12 research outputs found
Enhancing Radiotherapy by Lipid Nanocapsule-Mediated Delivery of Amphiphilic Gold Nanoparticles to Intracellular Membranes
Amphiphilic gold nanoparticles (amph-NPs), composed of gold cores surrounded by an amphiphilic mixed organic ligand shell, are capable of embedding within and traversing lipid membranes. Here we describe a strategy using crosslink-stabilized lipid nanocapsules (NCs) as carriers to transport such membrane-penetrating particles into tumor cells and promote their transfer to intracellular membranes for enhanced radiotherapy of cancer. We synthesized and characterized interbilayer-crosslinked multilamellar lipid vesicles (ICMVs) carrying amph-NPs embedded in the capsule walls, forming Au-NCs. Confocal and electron microscopies revealed that the intracellular distribution of amph-NPs within melanoma and breast tumor cells following uptake of free particles <i>vs</i> Au-NCs was quite distinct and that amph-NPs initially delivered into endosomes by Au-NCs transferred over a period of hours to intracellular membranes through tumor cells, with greater intracellular spread in melanoma cells than breast carcinoma cells. Clonogenic assays revealed that Au-NCs enhanced radiotherapeutic killing of melanoma cells. Thus, multilamellar lipid capsules may serve as an effective carrier to deliver amphiphilic gold nanoparticles to tumors, where the membrane-penetrating properties of these materials can significantly enhance the efficacy of frontline radiotherapy treatments
Effects of Surface Compositional and Structural Heterogeneity on NanoparticleâProtein Interactions: Different Protein Configurations
As nanoparticles (NPs) enter into biological systems, they are immediately exposed to a variety and concentration of proteins. The physicochemical interactions between proteins and NPs are influenced by the surface properties of the NPs. To identify the effects of NP surface heterogeneity, the interactions between bovine serum albumin (BSA) and gold NPs (AuNPs) with similar chemical composition but different surface structures were investigated. Different interaction modes and BSA conformations were studied by dynamic light scattering, circular dichroism spectroscopy, fluorescence quenching and isothermal titration calorimetry (ITC). Depending on the surface structure of AuNPs, BSA seems to adopt either a âside-onâ or an âend-onâ conformation on AuNPs. ITC demonstrated that the adsorption of BSA onto AuNPs with randomly distributed polar and nonpolar groups was primarily driven by electrostatic interaction, and all BSA were adsorbed in the same process. The adsorption of BSA onto AuNPs covered with alternating domains of polar and nonpolar groups was a combination of different interactions. Overall, the results of this study point to the potential for utilizing nanoscale manipulation of NP surfaces to control the resulting NPâprotein interactions
Electrical Method to Quantify Nanoparticle Interaction with Lipid Bilayers
Understanding as well as rapidly screening the interaction of nanoparticles with cell membranes is of central importance for biological applications such as drug and gene delivery. Recently, we have shown that âstripedâ mixed-monolayer-coated gold nanoparticles spontaneously penetrate a variety of cell membranes through a passive pathway. Here, we report an electrical approach to screen and readily quantify the interaction between nanoparticles and bilayer lipid membranes. Membrane adsorption is monitored through the capacitive increase of suspended planar lipid membranes upon fusion with nanoparticles. We adopt a Langmuir isotherm model to characterize the adsorption of nanoparticles by bilayer lipid membranes and extract the partition coefficient, <i>K</i>, and the standard free energy gain by this spontaneous process, for a variety of sizes of cell-membrane-penetrating nanoparticles. We believe that the method presented here will be a useful qualitative and quantitative tool to determine nanoparticle interaction with lipid bilayers and consequently with cell membranes
Microfluidic Print-to-Synthesis Platform for Efficient Preparation and Screening of Combinatorial Peptide Microarrays
In
this paper, we introduce a novel microfluidic combinatorial
synthesis platform, referred to as Microfluidic Print-to-Synthesis
(MPS), for custom high-throughput and automated synthesis of a large
number of unique peptides in a microarray format. The MPS method utilizes
standard Fmoc chemistry to link amino acids on a polyethylene glycol
(PEG)-functionalized microdisc array. The resulting peptide microarrays
permit rapid screening for interactions with molecular targets or
live cells, with low nonspecific binding. Such combinatorial peptide
microarrays can be reliably prepared at a spot size of 200 Îźm
with 1 mm center-to-center distance, dimensions that require only
minimal reagent consumption (less than 30 nL per spot per coupling
reaction). The MPS platform has a scalable design for extended multiplexibility,
allowing for 12 different building blocks and coupling reagents to
be dispensed in one microfluidic cartridge in the current format,
and could be further scaled up. As proof of concept for the MPS platform,
we designed and constructed a focused tetrapeptide library featuring
2560 synthetic peptide sequences, capped at the N-terminus with 4-[(<i>N</i>â˛-2-methylphenyl)Âureido]Âphenylacetic acid. We then
used live human T lymphocyte Jurkat cells as a probe to screen the
peptide microarrays for their interaction with ι4β1 integrin
overexpressed and activated on these cells. Unlike the one-bead-one-compound
approach that requires subsequent decoding of positive beads, each
spot in the MPS array is spatially addressable. Therefore, this platform
is an ideal tool for rapid optimization of lead compounds found in
nature or discovered from diverse combinatorial libraries, using either
biochemical or cell-based assays
Development and Characterization of Bioinspired Lipid Raft Nanovesicles for Therapeutic Applications
Lipid rafts are highly ordered regions of the plasma
membrane enriched
in signaling proteins and lipids. Their biological potential is realized
in exosomes, a subclass of extracellular vesicles (EVs) that originate
from the lipid raft domains. Previous studies have shown that EVs
derived from human placental mesenchymal stromal cells (PMSCs) possess
strong neuroprotective and angiogenic properties. However, clinical
translation of EVs is challenged by very low, impure, and heterogeneous
yields. Therefore, in this study, lipid rafts are validated as a functional
biomaterial that can recapitulate the exosomal membrane and then be
synthesized into biomimetic nanovesicles. Lipidomic and proteomic
analyses show that lipid raft isolates retain functional lipids and
proteins comparable to PMSC-EV membranes. PMSC-derived lipid raft
nanovesicles (LRNVs) are then synthesized at high yields using a facile,
extrusion-based methodology. Evaluation of biological properties reveals
that LRNVs can promote neurogenesis and angiogenesis through modulation
of lipid raft-dependent signaling pathways. A proof-of-concept methodology
further shows that LRNVs could be loaded with proteins or other bioactive
cargo for greater disease-specific functionalities, thus presenting
a novel
type of biomimetic nanovesicles that can be leveraged as targeted
therapeutics for regenerative medicine
Development and Characterization of Bioinspired Lipid Raft Nanovesicles for Therapeutic Applications
Lipid rafts are highly ordered regions of the plasma
membrane enriched
in signaling proteins and lipids. Their biological potential is realized
in exosomes, a subclass of extracellular vesicles (EVs) that originate
from the lipid raft domains. Previous studies have shown that EVs
derived from human placental mesenchymal stromal cells (PMSCs) possess
strong neuroprotective and angiogenic properties. However, clinical
translation of EVs is challenged by very low, impure, and heterogeneous
yields. Therefore, in this study, lipid rafts are validated as a functional
biomaterial that can recapitulate the exosomal membrane and then be
synthesized into biomimetic nanovesicles. Lipidomic and proteomic
analyses show that lipid raft isolates retain functional lipids and
proteins comparable to PMSC-EV membranes. PMSC-derived lipid raft
nanovesicles (LRNVs) are then synthesized at high yields using a facile,
extrusion-based methodology. Evaluation of biological properties reveals
that LRNVs can promote neurogenesis and angiogenesis through modulation
of lipid raft-dependent signaling pathways. A proof-of-concept methodology
further shows that LRNVs could be loaded with proteins or other bioactive
cargo for greater disease-specific functionalities, thus presenting
a novel
type of biomimetic nanovesicles that can be leveraged as targeted
therapeutics for regenerative medicine
Development and Characterization of Bioinspired Lipid Raft Nanovesicles for Therapeutic Applications
Lipid rafts are highly ordered regions of the plasma
membrane enriched
in signaling proteins and lipids. Their biological potential is realized
in exosomes, a subclass of extracellular vesicles (EVs) that originate
from the lipid raft domains. Previous studies have shown that EVs
derived from human placental mesenchymal stromal cells (PMSCs) possess
strong neuroprotective and angiogenic properties. However, clinical
translation of EVs is challenged by very low, impure, and heterogeneous
yields. Therefore, in this study, lipid rafts are validated as a functional
biomaterial that can recapitulate the exosomal membrane and then be
synthesized into biomimetic nanovesicles. Lipidomic and proteomic
analyses show that lipid raft isolates retain functional lipids and
proteins comparable to PMSC-EV membranes. PMSC-derived lipid raft
nanovesicles (LRNVs) are then synthesized at high yields using a facile,
extrusion-based methodology. Evaluation of biological properties reveals
that LRNVs can promote neurogenesis and angiogenesis through modulation
of lipid raft-dependent signaling pathways. A proof-of-concept methodology
further shows that LRNVs could be loaded with proteins or other bioactive
cargo for greater disease-specific functionalities, thus presenting
a novel
type of biomimetic nanovesicles that can be leveraged as targeted
therapeutics for regenerative medicine
Effect of Particle Diameter and Surface Composition on the Spontaneous Fusion of Monolayer-Protected Gold Nanoparticles with Lipid Bilayers
Anionic,
monolayer-protected gold nanoparticles (AuNPs) have been
shown to nondisruptively penetrate cellular membranes. Here, we show
that a critical first step in the penetration process is potentially
the fusion of such AuNPs with lipid bilayers. Free energy calculations,
experiments on unilamellar and multilamellar vesicles, and cell studies
all support this hypothesis. Furthermore, we show that fusion is only
favorable for AuNPs with core diameters below a critical size that
depends on the monolayer composition
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Combinatorial Library Screening with Liposomes for Discovery of Membrane Active Peptides
Membrane active peptides
(MAPs) represent a class of short biomolecules
that have shown great promise in facilitating intracellular delivery
without disrupting cellular plasma membranes. Yet their clinical application
has been stalled by numerous factors: off-target delivery, a requirement
for high local concentration near cells of interest, degradation en
route to the target site, and in the case of cell-penetrating peptides,
eventual entrapment in endolysosomal compartments. The current method
of deriving MAPs from naturally occurring proteins has restricted
the discovery of new peptides that may overcome these limitations.
Here, we describe a new branch of assays featuring high-throughput
functional screening capable of discovering new peptides with tailored
cell uptake and endosomal escape capabilities. The one-bead-one-compound
(OBOC) combinatorial method is used to screen libraries containing
millions of potential MAPs for binding to synthetic liposomes, which
can be adapted to mimic various aspects of limiting membranes. By
incorporating unnatural and d-amino acids in the library,
in addition to varying buffer conditions and liposome compositions,
we have identified several new highly potent MAPs that improve on
current standards and introduce motifs that were previously unknown
or considered unsuitable. Since small variations in pH and lipid composition
can be controlled during screening, peptides discovered using this
methodology could aid researchers building drug delivery platforms
with unique requirements, such as targeted intracellular localization
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Multispectral Optical Tweezers for Biochemical Fingerprinting of CD9-Positive Exosome Subpopulations
Extracellular
vesicles (EVs), including exosomes, are circulating
nanoscale particles heavily implicated in cell signaling and can be
isolated in vast numbers from human biofluids. Study of their molecular
profiling and materials properties is currently underway for purposes
of describing a variety of biological functions and diseases. However,
the large, and as yet largely unquantified, variety of EV subpopulations
differing in composition, size, and likely function necessitates characterization
schemes capable of measuring single vesicles. Here we describe the
first application of multispectral optical tweezers (MS-OTs) to single
vesicles for molecular fingerprinting of EV subpopulations. This versatile
imaging platform allows for sensitive measurement of Raman chemical
composition (e.g., variation in protein, lipid, cholesterol, nucleic
acids), coupled with discrimination by fluorescence markers. For exosomes
isolated by ultracentrifugation, we use MS-OTs to interrogate the
CD9-positive subpopulations via antibody fluorescence labeling and
Raman spectra measurement. We report that the CD9-positive exosome
subset exhibits reduced component concentration per vesicle and reduced
chemical heterogeneity compared to the total purified EV population.
We observed that specific vesicle subpopulations are present across
exosomes isolated from cell culture supernatant of several clonal
varieties of mesenchymal stromal cells and also from plasma and ascites
isolated from human ovarian cancer patients