11 research outputs found
Uptake and Retention of Nanoplastics in Quagga Mussels
Here, a set of experiments to assess the feasibility of using an invasive and widespread freshwater mussel (Dreissena rostrformis bugensis) as a sentinel species for nanoplastic detection is reported. Under laboratory experimental conditions, mussels ingest and retain fluorescent polystyrene (PS) beads with carboxylic acid (ĂŻÂŁÂżCOOH) termination over a size range of 200- 2000 nm. The number of beads the mussels ingested is quantified using fluorescence spectroscopy and the location of the beads in the mussels is imaged using fluorescence microscopy. PS beads of similar size (1000- 2000 nm) to musselsâ preferred food are trafficked in the ciliated food grooves of the gills. Beads of all sizes are observed in the musselsâ digestive tracts, indicating that the mussels do not efficiently reject the beads as unwanted foreign material, regardless of size. Fluorescence microscopy shows all sizes of beads are concentrated in the siphons and are retained there for longer than one month postexposure. Combined atomic force microscopy- infrared spectroscopy and photothermal infrared spectroscopy are used to locate, image, and chemically identify the beads in the mussel siphons. In sum, these experiments demonstrate the potential for using mussels, specifically their siphons, to monitor environmental accumulation of aquatic nanoplastics.Can quagga mussels (Dreissena rostriformis bugensis), a widespread and invasive freshwater species that alters local ecosystems, act as a sentinel species for detecting nanoplastics? In the laboratory, mussels ingest and retain 200- 2000 nm fluorescent polystyrene beads, which are in the size range for the musselsâ preferred food and are trafficked like food in the ciliated grooves of the gills.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/155884/1/gch2201800104-sup-0001-SuppMat.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/155884/2/gch2201800104.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/155884/3/gch2201800104_am.pd
Distributions: The Importance of the Chemistâs Molecular View for Biological Materials
Characterization
of materials with biological applications and
assessment of physiological effects of therapeutic interventions are
critical for translating research to the clinic and preventing adverse
reactions. Analytical techniques typically used to characterize targeted
nanomaterials and tissues rely on bulk measurement. Therefore, the
resulting data represent an <i>average</i> structure of
the sample, masking stochastic (randomly generated) distributions
that are commonly present. In this Perspective, we examine almost
20 years of work our group has done in different fields to characterize
and control distributions. We discuss the analytical techniques and
statistical methods we use and illustrate how we leverage them in
tandem with other bulk techniques. We also discuss the challenges
and time investment associated with taking such a detailed view of
distributions as well as the risks of not fully appreciating the extent
of heterogeneity present in many systems. Through three case studies
showcasing our research on conjugated polymers for drug delivery,
collagen in bone, and endogenous protein nanoparticles, we discuss
how identification and characterization of distributions, i.e., a
molecular view of the system, was critical for understanding the observed
biological effects. In all three cases, data would have been misinterpreted
and insights missed if we had only relied upon spatially averaged
data. Finally, we discuss how new techniques are starting to bridge
the gap between bulk and molecular level analysis, bringing more opportunity
and capacity to the research community to address the challenges of
distributions and their roles in biology, chemistry, and the translation
of science and engineering to societal challenges
Atomic Force Microscopy-Infrared Spectroscopy of Individual Atmospheric Aerosol Particles: Subdiffraction Limit Vibrational Spectroscopy and Morphological Analysis
Chemical
analysis of atmospheric aerosols is an analytical challenge,
as aerosol particles are complex chemical mixtures that can contain
hundreds to thousands of species in attoliter volumes at the most
abundant sizes in the atmosphere (âŒ100 nm). These particles
have global impacts on climate and health, but there are few methods
available that combine imaging and the detailed molecular information
from vibrational spectroscopy for individual particles <500 nm.
Herein, we show the first application of atomic force microscopy with
infrared spectroscopy (AFM-IR) to detect trace organic and inorganic
species and probe intraparticle chemical variation in individual particles
down to 150 nm. By detecting photothermal expansion at frequencies
where particle species absorb IR photons from a tunable laser, AFM-IR
can study particles smaller than the optical diffraction limit. Combining
strengths of AFM (ambient pressure, height, morphology, and phase
measurements) with photothermal IR spectroscopy, the potential of
AFM-IR is shown for a diverse set of single-component particles, liquidâliquid
phase separated particles (coreâshell morphology), and ambient
atmospheric particles. The spectra from atmospheric model systems
(ammonium sulfate, sodium nitrate, succinic acid, and sucrose) had
clearly identifiable features that correlate with absorption frequencies
for infrared-active modes. Additionally, molecular information was
obtained with <100 nm spatial resolution for phase separated particles
with a âŒ150 nm shell and 300 nm core. The subdiffraction limit
capability of AFM-IR has the potential to advance understanding of
particle impacts on climate and health by improving analytical capabilities
to study water uptake, heterogeneous reactivity, and viscosity
Conjugation Dependent Interaction of Folic Acid with Folate Binding Protein
Serum proteins play
a critical role in the transport, uptake, and
efficacy of targeted drug therapies, and here we investigate the interactions
between folic acidâpolymer conjugates and serum folate binding
protein (FBP), the soluble form of the cellular membrane-bound folate
receptor. We demonstrate that both choice of polymer and method of
ligand conjugation affect the interactions between folic acidâpolymer
conjugates and serum FBP, resulting in changes in the folic acid-induced
protein aggregation process. We have previously demonstrated that
individual FBP molecules self-aggregate into nanoparticles at physiological
concentrations. When polyÂ(amidoamine) dendrimerâfolic acid
conjugates bound to FBP, the distribution of nanoparticles was preserved.
However, the dendritic conjugates produced larger nanoparticles than
those formed in the presence of physiologically normal human levels
of folic acid, and the conjugation method affected particle size distribution.
In contrast, polyÂ(ethylene glycol)âfolic acid conjugates demonstrated
substantially reduced binding to FBP, did not cause folic acid-induced
aggregation, and fully disrupted FBP self-aggregation. On the basis
of these results, we discuss the potential implications for biodistribution,
trafficking, and therapeutic efficacy of targeted nanoscale therapeutics,
especially considering the widespread clinical use of polyÂ(ethylene
glycol) conjugates. We highlight the importance of considering specific
serum protein interactions in the rational design of similar nanocarrier
systems. Our results suggest that prebinding therapeutic nanocarriers
to serum FBP may allow folate-specific metabolic pathways to be exploited
for delivery while also affording benefits of utilizing an endogenous
protein as a vector
Quantitative Measurement of Cationic Polymer Vector and PolymerâpDNA Polyplex Intercalation into the Cell Plasma Membrane
Cationic gene delivery agents (vectors) are important for delivering nucleotides, but are also responsible for cytotoxicity. Cationic polymers (L-PEI, jetPEI, and G5 PAMAM) at 1Ă to 100Ă the concentrations required for translational activity (protein expression) induced the same increase in plasma membrane current of HEK 293A cells (30â50 nA) as measured by whole cell patch-clamp. This indicates saturation of the cell membrane by the cationic polymers. The increased currents induced by the polymers are not reversible for over 15 min. Irreversibility on this time scale is consistent with a polymer-supported pore or carpet model and indicates that the cell is unable to clear the polymer from the membrane. For polyplexes, although the charge concentration was the same (at N/P ratio of 10:1), G5 PAMAM and jetPEI polyplexes induced a much larger current increase (40- 50 nA) than L-PEI polyplexes (<20 nA). Both free cationic lipid and lipid polyplexes induced a lower increase in current than cationic polymers (<20 nA). To quantify the membrane bound material, partition constants were measured for both free vectors and polyplexes into the HEK 293A cell membrane using a dye influx assay. The partition constants of free vectors increased with charge density of the vectors. Polyplex partition constants did not show such a trend. The long lasting cell plasma permeability induced by exposure to the polymer vectors or the polyplexes provides a plausible mechanism for the toxicity and inflammatory response induced by exposure to these materials
Substrate-Triggered Exosite Binding: Synergistic Dendrimer/Folic Acid Action for Achieving Specific, Tight-Binding to Folate Binding Protein
Polymerâligand conjugates
are designed to bind proteins
for applications as drugs, imaging agents, and transport scaffolds.
In this work, we demonstrate a folic acid (FA)-triggered exosite binding
of a generation five polyÂ(amidoamine) (G5 PAMAM) dendrimer scaffold
to bovine folate binding protein (bFBP). The protein exosite is a
secondary binding site on the protein surface, separate from the FA
binding pocket, to which the dendrimer binds. Exosite binding is required
to achieve the greatly enhanced binding constants and protein structural
change observed in this study. The G5<sub>Ac</sub>-COG-FA<sub>1.0</sub> conjugate bound tightly to bFBP, was not displaced by a 28-fold
excess of FA, and quenched roughly 80% of the initial fluorescence.
Two-step binding kinetics were measured using the intrinsic fluorescence
of the FBP tryptophan residues to give a <i>K</i><sub>D</sub> in the low nanomolar range for formation of the initial G5<sub>Ac</sub>-COG-FA<sub>1.0</sub>/FBP* complex, and a slow conversion to the
tight complex formed between the dendrimer and the FBP exosite. The
extent of quenching was sensitive to the choice of FA-dendrimer linker
chemistry. Direct amide conjugation of FA to G5-PAMAM resulted in
roughly 50% fluorescence quenching of the FBP. The G5<sub>Ac</sub>-COG-FA, which has a longer linker containing a 1,2,3-triazole ring,
exhibited an âŒ80% fluorescence quenching. The binding of the
G5<sub>Ac</sub>-COG-FA<sub>1.0</sub> conjugate was compared to polyÂ(ethylene
glycol) (PEG) conjugates of FA (PEG<sub><i>n</i></sub>-FA).
PEG<sub>2k</sub>-FA had a binding strength similar to that of FA,
whereas other PEG conjugates with higher molecular weight showed weaker
binding. However, no PEG conjugates gave an increased degree of total
fluorescence quenching