7 research outputs found
Probing the Dynamic Nature of DNA Multilayer Films Using Förster Resonance Energy Transfer
DNA films are of interest for use in a number of areas,
including
sensing, diagnostics, and as drug/gene delivery carriers. The specific
base pairing of DNA materials can be used to manipulate their architecture
and degradability. The programmable nature of these materials leads
to complex and unexpected structures that can be formed from solution
assembly. Herein, we investigate the structure of DNA multilayer films
using Förster resonance energy transfer (FRET). The DNA films
are assembled on silica particles by depositing alternating layers
of homopolymeric diblocks (polyA<sub>15</sub>G<sub>15</sub> and polyT<sub>15</sub>C<sub>15</sub>) with fluorophore (polyA<sub>15</sub>G<sub>15</sub>-TAMRA) and quencher (polyT<sub>15</sub>C<sub>15</sub>-BHQ2)
layers incorporated at predesigned locations throughout the films.
Our results show that DNA films are dynamic structures that undergo
rearrangement. This occurs when the multilayer films are perturbed
during new layer formation through hybridization but can also take
place spontaneously when left over time. These films are anticipated
to be useful in drug delivery applications and sensing applications
Quantifying Cellular Internalization with a Fluorescent Click Sensor
The
ability to determine the amount of material endocytosed by
a cell is important for our understanding of cell biology and in the
design of effective carriers for drug delivery. To quantify internalization
by fluorescence, the signal from material remaining on the cell surface
must be differentiated from endocytosed material. Sensors for internalization
offer advantages over traditional methods for achieving this as they
exhibit improved sensitivity, allow for multiple fluorescent markers
to be used simultaneously, and are amenable to high-throughput analysis.
We have developed a small fluorescent internalization sensor, similar
in size to a standard fluorescent dye, that can be conjugated to proteins
and uses the rapid and highly specific bio-orthogonal reaction between
a tetrazine and a <i>trans</i>-cyclooctene group to switch
off the surface signal. The sensor can be attached to a variety of
materials using simple chemistry and is compatible with flow cytometry
and fluorescence microscopy, making it a useful tool to study the
uptake of material into cells
Fundamental Studies of Hybrid Poly(2-(diisopropylamino)ethyl methacrylate)/Poly(<i>N</i>‑vinylpyrrolidone) Films and Capsules
Hybrid and multicompartment carriers
are of significant interest
for the development of next-generation therapeutic drug carriers.
Herein, fundamental investigations on layer-by-layer (LbL) capsules
consisting of two different polymers are presented. The hybrid systems
were designed to have pH-responsive, charge-shifting polyÂ(2-(diisopropylamino)Âethyl
methacrylate) (PDPA) inner layers and low-fouling polyÂ(<i>N</i>-vinylpyrrolidone) (PVPON) outer layers. Planar hybrid films with
different layer ratios were studied by quartz crystal microgravimetry
(QCM) and atomic force microscopy (AFM). The information obtained
was translated to particulate templates to prepare hybrid capsules,
which were stabilized by click chemistry. The charge-shifting behavior
of PDPA improved the cargo encapsulation and initial retention of
a model CpG cargo, while outer layers of PVPON improved biofouling
properties compared to single-component PDPA capsules. The results
demonstrate the need to understand and design multifunctional systems
that can successfully embody different functionalities in a single,
stable construct for the fabrication of next-generation drug and gene
delivery carriers aimed at overcoming the challenges encountered in
biological systems
Tuning Particle Biodegradation through Polymer–Peptide Blend Composition
We
report the preparation of polymer–peptide blend replica
particles via the mesoporous silica (MS) templated assembly of polyÂ(ethylene
glycol)-<i>block</i>-polyÂ(2-diisopropylaminoethyl methacrylate-<i>co</i>-2-(2-(2-(prop-2-ynyloxy)Âethoxy)Âethoxy)Âethyl methacrylate)
(PEG<sub>45</sub>-<i>b</i>-PÂ(DPA<sub>55</sub>-<i>co</i>-PgTEGMA<sub>4</sub>)) and polyÂ(l-histidine) (PHis). PEG<sub>45</sub>-<i>b</i>-PÂ(DPA<sub>55</sub>-<i>co</i>-PgTEGMA<sub>4</sub>) was synthesized by atom transfer radical polymerization
(ATRP), and was coinfiltrated with PHis into polyÂ(methacrylic acid)
(PMA)-coated MS particles assembled from different peptide-to-polymer
ratios (1:1, 1:5, 1:10, or 1:15). Subsequent removal of the sacrificial
templates and PMA resulted in monodisperse, colloidally stable, noncovalently
cross-linked polymer–peptide blend replica particles that were
stabilized by a combination of hydrophobic interactions between the
PDPA and the PHis, hydrogen bonding between the PEG and PHis backbone,
and π–π stacking of the imidazole rings of PHis
side chains at physiological pH (pH ∼ 7.4). The synergistic charge-switchable properties of PDPA and PHis, and the enzymatic degradability of PHis, make these particles
responsive to pH and enzymes. In vitro studies, in simulated endosomal
conditions and inside cells, demonstrated that particle degradation
kinetics could be engineered (from 2 to 8 h inside dendritic cells)
based on simple adjustment of the peptide-to-polymer ratio used
Targeting Cancer Cells: Controlling the Binding and Internalization of Antibody-Functionalized Capsules
The development of nanoengineered particles, such as polymersomes, liposomes, and polymer capsules, has the potential to offer significant advances in vaccine and cancer therapy. However, the effectiveness of these carriers has the potential to be greatly improved if they can be specifically delivered to target cells. We describe a general method for functionalizing nanoengineered polymer capsules with antibodies using click chemistry and investigate their interaction with cancer cells <i>in vitro</i>. The binding efficiency to cells was found to be dependent on both the capsule-to-cell ratio and the density of antibody on the capsule surface. In mixed cell populations, more than 90% of target cells bound capsules when the capsule-to-target cell ratio was 1:1. Strikingly, greater than 50% of target cells exhibited capsules on the cell surface even when the target cells were present as less than 0.1% of the total cell population. Imaging flow cytometry was used to quantify the internalization of the capsules, and the target cells were found to internalize capsules efficiently. However, the role of the antibody in this process was determined to enhance accumulation of capsules on the cell surface rather than promote endocytosis. This represents a significant finding, as this is the first study into the role antibodies play in internalization of such capsules. It also opens up the possibility of targeting these capsules to cancer cells using targeting molecules that do not trigger an endocytic pathway. We envisage that this approach will be generally applicable to the specific targeting of a variety of nanoengineered materials to cells
Thiol-Reactive Star Polymers Display Enhanced Association with Distinct Human Blood Components
Directing
nanoparticles to specific cell types using nonantibody-based methods
is of increasing interest. Thiol-reactive nanoparticles can enhance
the efficiency of cargo delivery into specific cells through interactions
with cell-surface proteins. However, studies to date using this technique
have been largely limited to immortalized cell lines or rodents, and
the utility of this technology on primary human cells is unknown.
Herein, we used RAFT polymerization to prepare pyridyl disulfide (PDS)-functionalized
star polymers with a methoxy-polyÂ(ethylene glycol) brush corona and
a fluorescently labeled cross-linked core using an arm-first method.
PDS star polymers were examined for their interaction with primary
human blood components: six separate white blood cell subsets, as
well as red blood cells and platelets. Compared with control star
polymers, thiol-reactive nanoparticles displayed enhanced association
with white blood cells at 37 °C, particularly the phagocytic
monocyte, granulocyte, and dendritic cell subsets. Platelets associated
with more PDS than control nanoparticles at both 37 °C and on
ice, but they were not activated in the duration examined. Association
with red blood cells was minor but still enhanced with PDS nanoparticles.
Thiol-reactive nanoparticles represent a useful strategy to target
primary human immune cell subsets for improved nanoparticle delivery
Targeting of Cancer Cells Using Click-Functionalized Polymer Capsules
Targeted delivery of drugs to specific cells allows a high therapeutic dose to be delivered to the target site with minimal harmful side effects. Combining targeting molecules with nanoengineered drug carriers, such as polymer capsules, micelles and polymersomes, has significant potential to improve the therapeutic delivery and index of a range of drugs. We present a general approach for functionalization of low-fouling, nanoengineered polymer capsules with antibodies using click chemistry. We demonstrate that antibody (Ab)-functionalized capsules specifically bind to colorectal cancer cells even when the target cells constitute less than 0.1% of the total cell population. This precise targeting offers promise for drug delivery applications