spectroscopy data revealed that the extent and type of secondary structure of p5314-29 are controlled through size and hydrogen bond potential of the linker. In addition, the structure of the self-assembled micelles was influenced through linker-dependent altered headgroup interactions. This study provides insight into the mechanisms through which headgroup structuring occurs on peptide amphiphile micelles, with implications on the bioactivity, stability, and morphology of the self-assembled entities.

Single-molecule resolution of protein structure and interfacial dynamics on biomaterial surfaces

Significance Understanding the effect of near-surface environments on protein conformation is critical in many fields, including biosensing, cell culture, tissue engineering, biocatalysis, and pharmaceutical formulation. However, methods to elucidate both protein structure and interfacial dynamics in heterogeneous near-surface environments are virtually nonexistent. This article describes an approach to characterize changes in protein structure on surfaces using dynamic single-molecule microscopy. Specifically, this approach exploits single-molecule Förster resonance energy transfer tracking to elucidate changes in protein structure at the single-molecule level. Using this approach, structural changes in the protein organophosphorus hydrolase were monitored upon adsorption to fused silica in the presence of BSA on a molecule-by-molecule basis. This method, which is widely applicable to virtually any protein, provides the framework for developing surfaces and surface modifications with improved biocompatibility. A method was developed to monitor dynamic changes in protein structure and interfacial behavior on surfaces by single-molecule Förster resonance energy transfer. This method entails the incorporation of unnatural amino acids to site-specifically label proteins with single-molecule Förster resonance energy transfer probes for high-throughput dynamic fluorescence tracking microscopy on surfaces. Structural changes in the enzyme organophosphorus hydrolase (OPH) were monitored upon adsorption to fused silica (FS) surfaces in the presence of BSA on a molecule-by-molecule basis. Analysis of >30,000 individual trajectories enabled the observation of heterogeneities in the kinetics of surface-induced OPH unfolding with unprecedented resolution. In particular, two distinct pathways were observed: a majority population (∼ 85%) unfolded with a characteristic time scale of 0.10 s, and the remainder unfolded more slowly with a time scale of 0.7 s. Importantly, even after unfolding, OPH readily desorbed from FS surfaces, challenging the common notion that surface-induced unfolding leads to irreversible protein binding. This suggests that protein fouling of surfaces is a highly dynamic process because of subtle differences in the adsorption/desorption rates of folded and unfolded species. Moreover, such observations imply that surfaces may act as a source of unfolded (i.e., aggregation-prone) protein back into solution. Continuing study of other proteins and surfaces will examine whether these conclusions are general or specific to OPH in contact with FS. Ultimately, this method, which is widely applicable to virtually any protein, provides the framework to develop surfaces and surface modifications with improved biocompatibility.