9 research outputs found
Hypoxia-Responsive Polymersomes for Drug Delivery to Hypoxic Pancreatic Cancer Cells
Hypoxia in tumors
contributes to overall tumor progression by assisting
in epithelial-to-mesenchymal transition, angiogenesis, and metastasis
of cancer. In this study, we have synthesized a hypoxia-responsive,
diblock copolymer polyÂ(lactic acid)–azobenzene–polyÂ(ethylene
glycol), which self-assembles to form polymersomes in an aqueous medium.
The polymersomes did not release any encapsulated contents for 50
min under normoxic conditions. However, under hypoxia, 90% of the
encapsulated dye was released in 50 min. The polymersomes encapsulated
the combination of anticancer drugs gemcitabine and erlotinib with
entrapment efficiency of 40% and 28%, respectively. We used three-dimensional
spheroid cultures of pancreatic cancer cells BxPC-3 to demonstrate
hypoxia-mediated release of the drugs from the polymersomes. The vesicles
were nontoxic. However, a significant decrease in cell viability was
observed in hypoxic spheroidal cultures of BxPC-3 cells in the presence
of drug encapsulated polymersomes. These polymersomes have potential
for future applications in imaging and treatment of hypoxic tumors
Hypoxia Responsive, Tumor Penetrating Lipid Nanoparticles for Delivery of Chemotherapeutics to Pancreatic Cancer Cell Spheroids
Solid
tumors are often poorly irrigated due to structurally compromised
microcirculation. Uncontrolled multiplication of cancer cells, insufficient
blood flow, and the lack of enough oxygen and nutrients lead to the
development of hypoxic regions in the tumor tissues. As the partial
pressure of oxygen drops below the necessary level (10 psi), the cancer
cells modulate their genetic makeup to survive. Hypoxia triggers tumor
progression by enhancing angiogenesis, cancer stem cell production,
remodeling of the extracellular matrix, and epigenetic changes in
the cancer cells. However, the hypoxic regions are usually located
deep in the tumors and are usually inaccessible to the intravenously
injected drug carrier or the drug. Considering the designs of the
reported nanoparticles, it is likely that the drug is delivered to
the peripheral tumor tissues, close to the blood vessels. In this
study, we prepared lipid nanoparticles (LNs) comprising the synthesized
hypoxia-responsive lipid and a peptide–lipid conjugate. We
observed that the resultant LNs penetrated to the hypoxic regions
of the tumors. Under low oxygen partial pressure, the hypoxia-responsive
lipid undergoes reduction, destabilizing the lipid membrane, and releasing
encapsulated drugs from the nanoparticles. We demonstrated the results
employing spheroidal cultures of the pancreatic cancer cells BxPC-3.
We observed that the peptide-decorated, drug encapsulated LNs reduced
the viability of pancreatic cancer cells of the spheroids to 35% under
hypoxic conditions
Acridine Orange Conjugated Polymersomes for Simultaneous Nuclear Delivery of Gemcitabine and Doxorubicin to Pancreatic Cancer Cells
Considering
the systemic toxicity of chemotherapeutic agents, there
is an urgent need to develop new targeted drug delivery systems. Herein,
we have developed a new nuclear targeted, redox sensitive, drug delivery
vehicle to simultaneously deliver the anticancer drugs gemcitabine
and doxorubicin to the nuclei of pancreatic cancer cells. We prepared
polymeric bilayer vesicles (polymersomes), and actively encapsulated
the drug combination by the pH gradient method. A redox-sensitive
polymer (PEG–S–S–PLA) was incorporated to sensitize
the formulation to reducing agent concentration. Acridine orange (AO)
was conjugated to the surface of the polymersomes imparting nuclear
localizing property. The polymersomes’ toxicity and efficacy
were compared with those of a free drug combination using monolayer
and three-dimensional spheroid cultures of pancreatic cancer cells.
We observed that the redox sensitive, nuclear-targeted polymersomes
released more than 60% of their encapsulated contents in response
to 50 mM glutathione. The nanoparticles are nontoxic; however, the
drug encapsulated vesicles have significant toxicity. The prepared
formulation can increase the drug’s therapeutic index by delivering
the drugs directly to the cells’ nuclei, one of the key organelles
in the cells. This study is likely to initiate research in targeted
nuclear delivery using other drug formulations in other types of cancers
Dissecting Single-Molecule Signal Transduction in Carbon Nanotube Circuits with Protein Engineering
Single-molecule experimental methods have provided new
insights
into biomolecular function, dynamic disorder, and transient states
that are all invisible to conventional measurements. A novel, nonfluorescent
single-molecule technique involves attaching single molecules to single-walled
carbon nanotube field-effective transistors (SWNT FETs). These ultrasensitive
electronic devices provide long-duration, label-free monitoring of
biomolecules and their dynamic motions. However, generalization of
the SWNT FET technique first requires design rules that can predict
the success and applicability of these devices. Here, we report on
the transduction mechanism linking enzymatic processivity to electrical
signal generation by a SWNT FET. The interaction between SWNT FETs
and the enzyme lysozyme was systematically dissected using eight different
lysozyme variants synthesized by protein engineering. The data prove
that effective signal generation can be accomplished using a single
charged amino acid, when appropriately located, providing a foundation
to widely apply SWNT FET sensitivity to other biomolecular systems
Observing Lysozyme’s Closing and Opening Motions by High-Resolution Single-Molecule Enzymology
Single-molecule techniques can monitor
the kinetics of transitions
between enzyme open and closed conformations, but such methods usually
lack the resolution to observe the underlying transition pathway or
intermediate conformational dynamics. We have used a 1 MHz bandwidth
carbon nanotube transistor to electronically monitor single molecules
of the enzyme T4 lysozyme as it processes substrate. An experimental
resolution of 2 μs allowed the direct recording of lysozyme’s
opening and closing transitions. Unexpectedly, both motions required
37 ÎĽs, on average. The distribution of transition durations
was also independent of the enzyme’s state: either catalytic
or nonproductive. The observation of smooth, continuous transitions
suggests a concerted mechanism for glycoside hydrolysis with lysozyme’s
two domains closing upon the polysaccharide substrate in its active
site. We distinguish these smooth motions from a nonconcerted mechanism,
observed in approximately 10% of lysozyme openings and closings, in
which the enzyme pauses for an additional 40–140 μs in
an intermediate, partially closed conformation. During intermediate
forming events, the number of rate-limiting steps observed increases
to four, consistent with four steps required in the stepwise, arrow-pushing
mechanism. The formation of such intermediate conformations was again
independent of the enzyme’s state. Taken together, the results
suggest lysozyme operates as a Brownian motor. In this model, the
enzyme traces a single pathway for closing and the reverse pathway
for enzyme opening, regardless of its instantaneous catalytic productivity.
The observed symmetry in enzyme opening and closing thus suggests
that substrate translocation occurs while the enzyme is closed
Electronic Measurements of Single-Molecule Catalysis by cAMP-Dependent Protein Kinase A
Single-molecule
studies of enzymes open a window into their dynamics
and kinetics. A single molecule of the catalytic domain of cAMP-dependent
protein kinase A (PKA) was attached to a single-walled carbon nanotube
device for long-duration monitoring. The electronic recording clearly
resolves substrate binding, ATP binding, and cooperative formation
of PKA’s catalytically functional, ternary complex. Using recordings
of a single PKA molecule extending over 10 min and tens of thousands
of binding events, we determine the full transition probability matrix
and conversion rates governing formation of the apo, intermediate,
and closed enzyme configurations. We also observe kinetic rates varying
over 2 orders of magnitude from one second to another. Anti-correlation
of the on and off rates for PKA binding to the peptide substrate,
but not ATP, demonstrates that regulation of enzyme activity results
from altering the stability of the PKA–substrate complex, not
its binding to ATP. The results depict a highly dynamic enzyme offering
dramatic possibilities for regulated activity, an attribute useful
for an enzyme with crucial roles in cell signaling
Electronic Measurements of Single-Molecule Processing by DNA Polymerase I (Klenow Fragment)
Bioconjugating
single molecules of the Klenow fragment of DNA polymerase
I into electronic nanocircuits allowed electrical recordings of enzymatic
function and dynamic variability with the resolution of individual
nucleotide incorporation events. Continuous recordings of DNA polymerase
processing multiple homopolymeric DNA templates extended over 600
s and through >10 000 bond-forming events. An enzymatic
processivity
of 42 nucleotides for a template of the same length was directly observed.
Statistical analysis determined key kinetic parameters for the enzyme’s
open and closed conformations. Consistent with these nanocircuit-based
observations, the enzyme’s closed complex forms a phosphodiester
bond in a highly efficient process >99.8% of the time, with a mean
duration of only 0.3 ms for all four dNTPs. The rate-limiting step
for catalysis occurs during the enzyme’s open state, but with
a nearly 2-fold longer duration for dATP or dTTP incorporation than
for dCTP or dGTP into complementary, homopolymeric DNA templates.
Taken together, the results provide a wealth of new information complementing
prior work on the mechanism and dynamics of DNA polymerase I
Single-Molecule Dynamics of Lysozyme Processing Distinguishes Linear and Cross-Linked Peptidoglycan Substrates
The dynamic processivity of individual T4 lysozyme molecules
was
monitored in the presence of either linear or cross-linked peptidoglycan
substrates. Single-molecule monitoring was accomplished using a novel
electronic technique in which lysozyme molecules were tethered to
single-walled carbon nanotube field-effect transistors through pyrene
linker molecules. The substrate-driven hinge-bending motions of lysozyme
induced dynamic electronic signals in the underlying transistor, allowing
long-term monitoring of the same molecule without the limitations
of optical quenching or bleaching. For both substrates, lysozyme exhibited
processive low turnover rates of 20–50 s<sup>–1</sup> and rapid (200–400 s<sup>–1</sup>) nonproductive motions.
The latter nonproductive binding events occupied 43% of the enzyme’s
time in the presence of the cross-linked peptidoglycan but only 7%
with the linear substrate. Furthermore, lysozyme catalyzed the hydrolysis
of glycosidic bonds to the end of the linear substrate but appeared
to sidestep the peptide cross-links to zigzag through
the wild-type substrate
Nuclear Localizing Peptide-Conjugated, Redox-Sensitive Polymersomes for Delivering Curcumin and Doxorubicin to Pancreatic Cancer Microtumors
Improving the therapeutic index of
anticancer agents is an enormous challenge. Targeting decreases the
side effects of the therapeutic agents by delivering the drugs to
the intended destination. Nanocarriers containing the nuclear localizing
peptide sequences (NLS) translocate to the cell nuclei. However, the
nuclear localization peptides are nonselective and cannot distinguish
the malignant cells from the healthy counterparts. In this study,
we designed a “masked” NLS peptide which is activated
only in the presence of overexpressed matrix metalloproteinase-7 (MMP-7)
enzyme in the pancreatic cancer microenvironment. This peptide is
conjugated to the surface of redox responsive polymersomes to deliver
doxorubicin and curcumin to the pancreatic cancer cell nucleus. We
have tested the formulation in both two- and three-dimensional cultures
of pancreatic cancer and normal cells. Our studies revealed that the
drug-encapsulated polymeric vesicles are significantly more toxic
toward the cancer cells (shrinking the spheroids up to 49%) compared
to the normal cells (shrinking the spheroids up to 24%). This study
can lead to the development of other organelle targeted drug delivery
systems for various human malignancies