44 research outputs found
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Protein Transduction Domain Mimic (PTDM) Self-Assembly?
Intracellular protein delivery is an invaluable tool for biomedical research, as it enables fundamental studies of cellular processes and creates opportunities for novel therapeutic development. Protein delivery reagents such as cell penetration peptides (CPPs) and protein transduction domains (PTDs) are frequently used to facilitate protein delivery. Herein, synthetic polymer mimics of PTDs, called PTDMs, were studied for their ability to self-assemble in aqueous media as it was not known whether self-assembly plays a role in the protein binding and delivery process. The results obtained from interfacial tensiometry (IFT), transmission electron microscopy (TEM), transmittance assays (%T), and dynamic light scattering (DLS) indicated that PTDMs do not readily aggregate or self-assemble at application-relevant time scales and concentrations. However, additional DLS experiments were used to confirm that the presence of protein is required to induce the formation of PTDM-protein complexes and that PTDMs likely bind as single chains
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Bio-inspired Polymers that Bind and Deliver Protein Cargo
Delivering functional proteins and antibodies into cells can allow researchers to probe the intracellular environment, discover new cellular pathways, and pioneer new therapeutics. However, the entry of exogenous, charged molecules, like proteins, into the cell is usually restricted by the membrane, thereby hindering intracellular delivery. Membrane permeable molecules such as cell penetrating peptides (CPPs) and protein transduction domains (PTDs) can be used to bypass the cell membrane and deliver protein into the cell, but these peptides involve iterative and laborious syntheses and are limited in terms of their chemical diversity.
This dissertation work overall focuses on the design and synthesis of polymeric CPP or PTD mimics (CPPMs or PTDMs), which are easier to synthesize and more effective at non-covalently binding and delivering protein cargo into cells. (From here on and for consistency, these mimics will be referred to as PTDMs only.) Specifically, ring-opening metathesis polymerization was employed in this work to create highly tuned and optimized amphiphilic block copolymer PTDMs which can non-covalently bind and deliver protein cargo into difficult-to-transfect human T cells. From previous work on PTDMs, we learned that PTDMs require a hydrophobic block for effective protein delivery and that this block could be optimized for enhanced delivery. We also received early indications that the protein binding ability of PTDMs might be correlated with the ability to deliver protein cargo.
Herein, in an initial study, the hydrophobicity of PTDMs were carefully modulated by fine degrees to ascertain how precise changes in polymer hydrophobicity affected the ability to bind and deliver one model, protein cargo. Overall, changing the hydrophobicity in a systematic and significant way did not impact the ability to bind protein cargo, but it did reveal a requisite optimal hydrophobicity for maximal delivery in accordance with the results of related studies. Additionally, binding ability and delivery ability were uncorrelated in this study unlike the results of past research.
In a second study, the relationship between binding and delivery was explored further by first measuring the binding strength of PTDMs to various types of protein cargo. The results of this study established that PTDMs indeed exhibited preferential protein binding depending on the attributes of the protein. These preferences were manifested in measured dissociation constants (Kdâs) that differed by orders of magnitude, from weak to very tight binding. These substantial differences in binding ability were uncorrelated to the delivery outcomes further highlighting the importance of other factors, like PTDM hydrophobicity.
In a third study, the ability of a model PTDM to self-assemble at protein binding application relevant concentrations was probed in an effort to determine the importance of PTDM self-assembly to the protein cargo binding process. This study suggested that low molecular weight PTDMs act as weak surfactants and do not self-assemble, but can form detectable aggregates by dynamic light scattering (DLS) in the presence of protein. In further efforts to study the binding process, beyond the quantification of Kdâs and self-assembly, various PTDM-protein binding conditions, such as the presence of increased salt and urea, were explored to elucidate the underlying causes of binding. Future work in this field will fundamentally explore how PTDMs bind and unbind proteins, how these complexes are formed and organized, and how binding impacts the functionality of the protein
Protein Transduction Domain Mimic (PTDM) Self-Assembly?
Intracellular protein delivery is an invaluable tool for biomedical research, as it enables fundamental studies of cellular processes and creates opportunities for novel therapeutic development. Protein delivery reagents such as cell penetration peptides (CPPs) and protein transduction domains (PTDs) are frequently used to facilitate protein delivery. Herein, synthetic polymer mimics of PTDs, called PTDMs, were studied for their ability to self-assemble in aqueous media as it was not known whether self-assembly plays a role in the protein binding and delivery process. The results obtained from interfacial tensiometry (IFT), transmission electron microscopy (TEM), transmittance assays (%T), and dynamic light scattering (DLS) indicated that PTDMs do not readily aggregate or self-assemble at application-relevant time scales and concentrations. However, additional DLS experiments were used to confirm that the presence of protein is required to induce the formation of PTDM-protein complexes and that PTDMs likely bind as single chains
The Role of Cargo Binding Strength in Polymer-Mediated Intracellular Protein Delivery
Delivering
proteins into the intracellular environment is a critical
step toward probing vital cellular processes for the purposes of ultimately
developing new therapeutics. Polymeric carriers are widely used to
facilitate protein delivery with guanidinium-rich macromolecules leading
the way within this category. Although binding interactions between
natural proteins and synthetic polymers have been studied extensively,
the relationship between polymerâprotein binding and intracellular
delivery is seldom explored. Elucidating the role of cargo binding
in delivery is a promising direction that is expected to provide new
insights that further optimize intracellular protein delivery. Herein,
model polymeric carriers called protein transduction domain mimics
(PTDMs) were studied for their ability to bind to a variety of protein
cargoes, including an antibody, where the proteins encompassed a range
of sizes (âŒ16â151 kDa) and isoelectric points (4.7â11.4).
The PTDMâprotein complexes were also delivered into Jurkat
T cells in an attempt to establish a general correlation between binding
ability and delivery outcomes. Binding assays resulted in a vast range
of dissociation constants (K<sub>d</sub>), which spanned from 3.5
to 4820 nM and indicated a variety of binding strengths between PTDM
and protein. More significantly, PTDMs preferentially bound certain
types of proteins over others, such as the antibody fragment over
the whole antibody. Furthermore, increased PTDMâprotein binding
affinity did not correlate with protein delivery, suggesting that
the successful internalization of complexes is independent of binding
equilibrium. Although binding did not correlate with internalization
here, the potential for binding affinity to impact other aspects of
delivery, like cargo functionality inside the cell, remains an open
possibility
Optimal Hydrophobicity in Ring-Opening Metathesis Polymerization-Based Protein Mimics Required for siRNA Internalization
Functional Polyethylenes with Precisely Placed Thioethers and Sulfoniums through ThiolâEne Polymerization
The
precise functionalization of polyethylenes, often accomplished
through acyclic diene metathesis polymerization (ADMET), is a significant
area of research that has improved polyethylene properties and performance.
Here, the synthesis of precisely functionalized polyethylenes was
accomplished using the thiolâene step-growth (TES) polymerization.
The simplicity and versatility of this technique allowed for the synthesis
of a variety of polymers and enabled the study of carbon spacer length
between and repeat unit symmetry about the resulting backbone thioether
moiety. In addition, the backbone thioethers of some samples were
functionalized postpolymerization with methyl triflate to produce
polyethylenes containing sulfonium cations. All polymers were then
characterized for their thermal stability, crystallinity, and morphology
using differential scanning calorimetry (DSC) and X-ray scattering.
While the carbon spacer length and repeat unit symmetry had no effect
on polymer thermal stability, the incorporation of cationic sulfonium
groups reduced the degradation temperature. Most polymers were polymorphic
with respect to crystal structure, and increasing the carbon spacer
length led to an increase in polymer melting temperature and percent
crystallinity. Furthermore, the average carbon spacer length had a
larger effect on polymer percent crystallinity and crystal structure
than repeat unit symmetry, but the symmetry had a significant impact
on polymer crystal melting temperature, as symmetric polymers had
higher melting temperatures. Overall, TES polymerization was utilized
to fabricate precisely functionalized polyethylenes, where the repeat
unit symmetry improved polymer crystal perfection
Optimal Hydrophobicity in Ring-Opening Metathesis Polymerization-Based Protein Mimics Required for siRNA Internalization
Exploring
the role of polymer structure for the internalization
of biologically relevant cargo, specifically siRNA, is of critical
importance to the development of improved delivery reagents. Herein,
we report guanidinium-rich protein transduction domain mimics (PTDMs)
based on a ring-opening metathesis polymerization scaffold containing
tunable hydrophobic moieties that promote siRNA internalization. Structureâactivity
relationships using Jurkat T cells and HeLa cells were explored to
determine how the length of the hydrophobic block and the hydrophobic
side chain compositions of these PTDMs impacted siRNA internalization.
To explore the hydrophobic block length, two different series of diblock
copolymers were synthesized: one series with symmetric block lengths
and one with asymmetric block lengths. At similar cationic block lengths,
asymmetric and symmetric PTDMs promoted siRNA internalization in the
same percentages of the cell population regardless of the hydrophobic
block length; however, with 20 repeat units of cationic charge, the
asymmetric block length had greater siRNA internalization, highlighting
the nontrivial relationships between hydrophobicity and overall cationic
charge. To further probe how the hydrophobic side chains impacted
siRNA internalization, an additional series of asymmetric PTDMs was
synthesized that featured a fixed hydrophobic block length of five
repeat units that contained either dimethyl (<b>dMe</b>), methyl
phenyl (<b>MePh</b>), or diphenyl (<b>dPh</b>) side chains
and varied cationic block lengths. This series was further expanded
to incorporate hydrophobic blocks consisting of diethyl (<b>dEt</b>), diisobutyl (<b>diBu</b>), and dicyclohexyl (<b>dCy</b>) based repeat units to better define the hydrophobic window for
which our PTDMs had optimal activity. High-performance liquid chromatography
retention times quantified the relative hydrophobicities of the noncationic
building blocks. PTDMs containing the <b>MePh</b>, <b>diBu</b>, and <b>dPh</b> hydrophobic blocks were shown to have superior
siRNA internalization capabilities compared to their more and less
hydrophobic counterparts, demonstrating a critical window of relative
hydrophobicity for optimal internalization. This better understanding
of how hydrophobicity impacts PTDM-induced internalization efficiencies
will help guide the development of future delivery reagents
RNA sequencing of pancreatic adenocarcinoma tumors yields novel expression patterns associated with longâterm survival and reveals a role for ANGPTL4
Background Pancreatic adenocarcinoma patients have low survival rates due to lateâstage diagnosis and high rates of cancer recurrence even after surgical resection. It is important to understand the molecular characteristics associated with survival differences in pancreatic adenocarcinoma tumors that may inform patient care. Results RNA sequencing was performed for 51 patient tumor tissues extracted from patients undergoing surgical resection, and expression was associated with overall survival time from diagnosis. Our analysis uncovered 323 transcripts whose expression correlates with survival time in our pancreatic patient cohort. This genomic signature was validated in an independent RNAâseq dataset of 68 additional patients from the International Cancer Genome Consortium. We demonstrate that this transcriptional profile is largely independent of markers of cellular division and present a 19âtranscript predictive model built from a subset of the 323 transcripts that can distinguish patients with differing survival times across both the training and validation patient cohorts. We present evidence that a subset of the survivalâassociated transcripts is associated with resistance to gemcitabine treatment in vitro, and reveal that reduced expression of one of the survivalâassociated transcripts, Angiopoietinâlike 4, impairs growth of a gemcitabineâresistant pancreatic cancer cell line. Conclusions Gene expression patterns in pancreatic adenocarcinoma tumors can distinguish patients with differing survival outcomes after undergoing surgical resection, and the survival difference could be associated with the intrinsic gemcitabine sensitivity of primary patient tumors. Thus, these transcriptional differences may impact patient care by distinguishing patients who would benefit from a nonâgemcitabine based therapy