Ensembles of synthetic polymers mimic biological fluids.

Recently a report by Ruan et al. in Nature described how relatively simple random heteropolymers can replicate the properties of biological fluids. These polymers capture the segmental-level interactions between proteins and could enhance folding of membrane proteins, improve stability, and enable DNA sequestration in a chemistry specific manner.

Self-Assembling Polypeptides in Complex Coacervation.

Intracellular compartmentalization plays a pivotal role in cellular function, with membrane-bound organelles and membrane-less biomolecular "condensates" playing key roles. These condensates, formed through liquid-liquid phase separation (LLPS), enable selective compartmentalization without the barrier of a lipid bilayer, thereby facilitating rapid formation and dissolution in response to stimuli. Intrinsically disordered proteins (IDPs) or proteins with intrinsically disordered regions (IDRs), which are often rich in charged and polar amino acid sequences, scaffold many condensates, often in conjunction with RNA.Comprehending the impact of IDP/IDR sequences on phase separation poses a challenge due to the extensive chemical diversity resulting from the myriad amino acids and post-translational modifications. To tackle this hurdle, one approach has been to investigate LLPS in simplified polypeptide systems, which offer a narrower scope within the chemical space for exploration. This strategy is supported by studies that have demonstrated how IDP function can largely be understood based on general chemical features, such as clusters or patterns of charged amino acids, rather than residue-level effects, and the ways in which these kinds of motifs give rise to an ensemble of conformations.Our laboratory has utilized complex coacervates assembled from oppositely charged polypeptides as a simplified material analogue to the complexity of liquid-liquid phase separated biological condensates. Complex coacervation is an associative LLPS that occurs due to the electrostatic complexation of oppositely charged macro-ions. This process is believed to be driven by the entropic gains resulting from the release of bound counterions and the reorganization of water upon complex formation. Apart from their direct applicability to IDPs, polypeptides also serve as excellent model polymers for investigating molecular interactions due to the wide range of available side-chain functionalities and the capacity to finely regulate their sequence, thus enabling precise control over interactions with guest molecules.Here, we discuss fundamental studies examining how charge patterning, hydrophobicity, chirality, and architecture affect the phase separation of polypeptide-based complex coacervates. These efforts have leveraged a combination of experimental and computational approaches that provide insight into molecular level interactions. We also examine how these parameters affect the ability of complex coacervates to incorporate globular proteins and viruses. These efforts couple directly with our fundamental studies into coacervate formation, as such "guest" molecules should not be considered as experiencing simple encapsulation and are instead active participants in the electrostatic assembly of coacervate materials. Interestingly, we observed trends in the incorporation of proteins and viruses into coacervates formed using different chain length polypeptides that are not well explained by simple electrostatic arguments and may be the result of more complex interactions between globular and polymeric species. Additionally, we describe experimental evidence supporting the potential for complex coacervates to improve the thermal stability of embedded biomolecules, such as viral vaccines.Ultimately, peptide-based coacervates have the potential to help unravel the physics behind biological condensates, while paving the way for innovative methods in compartmentalization, purification, and biomolecule stabilization. These advancements could have implications spanning medicine to biocatalysis.

Design Rules for the Sequestration of Viruses into Polypeptide Complex Coacervates.

Encapsulation is a strategy that has been used to facilitate the delivery and increase the stability of proteins and viruses. Here, we investigate the encapsulation of viruses via complex coacervation, which is a liquid-liquid phase separation resulting from the complexation of oppositely charged polymers. In particular, we utilized polypeptide-based coacervates and explored the effects of peptide chemistry, chain length, charge patterning, and hydrophobicity to better understand the effects of the coacervating polypeptides on virus incorporation. Our study utilized two nonenveloped viruses, porcine parvovirus (PPV) and human rhinovirus (HRV). PPV has a higher charge density than HRV, and they both appear to be relatively hydrophobic. These viruses were compared to characterize how the charge, hydrophobicity, and patterning of chemistry on the surface of the virus capsid affects encapsulation. Consistent with the electrostatic nature of complex coacervation, our results suggest that electrostatic effects associated with the net charge of both the virus and polypeptide dominated the potential for incorporating the virus into a coacervate, with clustering of charges also playing a significant role. Additionally, the hydrophobicity of a virus appears to determine the degree to which increasing the hydrophobicity of the coacervating peptides can enhance virus uptake. Nonintuitive trends in uptake were observed with regard to both charge patterning and polypeptide chain length, with these parameters having a significant effect on the range of coacervate compositions over which virus incorporation was observed. These results provide insights into biophysical mechanisms, where sequence effects can control the uptake of proteins or viruses into biological condensates and provide insights for use in formulation strategies.

Conjugated Polyelectrolyte-Based Complex Fluids as Aqueous Exciton Transport Networks.

The ability to assemble artificial systems that mimic aspects of natural light-harvesting functions is fascinating and attractive for materials design. Given the complexity of such a system, a simple design pathway is desirable. Here, we argue that associative phase separation of oppositely charged conjugated polyelectrolytes (CPEs) can provide such a path in an environmentally benign medium: water. We find that complexation between an exciton-donor and acceptor CPE leads to formation of a complex fluid. We interrogate exciton transfer from the donor to the acceptor CPE within the complex fluid and find that transfer is highly efficient. We also find that excess molecular ions can tune the modulus of the inter-CPE complex fluid. Even at high ion concentrations, CPEs remain complexed with significantly delocalized electronic wavefunctions. Our work lays the rational foundation for complex, tunable aqueous light-harvesting systems via the intrinsic thermodynamics of associative phase separation.

The utility of c-Met as a diagnostic tissue biomarker in primary colorectal cancer.

The transmembrane protein, c-Met, is thought to be overexpressed and activated in colorectal cancer (CRC). This study explored its potential as a diagnostic tissue biomarker for CRC in a large human CRC tissue collection obtained from a randomized clinical trial. Tissue microarrays of matched normal colorectal epithelium and primary cancer were prepared from specimens obtained from 280 patients recruited to the MRC CLASICC trial (ISRCTN 74883561) and interrogated using immunohistochemistry for c-Met expression. The distribution and intensity of immunopositivity was graded using a validated, semi-quantifiable score, and differences in median scores analysed using the Wilcoxon signed-rank test. A receiver operating characteristic (ROC) curve was plotted to measure the diagnostic accuracy of c-Met as a biomarker in CRC. Epithelial cell membrane expression of c-Met differed significantly between CRC and normal colorectal tissue: median 12.00 (Interquartile range (IQR) 6-15) versus median 6.00 (IQR 2.70-12.00) respectively (P = <.0001). ROC-AUC analysis of c-Met expression yielded a CRC diagnostic probability of 0.66 (95% CI: 0.61 to 0.70; P < .0001). A score of ≥14.50 showed high specificity at 85.32% (95% CI 80.33%-89.45%) but sensitivity of only 30.92% (CI 25.37%-36.90%). Thus c-Met is consistently overexpressed in human CRC as compared to normal colorectal epithelium tissue. c-Met expression may have a role in diagnosis and prognostication if combined with other biomarkers.

Luminal Bioavailability of Orally Administered ω-3 PUFAs in the Distal Small Intestine, and Associated Changes to the Ileal Microbiome, in Humans with a Temporary Ileostomy.

BACKGROUND: Oral administration of purified omega-3 (ω-3) PUFAs is associated with changes to the fecal microbiome. However, it is not known whether this effect is associated with increased PUFA concentrations in the gut. OBJECTIVES: We investigated the luminal bioavailability of oral ω-3 PUFAs (daily dose 1 g EPA and 1g DHA free fatty acid equivalents as triglycerides in soft-gel capsules, twice daily) and changes to the gut microbiome, in the ileum. METHODS: Ileostomy fluid (IF) and blood were obtained at baseline, after first capsule dosing (median 2 h), and at a similar time after final dosing on day 28, in 11 individuals (median age 63 y) with a temporary ileostomy. Fatty acids were measured by LC-tandem MS. The ileal microbiome was characterized by 16S rRNA PCR and Illumina sequencing. RESULTS: There was a mean 6.0 ± 9.8-fold and 6.6 ± 9.6-fold increase in ileal EPA and DHA concentrations (primary outcome), respectively, at 28 d, which was associated with increased RBC ω-3 PUFA content (P ≤ 0.05). The first oral dose did not increase the ileal ω-3 PUFA concentration except in 4 individuals, who displayed high luminal EPA and DHA concentrations, which reduced to concentrations similar to the overall study population at day 28, suggesting physiological adaptation. Bacteroides, Clostridium, and Streptococcus were abundant bacterial genera in the ileum. Ileal microbiome variability over time and between individuals was large, with no consistent change associated with acute ω-3 PUFA dosing. However, high concentrations of EPA and DHA in IF on day 28 were associated with higher abundance of Bacteroides (r2 > 0.86, P < 0.05) and reduced abundance of other genera, including Actinomyces (r2 > 0.94, P < 0.05). CONCLUSIONS: Oral administration of ω-3 PUFAs leads to increased luminal ω-3 PUFA concentrations and changes to the microbiome, in the ileum of individuals with a temporary ileostomy. This study is registered on the ISRCTN registry as ISRCTN14530452.

Protein Encapsulation Using Complex Coacervates: What Nature Has to Teach Us.

Protein encapsulation is a growing area of interest, particularly in the fields of food science and medicine. The sequestration of protein cargoes is achieved using a variety of methods, each with benefits and drawbacks. One of the most significant challenges associated with protein encapsulation is achieving high loading while maintaining protein viability. This difficulty is exacerbated because many encapsulant systems require the use of organic solvents. By contrast, nature has optimized strategies to compartmentalize and protect proteins inside the cell-a purely aqueous environment. Although the mechanisms whereby aspects of the cytosol is able to stabilize proteins are unknown, the crowded nature of many newly discovered, liquid phase separated "membraneless organelles" that achieve protein compartmentalization suggests that the material environment surrounding the protein may be critical in determining stability. Here, encapsulation strategies based on liquid-liquid phase separation, and complex coacervation in particular, which has many of the key features of the cytoplasm as a material, are reviewed. The literature on protein encapsulation via coacervation is also reviewed and the parameters relevant to creating protein-containing coacervate formulations are discussed. Additionally, potential opportunities associated with the creation of tailored materials to better facilitate protein encapsulation and stabilization are highlighted.

Recent progress in the science of complex coacervation.

Complex coacervation is an associative, liquid-liquid phase separation that can occur in solutions of oppositely-charged macromolecular species, such as proteins, polymers, and colloids. This process results in a coacervate phase, which is a dense mix of the oppositely-charged components, and a supernatant phase, which is primarily devoid of these same species. First observed almost a century ago, coacervates have since found relevance in a wide range of applications; they are used in personal care and food products, cutting edge biotechnology, and as a motif for materials design and self-assembly. There has recently been a renaissance in our understanding of this important class of material phenomena, bringing the science of coacervation to the forefront of polymer and colloid science, biophysics, and industrial materials design. In this review, we describe the emergence of a number of these new research directions, specifically in the context of polymer-polymer complex coacervates, which are inspired by a number of key physical and chemical insights and driven by a diverse range of experimental, theoretical, and computational approaches.

The benefit of poor mixing: kinetics of coacervation.

Complex coacervation has become a prominent area of research in the fields of food science, personal care, drug stabilization, and more. However, little has been reported on the kinetics of assembly of coacervation itself. Here, we describe a simple, low-cost way of looking at the kinetics of coacervation by creating poorly mixed samples. In particular, we examine how polymer chain length, the patterning and symmetry of charges on the oppositely charged polyelectrolytes, and the presence of salt and a zwitterionic buffer affect the kinetics of complex coacervation. Our results suggest an interesting relationship between the time for equilibration and the order of addition of polymers with asymmetric patterns of charge. Furthermore, we demonstrated that increasing polymer chain length resulted in a non-monotonic trend in the sample equilibration times as a result of opposing factors such as excluded volume and diffusion. We also observed differences in the rate of sample equilibration based on the presence of a neutral, zwitterionic buffer, as well as the presence and identity of added salt, consistent with previous reports of salt-specific effects on the rheology of complex coacervates. While not a replacement for more advanced characterization strategies, this turbidity-based method could serve as a screening tool to identify interesting and unique phenomena for further study.

Recent Advances in Encapsulation, Protection, and Oral Delivery of Bioactive Proteins and Peptides using Colloidal Systems.

There are many areas in medicine and industry where it would be advantageous to orally deliver bioactive proteins and peptides (BPPs), including ACE inhibitors, antimicrobials, antioxidants, hormones, enzymes, and vaccines. A major challenge in this area is that many BPPs degrade during storage of the product or during passage through the human gut, thereby losing their activity. Moreover, many BPPs have undesirable taste profiles (such as bitterness or astringency), which makes them unpleasant to consume. These challenges can often be overcome by encapsulating them within colloidal particles that protect them from any adverse conditions in their environment, but then release them at the desired site-of-action, which may be inside the gut or body. This article begins with a discussion of BPP characteristics and the hurdles involved in their delivery. It then highlights the characteristics of colloidal particles that can be manipulated to create effective BPP-delivery systems, including particle composition, size, and interfacial properties. The factors impacting the functional performance of colloidal delivery systems are then highlighted, including their loading capacity, encapsulation efficiency, protective properties, retention/release properties, and stability. Different kinds of colloidal delivery systems suitable for encapsulation of BPPs are then reviewed, such as microemulsions, emulsions, solid lipid particles, liposomes, and microgels. Finally, some examples of the use of colloidal delivery systems for delivery of specific BPPs are given, including hormones, enzymes, vaccines, antimicrobials, and ACE inhibitors. An emphasis is on the development of food-grade colloidal delivery systems, which could be used in functional or medical food applications. The knowledge presented should facilitate the design of more effective vehicles for the oral delivery of bioactive proteins and peptides.

Electrospinning Nanofibers from Chitosan/Hyaluronic Acid Complex Coacervates.

Electrospun biopolyelectrolyte nanofibers hold potential for use in a range of biomedical applications, but eliminating toxic chemicals involved in their production remains a key challenge. In this study, we successfully electrospun nanofibers from an aqueous complex coacervate solution composed of chitosan and hyaluronic acid. Experimentally, we investigated the effect of added salt and electrospinning apparatus parameters, such as how applied voltage affected fiber formation. We also studied how the addition of alcohol cosolvents affected the properties of the coacervate solution and the resulting nanofibers. Overall, we observed a trade-off in how the addition of salt and alcohol affected the phase behavior and rheology of the coacervates and, consequently, the size of the resulting fibers. While salt served to weaken electrostatic associations within the coacervate and decrease the precursor solution viscosity, the addition of alcohol lowered the dielectric constant of the system and strengthened these interactions. We hypothesize that the optimized concentration of alcohol accelerated the solvent evaporation during the electrospinning process to yield desirable nanofiber morphology. The smallest average nanofiber diameter was determined to be 115 ± 30 nm when coacervate samples were electrospun using an aqueous solvent containing 3 wt % ethanol and an applied voltage of 24 kV. These results demonstrate a potentially scalable strategy to manufacture electrospun nanofibers from biopolymer complex coacervates that eliminate the need for toxic solvents and could enable the use of these materials across a range of biomedical applications.

Correction: Design rules for encapsulating proteins into complex coacervates.

Correction for 'Design rules for encapsulating proteins into complex coacervates' by Whitney C. Blocher McTigue et al., Soft Matter, 2019, 15, 3089-3103.

Design rules for encapsulating proteins into complex coacervates.

We investigated the encapsulation of the model proteins bovine serum albumin (BSA), human hemoglobin (Hb), and hen egg white lysozyme (HEWL) into two-polymer complex coacervates as a function of polymer and solution conditions. Electrostatic parameters such as pH, protein net charge, salt concentration, and polymer charge density can be used to modulate protein uptake. While the use of a two-polymer coacervation system enables the encapsulation of weakly charged proteins that would otherwise require chemical modification to facilitate electrostatic complexation, we observed significantly higher uptake for proteins whose structure includes a cluster of like-charged residues on the protein surface. In addition to enhancing uptake, the presence of a charge patch also increased the sensitivity of the system to modulation by other parameters, including the length of the complexing polymers. Lastly, our results suggest that the distribution of charge on a protein surface may lead to different scaling behaviour for both the encapsulation efficiency and partition coefficient as a function of the absolute difference between the protein isoelectric point and the solution pH. These results provide insight into possible biophysical mechanisms whereby cells can control the uptake of proteins into coacervate-like granules, and suggest future utility in applications ranging from medicine and sensing to remediation and biocatalysis.

A randomised trial of the effect of omega-3 polyunsaturated fatty acid supplements on the human intestinal microbiota.

OBJECTIVE: Omega-3 polyunsaturated fatty acids (PUFAs) have anticolorectal cancer (CRC) activity. The intestinal microbiota has been implicated in colorectal carcinogenesis. Dietary omega-3 PUFAs alter the mouse intestinal microbiome compatible with antineoplastic activity. Therefore, we investigated the effect of omega-3 PUFA supplements on the faecal microbiome in middle-aged, healthy volunteers (n=22). DESIGN: A randomised, open-label, cross-over trial of 8 weeks' treatment with 4 g mixed eicosapentaenoic acid/docosahexaenoic acid in two formulations (soft-gel capsules and Smartfish drinks), separated by a 12-week 'washout' period. Faecal samples were collected at five time-points for microbiome analysis by 16S ribosomal RNA PCR and Illumina MiSeq sequencing. Red blood cell (RBC) fatty acid analysis was performed by liquid chromatography tandem mass spectrometry. RESULTS: Both omega-3 PUFA formulations induced similar changes in RBC fatty acid content, except that drinks were associated with a larger, and more prolonged, decrease in omega-6 PUFA arachidonic acid than the capsule intervention (p=0.02). There were no significant changes in α or ß diversity, or phyla composition, associated with omega-3 PUFA supplementation. However, a reversible increased abundance of several genera, including Bifidobacterium, Roseburia and Lactobacillus was observed with one or both omega-3 PUFA interventions. Microbiome changes did not correlate with RBC omega-3 PUFA incorporation or development of omega-3 PUFA-induced diarrhoea. There were no treatment order effects. CONCLUSION: Omega-3 PUFA supplementation induces a reversible increase in several short-chain fatty acid-producing bacteria, independently of the method of administration. There is no simple relationship between the intestinal microbiome and systemic omega-3 PUFA exposure. TRIAL REGISTRATION NUMBER: ISRCTN18662143.

Partitioning and Enhanced Self-Assembly of Actin in Polypeptide Coacervates.

Biomolecules exist and function in cellular microenvironments that control their spatial organization, local concentration, and biochemical reactivity. Due to the complexity of native cytoplasm, the development of artificial bioreactors and cellular mimics to compartmentalize, concentrate, and control the local physico-chemical properties is of great interest. Here, we employ self-assembling polypeptide coacervates to explore the partitioning of the ubiquitous cytoskeletal protein actin into liquid polymer-rich droplets. We find that actin spontaneously partitions into coacervate droplets and is enriched by up to ∼30-fold. Actin polymerizes into micrometer-long filaments and, in contrast to the globular protein BSA, these filaments localize predominately to the droplet periphery. We observe up to a 50-fold enhancement in the actin filament assembly rate inside coacervate droplets, consistent with the enrichment of actin within the coacervate phase. Together these results suggest that coacervates can serve as a versatile platform in which to localize and enrich biomolecules to study their reactivity in physiological environments.

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 poly(propylene oxide) (PPO) macroinitiator. Well-defined, water-dispersible zwitterionic triblock copolymers, or "zwitteronics", 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 hydrophilic-lipophilic 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 thiol-yne "click" chemistry.

Rheological characterization of liquid-to-solid transitions in bulk polyelectrolyte complexes.

Polyelectrolyte complexation has long been known to result in both liquid and solid complexes. However, the exact nature of the liquid-to-solid transition remains an open question. We have used rheology to explain this phenomenon for the model system of poly(4-styrenesulfonic acid, sodium salt) (PSS) and poly(diallyldimethyl ammonium chloride) (PDADMAC) in the presence of potassium bromide (KBr). The use of a time-salt superposition allows for a detailed analysis of changes in the linear viscoelastic response for both liquid complex coacervates and solid polyelectrolyte complexes as a function of salt concentration, and facilitates unambiguous determination of the mechanism for this phase transition. Decreasing salt concentration, and the commensurate decrease in the water content of PSS/PDADMAC/KBr complexes is shown to lead to the formation of a physical gel due to the development of a network with trapped electrostatic crosslinks that percolates the sample at a critical salt concentration.

The effect of comb architecture on complex coacervation.

Complex coacervation is a widely utilized technique for effecting phase separation, though predictive understanding of molecular-level details remains underdeveloped. Here, we couple coarse-grained Monte Carlo simulations with experimental efforts using a polypeptide-based model system to investigate how a comb-like architecture affects complex coacervation and coacervate stability. Specifically, the phase separation behavior of linear polycation-linear polyanion pairs was compared to that of comb polycation-linear polyanion and comb polycation-comb polyanion pairs. The comb architecture was found to mitigate cooperative interactions between oppositely charged polymers, as no discernible phase separation was observed for comb-comb pairs and complex coacervation of linear-linear pairs yielded stable coacervates at higher salt concentration than linear-comb pairs. This behavior was attributed to differences in counterion release by linear vs. comb polymers during polyeletrolyte complexation. Additionally, the comb polycation formed coacervates with both stereoregular poly(l-glutamate) and racemic poly(d,l-glutamate), whereas the linear polycation formed coacervates only with the racemic polyanion. In contrast, solid precipitates were obtained from mixtures of stereoregular poly(l-lysine) and poly(l-glutamate). Moreover, the formation of coacervates from cationic comb polymers incorporating up to ∼90% pendant zwitterionic groups demonstrated the potential for inclusion of comonomers to modulate the hydrophilicity and/or other properties of a coacervate-forming polymer. These results provide the first detailed investigation into the role of polymer architecture on complex coacervation using a chemically and architecturally well-defined model system, and highlight the need for additional research on this topic.

Are there biological differences between screen-detected and interval colorectal cancers in the English Bowel Cancer Screening Programme?

BACKGROUND: We measured biomarkers of tumour growth and vascularity in interval and screen-detected colorectal cancers (CRCs) in the English Bowel Cancer Screening Programme in order to determine whether rapid tumour growth might contribute to interval CRC (a CRC diagnosed between a negative guaiac stool test and the next scheduled screening episode). METHODS: Formalin-fixed, paraffin-embedded sections from 71 CRCs (screen-detected 43, interval 28) underwent immunohistochemistry for CD31 and Ki-67, in order to measure the microvessel density (MVD) and proliferation index (PI), respectively, as well as microsatellite instability (MSI) testing. RESULTS: Interval CRCs were larger (P=0.02) and were more likely to exhibit venous invasion (P=0.005) than screen-detected tumours. There was no significant difference in MVD or PI between interval and screen-detected CRCs. More interval CRCs displayed MSI-high (14%) compared with screen-detected tumours (5%). A significantly (P=0.005) higher proportion (51%) of screen-detected CRC resection specimens contained at least one polyp compared with interval CRC (18%) resections. CONCLUSIONS: We found no evidence of biological differences between interval and screen-detected CRCs, consistent with the low sensitivity of guaiac stool testing as the main driver of interval CRC. The contribution of synchronous adenomas to occult blood loss for screening requires further investigation.

Chemotherapy induces Notch1-dependent MRP1 up-regulation, inhibition of which sensitizes breast cancer cells to chemotherapy.

BACKGROUND: Multi-drug Resistance associated Protein-1 (MRP1) can export chemotherapeutics from cancer cells and is implicated in chemoresistance, particularly as is it known to be up-regulated by chemotherapeutics. Our aims in this study were to determine whether activation of Notch signalling is responsible for chemotherapy-induced MRP1 expression Notch in breast cancers, and whether this pathway can be manipulated with an inhibitor of Notch activity. METHODS: MRP1 and Notch1 were investigated in 29 patients treated with neoadjuvant chemotherapy (NAC) for breast cancer, using immunohistochemistry on matched biopsy (pre-NAC) and surgical samples (post-NAC). Breast epithelial cell cultures (T47D, HB2) were treated with doxorubicin in the presence and absence of functional Notch1, and qPCR, siRNA, Western blots, ELISAs and flow-cytometry were used to establish interactions. RESULTS: In clinical samples, Notch1 was activated by neoadjuvant chemotherapy (Wilcoxon signed-rank p < 0.0001) and this correlated with induction of MRP1 expression (rho = 0.6 p = 0.0008). In breast cell lines, doxorubicin induced MRP1 expression and function (non-linear regression p < 0.004). In the breast cancer line T47D, doxorubicin activated Notch1 and, critically, inhibition of Notch1 activation with the γ-secretase inhibitor DAPT abolished the doxorubicin-induced increase in MRP1 expression and function (t-test p < 0.05), resulting in enhanced cellular retention of doxorubicin and increased doxorubicin-induced apoptosis (t-test p = 0.0002). In HB2 cells, an immortal but non-cancer derived breast cell line, Notch1-independent MRP1 induction was noted and DAPT did not enhance doxorubicin-induced apoptosis. CONCLUSIONS: Notch inhibitors may have potential in sensitizing breast cancer cells to chemotherapeutics and therefore in tackling chemoresistance.