Functionalized Nanomaterials for Medical Applications

For the PowerPoint, click here.

For the paper, click here.

Role of target geometry in phagocytosis

by Julie A. Champion & Samir Mitragotri. PNAS (2006) 103(13): 4930-4934.

My choice of this paper was based on the idea that cell activities and behavior is based on the macroscopic, cumulative effect of individual nanoscopic actin fibers acting within the cell. Actin fibers can rapidly polymerize/depolymerize, allowing for cell motility, phagocytosis (cell eating), and more. In this case, the role of the actin cytoskeleton in phagocytosis is being examined. While I do think that the authors’ discussion of the results was questionable at times, it is of note that the local orientation of a particle as a cell approaches it can spell the difference between internalization or being seen as a substrate for spreading.

Purpose
To examine the cellular uptake of polystyrene microparticulates of different shapes.
-Determination of the effect of microparticle shape on phagocytosis efficiency
-Establish a secondary role of particle size on phagocytosis efficiency

Methods
-Purchase non-crosslinked polystyrene microspheres, and with a custom apparatus, mechanically stretch into various shapes (ellipses, discs, UFOs, etc.)
-Incubated macrophages in the presence of opsonized/nonopsonized microparticles
-Used time-lapse microscopy, actin staining to visualize cytoskeleton during interactions between cell and microparticles.

Key Findings
-Regardless of opsonization, phagocytosis of microparticles was dependent on local orientation of the microparticle upon contact with the macrophages
-Local orientation at point of contact was formulated into a long equation in the paper that resulted in an angle Ω, which is dependent only on the particle shape. Interesting relationship between Ω and phagocytosis efficiency.
-Observed some differences in cytoskeletal organization when phagocytosis was seen vs. the alternative behavior – cell spreading.

Definitions
-Macrophages: immune cell line responsible for nonspecific clearance of foreign bodies.
-Opsonization: process of coating substances with a layer of immunogenic substances, increasing the likelihood of an immune reaction to the substance.

Multifunctional Encoded Particles for High-Throughput Biomolecule Analysis

by Daniel C. Pregibon, Mehmet Toner, Patrick S. Doyle, Science (2007) 315: 1393-1396

Purpose
To build a platform that allows for high-throughput screening of oligonucleotide analytes, with multiplexing capabilities.

Methods
Previous work by Patrick Doyle’s group allowed for the formation of microparticles in a microfluidic system by flowing a polymer/photoinitiator mixture through the chamber, and then pulsing UV irradiation through a photomask (placed on light source between condenser and objective) through a method they called “continuous flow lithography” (CFL).

In this work, the group used CFL (and its ability to rapidly form microparticles, ~100/second) to create multifunctional microparticles. This was done by exploiting laminar flow of multiple polymer chemistries and polymerizing particles the shape of pills that were perpendicular to the direction of the flow. By incorporating oligonucleotide probes into the flowed polymer mixtures, particles were created that displayed multiple probes in well-defined regions. The pattern also allowed for the incorporation of a barcode tag to help identify the oligonucleotide probes that were present on each specific microparticle.

The target sequences for the probes can be easily fluorescently labeled. Incubation of a mixture of microparticles with these fluorescently labeled targets allows the target to attach to their probes on the microparticles with high specificity. Another microfluidic device and a custom computer program was written to be able to analyze the particles for the presence of fluorescent signal at high rates.

Key Findings
-The scheme for microparticle synthesis (bar codes) and the well-written software allows for the formulation of a vast amount of different probes, each of which by themselves have multiplexing capabilities due to the fabrication method.

-Accurate and reproducible detection of analytes with sensitivity on the order of hundreds of attomoles, though this required longer incubation time between microparticles and the target mixture.

Definitions
-Oligonucleotide Probe: a single strand of DNA containing (rough estimate) 10-100 nucleotides, which can be used to probe for the presence of its complementary sequence in solution.

Oligonucleotide Loading Determines Cellular Uptake of DNA-Modified Gold Nanoparticles

Purpose
To examine the cellular uptake of gold nanoparticles (~13nm) densely functionalized with antisense oligonucleotide sequences.
-Previously shown that these NPs can be used as agents for transfection or gene regulation
-Do not require additives/other reagents to enter cells effectively
-Antisense oligonucleotides can bind their targets with high affinity

Methods
-Cofunctionalized Au NPs with OEG (oligoethylene glycol) and antisense DNA to form “ASNPs” with varying surface concentrations of single-stranded DNA (ssDNA)
-Incubated three cell lines (differing species, tissue types represented) with ASNPs, investigated uptake of particles by digesting cells and measuring Au content by ICP-MS
-Analyzed ASNP sizes before and after exposure to media by DLS & fluorescence-based protein quantification assay

Key Findings
-Cell uptake of ASNPs was dependent on surface concentrations of ssDNA on the ASNPs.
-Protein adsorption on ASNP surface dependent on surface concentration of ssDNA, as confirmed by DLS & the fluorescence-based protein quantification assay
-Even at high doses, OEG-functionalized NPs (no ssDNA) did not demonstrate significant uptake by cells, indicating that oligonucleotides provided uptake ability.’
-Mechanism of ASNP uptake remains to be studied, but protein adsorption is likely an important factor.

Definitions
-Antisense oligonucleotide: ssDNA or single-stranded RNA that can be targeted to a complementary strand of oligonucleotide.
-Oligoethylene glycol: a oligomer of ethylene glycol that, when immobilized on a surface, yields a hydrophilic, biologically inert, and neutrally charged monolayer.
-ICP-MS: inductively coupled plasma mass spectroscopy; very sensitive

The “Music” of Core-Shell Spheres and Hollow Capsules: Influence of the Architecture on the Mechanical Properties at the Nanoscale

by: T. Still, R. Sainidou, M. Retsch, U. Jonas, P. Spahn, G.P. Hellmann, and G. Fytas
in Nano Letters (in press, Web published 2008-09-04)

Purpose
Use Brillouin light scattering (BLS) to measure elastic vibrations/resonance modes in silica-PMMA core-shell nanoparticles as opposed to hollow-core PMMA shells. Using numerical calculations, determine mechanical moduli of nanoparticle components (silica and PMMA).

Motivation
In a wide range of applications, mechanical properties of nanoparticles need to be tunable. Authors claim currently available methods are insufficient in that they treat nanomaterials like bulk materials and do not take a number of quantum effects into consideration (ie, confinement, depletion, surface/interfacial effects).

Methods
- Obtain BLS spectra of a variety of different nanoparticles (bare silica core, silica core with PMMA shells of increasing thicknesses, PMMA shell only), and curve fit to Lorentzian functions to determine experimental resonance frequencies (peaks in the BLS spectra after curve fitting).
- Compare experimental resonance frequencies to theoretical resonance frequencies obtained through calculated density-of-states (DOS) spectra for each NP system.
- For NPs containing >2 BLS peaks, formulas exist to enable calculation of Young’s modulus and shear modulus for the nanoparticle (authors referred to other sources for these formulations). Keep in mind these moduli may differ at the nanoscale from values in bulk due to quantum effects.

Key Findings
1) For silica-core/PMMA-shell nanoparticles, starting from bare silica and increasing the thickness of the PMMA shell, increasing number of resonance frequencies can be seen.

2) Coating silica cores with PMMA decreases resonance frequencies as thickness of the PMMA layer is increased. While bare silica exhibits mechanical moduli below that seen with bulk silica, coating with PMMA also seems to increase mechanical moduli a little above standard levels in bulk.

3) Removing the silica core does not seem to have much effect on PMMA mechanical moduli, which was already higher than that seen in bulk to begin with. PMMA shell resonance frequencies are directly related to shell thickness.

4) Demonstrated the creation of hybrid nanoparticles that experience heterogeneous elastic behavior.

Key Terms Defined
BLS: a method similar to Raman spectroscopy used to measure changes in light scattering due to acoustic modes (such as phonons and resonance frequencies) and magnetic effects in a material.

Phonons: quantized mode of vibration within a crystal lattice structure.