8 research outputs found
Protein Encapsulation via Polypeptide Complex Coacervation
Proteins
have gained increasing success as therapeutic agents;
however, challenges exist in effective and efficient delivery. In
this work, we present a simple and versatile method for encapsulating
proteins via complex coacervation with oppositely charged polypeptides,
polyÂ(l-lysine) (PLys) and polyÂ(d/l-glutamic
acid) (PGlu). A model protein system, bovine serum albumin (BSA),
was incorporated efficiently into coacervate droplets via electrostatic
interaction up to a maximum loading of one BSA per PLys/PGlu pair
and could be released under conditions of decreasing pH. Additionally,
encapsulation within complex coacervates did not alter the secondary
structure of the protein. Lastly the complex coacervate system was
shown to be biocompatible and interact well with cells in vitro. A
simple, modular system for encapsulation such as the one presented
here may be useful in a range of drug delivery applications
Determination of the Phase Diagram for Soluble and Membrane Proteins
Methods to efficiently determine the phase behavior of novel proteins have the potential to significantly benefit structural biology efforts. Here, we present protocols to determine both the solubility boundary and the supersolubility boundary for protein/precipitant systems using an evaporation-based crystallization platform. This strategy takes advantage of the well-defined rates of evaporation that occur in this platform to determine the state of the droplet at any point in time without relying on an equilibrium-based end point. The dynamic nature of this method efficiently traverses phase space along a known path, such that a solubility diagram can be mapped out for both soluble and membrane proteins while using a smaller amount of protein than what is typically used in optimization screens. Furthermore, a variation on this method can be used to decouple crystal nucleation and growth events, so fewer and larger crystals can be obtained within a given droplet. The latter protocol can be used to rescue a crystallization trial where showers of tiny crystals were observed. We validated both of the protocols to determine the phase behavior and the protocol to optimize crystal quality using the soluble proteins lysozyme and ribonuclease A as well as the membrane protein bacteriorhodopsin
Synthesis of Zwitterionic Pluronic Analogs
Novel polymer amphiphiles
with chemical structures designed as
zwitterionic analogs of Pluronic block copolymers were prepared by
controlled free radical polymerization of phosphorylcholine (PC) or
choline phosphate (CP) methacrylate monomers from a difunctional polyÂ(propylene
oxide) (PPO) macroinitiator. Well-defined, water-dispersible zwitterionic
triblock copolymers, or âzwitteronicsâ, were prepared
with PC content ranging from 5 to 47 mol percent and composition-independent
surfactant characteristics in water, which deviate from the properties
of conventional Pluronic amphiphiles. These PC-zwitteronics assembled
into nanoparticles in water, with tunable sizes and critical aggregation
concentrations (CACs) based on their hydrophilicâlipophilic
balance (HLB). Owing to the lower critical solution temperature (LCST)
miscibility of the hydrophobic PPO block in water, PC-zwitteronics
exhibited thermoreversible aqueous solubility tuned by block copolymer
composition. The chemical versatility of this approach was demonstrated
by embedding functionality, in the form of alkyne groups, directly
into the zwitterion moieties. These alkynes proved ideal for cross-linking
the zwitteronic nanoparticles and for generating nanoparticle-cross-linked
hydrogels using UV-initiated thiolâyne âclickâ
chemistry
Ternary, Tunable Polyelectrolyte Complex Fluids Driven by Complex Coacervation
Complex coacervation was achieved
by combining polyÂ(allylamine)
(PAH) or branched polyÂ(ethylenimine) (PEI) with polyÂ(acrylic acid)
(PAA) and polyÂ(<i><i>N,N</i></i>-dimethylaminoethyl
methacrylate) (PDMAEMA). We systematically investigated the effects
of stoichiometry, salt concentration, and pH. Ternary coacervates
formed over a broader range of stoichiometries compared to the base
PAA/PDMAEMA system. An enhanced resistance to salt, that is, resistance
to dissolution of the complex with added salt, was observed for ternary
coacervates. PEI-containing systems showed a considerable difference
in salt resistance at pH 6â8 due to the dramatic change in
charge density. This change was interpreted in the context of a theoretical
treatment that relies on the VoornâOverbeek model for free
energy. Coacervate stability and viscoelastic behavior were affected
by stoichiometry, salt, and pH. Ternary coacervates maintain the characteristics
and tunability of typical binary coacervates, but the choice of the
third component is important, as it significantly affects the response
and material properties
Neoadjuvant Chemotherapy Induces Expression Levels of Breast Cancer Resistance Protein That Predict Disease-Free Survival in Breast Cancer
<div><p>Three main xenobiotic efflux pumps have been implicated in modulating breast cancer chemotherapy responses. These are P-glycoprotein (Pgp), Multidrug Resistance-associated Protein 1 (MRP1), and Breast Cancer Resistance Protein (BCRP). We investigated expression of these proteins in breast cancers before and after neoadjuvant chemotherapy (NAC) to determine whether their levels define response to NAC or subsequent survival. Formalin-fixed paraffin-embedded tissues were collected representing matched pairs of core biopsy (pre-NAC) and surgical specimen (post-NAC) from 45 patients with invasive ductal carcinomas. NAC regimes were anthracyclines +/â taxanes. Immunohistochemistry was performed for Pgp, MRP1 and BCRP and expression was quantified objectively using computer-aided scoring. Pgp and MRP1 were significantly up-regulated after exposure to NAC (Wilcoxon signed-rank pâ=â0.0024 and p<0.0001), while BCRP showed more variation in response to NAC, with frequent up- (59% of cases) and down-regulation (41%) contributing to a lack of significant difference overall. Pre-NAC expression of all markers, and post-NAC expression of Pgp and MRP1 did not correlate with NAC response or with disease-free survival (DFS). Post-NAC expression of BCRP did not correlate with NAC response, but correlated significantly with DFS (Log rank pâ=â0.007), with longer DFS in patients with low post-NAC BCRP expression. In multivariate Cox regression analyses, post-NAC BCRP expression levels proved to predict DFS independently of standard prognostic factors, with high expression associated with a hazard ratio of 4.04 (95% confidence interval 1.3â12.2; pâ=â0.013). We conclude that NAC-induced expression levels of BCRP predict survival after NAC for breast cancer, while Pgp and MRP1 expression have little predictive value.</p></div
Clinical and pathological features of the patients.
<p>Clinical and pathological features of the patients.</p
Post-NAC expression of BCRP predicts disease-free survival.
<p>KaplanâMeier survival analyses for disease-free survival in patient groups with tumours with high or low post-NAC expression levels of Pgp (A), MRP1 (B) or BCRP (C). Cut-off used to dichotomise expression into low and high groups (Pgp: 90; MRP1â¶21; BCRP: 47) were determined by ROC curve analyses (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062766#pone.0062766.s001" target="_blank">Fig. S1</a>).</p
Representative staining patterns for Pgp, MRP1 and BCRP in matched breast tumour tissues pre- and post-neoadjuvant chemotherapy.
<p>Individual expression scores for each tissue shown, using a semi-automated scoring system, were: Pgp pre, 10.6; Pgp post, 117.9; MRP1 pre, 3.2; MRP1 post, 55; BCRP pre, 64.5; BCRP post, 89.7. Scale bar: 40 ”m.</p