11 research outputs found
Multifunctional Liquid Crystal Nanoparticles for Intracellular Fluorescent Imaging and Drug Delivery
A continuing goal of nanoparticle (NP)-mediated drug delivery (NMDD) is the simultaneous improvement of drug efficacy coupled with tracking of the intracellular fate of the nanoparticle delivery vehicle and its drug cargo. Here, we present a robust multifunctional liquid crystal NP (LCNP)-based delivery system that affords facile intracellular fate tracking coupled with the efficient delivery and modulation of the anticancer therapeutic doxorubicin (Dox), employed here as a model drug cargo. The LCNPs consist of (1) a liquid crystal cross-linking agent, (2) a homologue of the organic chromophore perylene, and (3) a polymerizable surfactant containing a carboxylate headgroup. The NP core provides an environment to both incorporate fluorescent dye for spectrally tuned particle tracking and encapsulation of amphiphilic and/or hydrophobic agents for intracellular delivery. The carboxylate head groups enable conjugation to biologicals to facilitate the cellular uptake of the particles. Upon functionalization of the NPs with transferrin, we show the ability to differentially label the recycling endocytic pathway in HEK 293T/17 cells in a time-resolved manner with minimal cytotoxicity and with superior dye photostability compared to traditional organic fluorophores. Further, when passively loaded with Dox, the NPs mediate the rapid uptake and subsequent sustained release of Dox from within endocytic vesicles. We demonstrate the ability of the LCNPs to simultaneously serve as both an efficient delivery vehicle for Dox as well as a modulator of the drug’s cytotoxicity. Specifically, the delivery of Dox as a LCNP conjugate results in a ∼40-fold improvement in its IC<sub>50</sub> compared to free Dox in solution. Cumulatively, our results demonstrate the utility of the LCNPs as an effective nanomaterial for simultaneous cellular imaging, tracking, and delivery of drug cargos
Lipid Raft-Mediated Membrane Tethering and Delivery of Hydrophobic Cargos from Liquid Crystal-Based Nanocarriers
A main goal of bionanotechnology and nanoparticle (NP)-mediated
drug delivery (NMDD) continues to be the development of novel biomaterials
that can controllably modulate the activity of the NP-associated therapeutic
cargo. One of the desired subcellular locations for targeted delivery
in NMDD is the plasma membrane. However, the controlled delivery of
hydrophobic cargos to the membrane bilayer poses significant challenges
including cargo precipitation and lack of specificity. Here, we employ
a liquid crystal NP (LCNP)-based delivery system for the controlled
partitioning of a model dye cargo from within the NP core into the
plasma membrane bilayer. During synthesis of the NPs, the water-insoluble
model dye cargo, 3,3′-dioctadecyloxacarbocyanine perchlorate
(DiO), was efficiently incorporated into the hydrophobic LCNP core
as confirmed by multiple spectroscopic analyses. Conjugation of a
PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol)
facilitated the localization of the dye-loaded NPs to lipid raft microdomains
in the plasma membrane in HEK 293T/17 cell. Analysis of DiO cellular
internalization kinetics revealed that when delivered as a LCNP-PEG-Chol
NP, the half-life of DiO membrane residence time (30 min) was twice
that of free DiO (DiO<sub>free</sub>) (15 min) delivered from bulk
solution. Time-resolved laser scanning confocal microscopy was employed
to visualize the passive efflux of DiO from the LCNP core and its
insertion into the plasma membrane bilayer as confirmed by Förster
resonance energy transfer (FRET) imaging. Finally, the delivery of
DiO as a LCNP-PEG-Chol complex resulted in the attenuation of its
cytotoxicity; the NP form of DiO exhibited ∼30–40% less
toxicity compared to DiO<sub>free</sub>. Our data demonstrate the
utility of the LCNP platform as an efficient vehicle for the combined
membrane-targeted delivery and physicochemical modulation of molecular
cargos using lipid raft-mediated tethering
Barnacle Balanus amphitrite Adheres by a Stepwise Cementing Process
Barnacles adhere permanently to surfaces by secreting
and curing
a thin interfacial adhesive underwater. Here, we show that the acorn
barnacle Balanus amphitrite adheres
by a two-step fluid secretion process, both contributing to adhesion.
We found that, as barnacles grow, the first barnacle cement secretion
(BCS1) is released at the periphery of the expanding base plate. Subsequently,
a second, autofluorescent fluid (BCS2) is released. We show that secretion
of BCS2 into the interface results, on average, in a 2-fold increase
in adhesive strength over adhesion by BCS1 alone. The two secretions
are distinguishable both spatially and temporally, and differ in morphology,
protein conformation, and chemical functionality. The short time window
for BCS2 secretion relative to the overall area increase demonstrates
that it has a disproportionate, surprisingly powerful, impact on adhesion.
The dramatic change in adhesion occurs without measurable changes
in interface thickness and total protein content. A fracture mechanics
analysis suggests the interfacial material’s modulus or work
of adhesion, or both, were substantially increased after BCS2 secretion.
Addition of BCS2 into the interface generates highly networked amyloid-like
fibrils and enhanced phenolic content. Both intertwined fibers and
phenolic chemistries may contribute to mechanical stability of the
interface through physically or chemically anchoring interface proteins
to the substrate and intermolecular interactions. Our experiments
point to the need to reexamine the role of phenolic components in
barnacle adhesion, long discounted despite their prevalence in structural
membranes of arthropods and crustaceans, as they may contribute to
chemical processes that strengthen adhesion through intermolecular
cross-linking
Barnacle Balanus amphitrite Adheres by a Stepwise Cementing Process
Barnacles adhere permanently to surfaces by secreting
and curing
a thin interfacial adhesive underwater. Here, we show that the acorn
barnacle Balanus amphitrite adheres
by a two-step fluid secretion process, both contributing to adhesion.
We found that, as barnacles grow, the first barnacle cement secretion
(BCS1) is released at the periphery of the expanding base plate. Subsequently,
a second, autofluorescent fluid (BCS2) is released. We show that secretion
of BCS2 into the interface results, on average, in a 2-fold increase
in adhesive strength over adhesion by BCS1 alone. The two secretions
are distinguishable both spatially and temporally, and differ in morphology,
protein conformation, and chemical functionality. The short time window
for BCS2 secretion relative to the overall area increase demonstrates
that it has a disproportionate, surprisingly powerful, impact on adhesion.
The dramatic change in adhesion occurs without measurable changes
in interface thickness and total protein content. A fracture mechanics
analysis suggests the interfacial material’s modulus or work
of adhesion, or both, were substantially increased after BCS2 secretion.
Addition of BCS2 into the interface generates highly networked amyloid-like
fibrils and enhanced phenolic content. Both intertwined fibers and
phenolic chemistries may contribute to mechanical stability of the
interface through physically or chemically anchoring interface proteins
to the substrate and intermolecular interactions. Our experiments
point to the need to reexamine the role of phenolic components in
barnacle adhesion, long discounted despite their prevalence in structural
membranes of arthropods and crustaceans, as they may contribute to
chemical processes that strengthen adhesion through intermolecular
cross-linking
FRET from Multiple Pathways in Fluorophore-Labeled DNA
Because of their ease of design and
assembly, DNA scaffolds provide
a valuable means for organizing fluorophores into complex light harvesting
antennae. However, as the size and complexity of the DNA–fluorophore
network grows, it can be difficult to fully understand energy transfer
properties because of the large number of dipolar interactions between
fluorophores. Here, we investigate simple DNA–fluorophore networks
that represent elements of the more complex networks and provide insight
into the Förster Resonance Energy Transfer (FRET) processes
in the presence of multiple pathways. These FRET networks consist
of up to two Cy3 donor fluorophores and two Cy3.5 acceptor fluorophores
that are linked to a rigid dual-rail DNA scaffold with short interfluorophore
separation corresponding to 10 DNA base pairs (∼34 Å).
This configuration results in five FRET pathways: four hetero-FRET
and one homo-FRET pathway. The FRET properties are characterized using
a combination of steady-state and time-resolved spectroscopy and understood
using Förster theory. We show that the multiple FRET pathways
lead to an increase in FRET efficiency, in part because homo-FRET
between donor fluorophores provides access to parallel pathways to
the acceptor and thereby compensates for low FRET efficiency channels
caused by a static transition dipole distribution. More generally,
the results show that multiple pathways may be used in the design
of artificial light harvesting devices to compensate for inhomogeneities
and nonideal ensemble effects that degrade FRET efficiency
Achieving Effective Terminal Exciton Delivery in Quantum Dot Antenna-Sensitized Multistep DNA Photonic Wires
Assembling DNA-based photonic wires around semiconductor quantum dots (QDs) creates optically active hybrid architectures that exploit the unique properties of both components. DNA hybridization allows positioning of multiple, carefully arranged fluorophores that can engage in sequential energy transfer steps while the QDs provide a superior energy harvesting antenna capacity that drives a Förster resonance energy transfer (FRET) cascade through the structures. Although the first generation of these composites demonstrated four-sequential energy transfer steps across a distance >150 Å, the exciton transfer efficiency reaching the final, terminal dye was estimated to be only ∼0.7% with no concomitant sensitized emission observed. Had the terminal Cy7 dye utilized in that construct provided a sensitized emission, we estimate that this would have equated to an overall end-to-end ET efficiency of ≤0.1%. In this report, we demonstrate that overall energy flow through a second generation hybrid architecture can be significantly improved by reengineering four key aspects of the composite structure: (1) making the initial DNA modification chemistry smaller and more facile to implement, (2) optimizing donor–acceptor dye pairings, (3) varying donor–acceptor dye spacing as a function of the Förster distance <i>R</i><sub>0</sub>, and (4) increasing the number of DNA wires displayed around each central QD donor. These cumulative changes lead to a <i>2 orders of magnitude</i> improvement in the exciton transfer efficiency to the final terminal dye in comparison to the first-generation construct. The overall end-to-end efficiency through the optimized, five-fluorophore/four-step cascaded energy transfer system now approaches 10%. The results are analyzed using Förster theory with various sources of randomness accounted for by averaging over ensembles of modeled constructs. Fits to the spectra suggest near-ideal behavior when the photonic wires have two sequential acceptor dyes (Cy3 and Cy3.5) and exciton transfer efficiencies approaching 100% are seen when the dye spacings are 0.5 × <i>R</i><sub>0</sub>. However, as additional dyes are included in each wire, strong nonidealities appear that are suspected to arise predominantly from the poor photophysical performance of the last two acceptor dyes (Cy5 and Cy5.5). The results are discussed in the context of improving exciton transfer efficiency along photonic wires and the contributions these architectures can make to understanding multistep FRET processes
Growth and development of the barnacle <i>Amphibalanus amphitrite</i>: time and spatially resolved structure and chemistry of the base plate
<div><p>The radial growth and advancement of the adhesive interface to the substratum of many species of acorn barnacles occurs underwater and beneath an opaque, calcified shell. Here, the time-dependent growth processes involving various autofluorescent materials within the interface of live barnacles are imaged for the first time using 3D time-lapse confocal microscopy. Key features of the interface development in the striped barnacle, <i>Amphibalanus</i> (= <i>Balanus</i>) <i>amphitrite</i> were resolved <i>in situ</i> and include advancement of the barnacle/substratum interface, epicuticle membrane development, protein secretion, and calcification. Microscopic and spectroscopic techniques provide <i>ex situ</i> material identification of regions imaged by confocal microscopy. <i>In situ</i> and <i>ex situ</i> analysis of the interface support the hypothesis that barnacle interface development is a complex process coupling sequential, timed secretory events and morphological changes. This results in a multi-layered interface that concomitantly fulfills the roles of strongly adhering to a substratum while permitting continuous molting and radial growth at the periphery.</p></div
Imaging Active Surface Processes in Barnacle Adhesive Interfaces
Surface plasmon resonance
imaging (SPRI) and voltammetry were used
simultaneously to monitor <i>Amphibalanus (=Balanus) amphitrite</i> barnacles reattached and grown on gold-coated glass slides in artificial
seawater. Upon reattachment, SPRI revealed rapid surface adsorption
of material with a higher refractive index than seawater at the barnacle/gold
interface. Over longer time periods, SPRI also revealed secretory
activity around the perimeter of the barnacle along the seawater/gold
interface extending many millimeters beyond the barnacle and varying
in shape and region with time. Ex situ experiments using attenuated
total reflectance infrared (ATR-IR) spectroscopy confirmed that reattachment
of barnacles was accompanied by adsorption of protein to surfaces
on similar time scales as those in the SPRI experiments. Barnacles
were grown through multiple molting cycles. While the initial reattachment
region remained largely unchanged, SPRI revealed the formation of
sets of paired concentric rings having alternately darker/lighter
appearance (corresponding to lower and higher refractive indices,
respectively) at the barnacle/gold interface beneath the region of
new growth. Ex situ experiments coupling the SPRI imaging with optical
and FTIR microscopy revealed that the paired rings coincide with molt
cycles, with the brighter rings associated with regions enriched in
amide moieties. The brighter rings were located just beyond orifices
of cement ducts, consistent with delivery of amide-rich chemistry
from the ducts. The darker rings were associated with newly expanded
cuticle. In situ voltammetry using the SPRI gold substrate as the
working electrode revealed presence of redox active compounds (oxidation
potential approx 0.2 V vs Ag/AgCl) after barnacles were reattached
on surfaces. Redox activity persisted during the reattachment period.
The results reveal surface adsorption processes coupled to the complex
secretory and chemical activity under barnacles as they construct
their adhesive interfaces
Imaging Active Surface Processes in Barnacle Adhesive Interfaces
Surface plasmon resonance
imaging (SPRI) and voltammetry were used
simultaneously to monitor <i>Amphibalanus (=Balanus) amphitrite</i> barnacles reattached and grown on gold-coated glass slides in artificial
seawater. Upon reattachment, SPRI revealed rapid surface adsorption
of material with a higher refractive index than seawater at the barnacle/gold
interface. Over longer time periods, SPRI also revealed secretory
activity around the perimeter of the barnacle along the seawater/gold
interface extending many millimeters beyond the barnacle and varying
in shape and region with time. Ex situ experiments using attenuated
total reflectance infrared (ATR-IR) spectroscopy confirmed that reattachment
of barnacles was accompanied by adsorption of protein to surfaces
on similar time scales as those in the SPRI experiments. Barnacles
were grown through multiple molting cycles. While the initial reattachment
region remained largely unchanged, SPRI revealed the formation of
sets of paired concentric rings having alternately darker/lighter
appearance (corresponding to lower and higher refractive indices,
respectively) at the barnacle/gold interface beneath the region of
new growth. Ex situ experiments coupling the SPRI imaging with optical
and FTIR microscopy revealed that the paired rings coincide with molt
cycles, with the brighter rings associated with regions enriched in
amide moieties. The brighter rings were located just beyond orifices
of cement ducts, consistent with delivery of amide-rich chemistry
from the ducts. The darker rings were associated with newly expanded
cuticle. In situ voltammetry using the SPRI gold substrate as the
working electrode revealed presence of redox active compounds (oxidation
potential approx 0.2 V vs Ag/AgCl) after barnacles were reattached
on surfaces. Redox activity persisted during the reattachment period.
The results reveal surface adsorption processes coupled to the complex
secretory and chemical activity under barnacles as they construct
their adhesive interfaces
Oxidase Activity of the Barnacle Adhesive Interface Involves Peroxide-Dependent Catechol Oxidase and Lysyl Oxidase Enzymes
Oxidases
are found to play a growing role in providing functional chemistry
to marine adhesives for the permanent attachment of macrofouling organisms.
Here, we demonstrate active peroxidase and lysyl oxidase enzymes in
the adhesive layer of adult Amphibalanus amphitrite barnacles through live staining, proteomic analysis, and competitive
enzyme assays on isolated cement. A novel full-length peroxinectin
(AaPxt-1) secreted by barnacles is largely responsible for oxidizing
phenolic chemistries; AaPxt-1 is driven by native hydrogen peroxide
in the adhesive and oxidizes phenolic substrates typically preferred
by phenoloxidases (POX) such as laccase and tyrosinase. A major cement
protein component AaCP43 is found to contain ketone/aldehyde modifications
via 2,4-dinitrophenylhydrazine (DNPH) derivatization, also called
Brady’s reagent, of cement proteins and immunoblotting with
an anti-DNPH antibody. Our work outlines the landscape of molt-related
oxidative pathways exposed to barnacle cement proteins, where ketone-
and aldehyde-forming oxidases use peroxide intermediates to modify
major cement components such as AaCP43