The effect of polymer end-group on the formation of styrene - maleic acid lipid particles (SMALPs).

A series of block copolymers comprising styrene and maleic acid (SMA) has been prepared using RAFT polymerisation. RAFT often results in a large hydrophobic alkylthiocarbonylthio end group and this work examines its effect on the solution behaviour of the copolymers. SMA variants with, and without, this end group were synthesised and their behaviour compared with a commercially-available random copolymer of similar molecular weight. Dynamic light scattering and surface tension measurements found the RAFT-copolymers preferentially self-assembled into higher-order aggregates in aqueous solution. Small angle neutron scattering using deuterated styrene varients add support to the accepted model that these agreggates comprise a solvent-protected styrenic core with an acid-rich shell. Replacing the hydrophobic RAFT end group with a more hydrophilic nitrile caused differences in the resulting surface activity, attributed to the ability of the adjoining styrene homoblock to drive aggregation. Each of the copolymers formed SMALP nanodiscs with DMPC lipids, which were found to encapsulate a model membrane protein, gramicidin. However, end group variation affected solubilisition of DPPC, a lipid with a higher phase transition temperature. When using RAFT-copolymers terminated with a hydrophobic group, swelling of the bilayer and greater penetration of the homoblock into the nanodisc core occurred with increasing homoblock length. Conversely, commercial and nitrile-terminated RAFT-copolymers produced nanodisc sizes that stayed constant, instead indicating interaction at the edge of the lipid patch. The results highlight how even minor changes to the copolymer can modify the amphiphilic balance between regions, knowledge useful towards optimising copolymer structure to enhance and control nanodisc formation.

Bayesian methods provide a practical real-world evidence framework for evaluating the impact of changes in radiotherapy.

PURPOSE: Retrospective studies have identified a link between the average set-up error of lung cancer patients treated with image-guided radiotherapy (IGRT) and survival. The IGRT protocol was subsequently changed to reduce the action threshold. In this study, we use a Bayesian approach to evaluate the clinical impact of this change to practice using routine 'real-world' patient data. METHODS AND MATERIALS: Two cohorts of NSCLC patients treated with IGRT were compared: pre-protocol change (N = 780, 5 mm action threshold) and post-protocol change (N = 411, 2 mm action threshold). Survival models were fitted to each cohort and changes in the hazard ratios (HR) associated with residual set-up errors was assessed. The influence of using an uninformative and a skeptical prior in the model was investigated. RESULTS: Following the reduction of the action threshold, the HR for residual set-up error towards the heart was reduced by up to 10%. Median patient survival increased for patients with set-up errors towards the heart, and remained similar for patients with set-up errors away from the heart. Depending on the prior used, a residual hazard ratio may remain. CONCLUSIONS: Our analysis found a reduced hazard of death and increased survival for patients with residual set-up errors towards versus away from the heart post-protocol change. This study demonstrates the value of a Bayesian approach in the assessment of technical changes in radiotherapy practice and supports the consideration of adopting this approach in further prospective evaluations of changes to clinical practice.

The interaction of styrene maleic acid copolymers with phospholipids in Langmuir monolayers, vesicles and nanodiscs; a structural study.

HYPOTHESIS: Self-assembly of amphipathic styrene maleic acid copolymers with phospholipids in aqueous solution results in the formation of 'nanodiscs' containing a planar segment of phospholipid bilayer encapsulated by a polymer belt. Recently, studies have reported that lipids rapidly exchange between both nanodiscs in solution and external sources of lipids. Outstanding questions remain regarding details of polymer-lipid interactions, factors influencing lipid exchange and structural effects of such exchange processes. Here, the dynamic behaviour of nanodiscs is investigated, specifically the role of membrane charge and polymer chemistry. EXPERIMENTS: Two model systems are investigated: fluorescently labelled phospholipid vesicles, and Langmuir monolayers of phospholipids. Using fluorescence spectroscopy and time-resolved neutron reflectometry, the membrane potential, monolayer structure and composition are monitored with respect to time upon polymer and nanodisc interactions. FINDINGS: In the presence of external lipids, polymer chains embed throughout lipid membranes, the extent of which is governed by the net membrane charge. Nanodiscs stabilised by three different polymers will all exchange lipids and polymer with monolayers to differing extents, related to the properties of the stabilising polymer belt. These results demonstrate the dynamic nature of nanodiscs which interact with the local environment and are likely to deposit both lipids and polymer at all stages of use.

Causal relation between heart irradiation and survival of lung cancer patients after radiotherapy.

INTRODUCTION: In a recent study, setup uncertainties in the direction of the heart were shown to impact the overall survival of non-small cell lung cancer (NSCLC) patients after radiotherapy, indicating the causal effect between heart irradiation and survival. The current study aims to externally evaluate this observation within a patient cohort treated using daily IGRT. METHOD: NSCLC patients with locally-advanced disease and daily CBCT were included. For all treatment fractions, the distance between the isocenter and the heart was evaluated based on the clinical setup registrations. The variation in heart position between planning and treatment (DeltaDistance) was estimated from these registrations. The possible impact of DeltaDistance on survival was analysed by a multivariable Cox model of overall survival, allowing for a time-dependent impact of DeltaDistance to allow for toxicity latency. RESULTS: Daily CBCT information was available for 489 patients at Odense University Hospital. The primary Cox model contained GTV volume, patient age, performance status, and DeltaDistance. DeltaDistance significantly impacted overall survival approximately 50 months after radiotherapy. Subanalyses indicated that the observed effect is mainly present among the patients with the least clinical risk factors. CONCLUSION: Our results confirm the impact of setup variations in the direction of the heart on the survival of NSCLC patients, even within a cohort using daily CBCT setup guidance. This result indicates a causal effect between heart irradiation and survival. It will be challenging to reduce the setup uncertainty even further; thus, increased focus on dose constraints on the heart seems warranted.

A preclinical radiotherapy dosimetry audit using a realistic 3D printed murine phantom.

Preclinical radiation research lacks standardized dosimetry procedures that provide traceability to a primary standard. Consequently, ensuring accuracy and reproducibility between studies is challenging. Using 3D printed murine phantoms we undertook a dosimetry audit of Xstrahl Small Animal Radiation Research Platforms (SARRPs) installed at 7 UK centres. The geometrically realistic phantom accommodated alanine pellets and Gafchromic EBT3 film for simultaneous measurement of the dose delivered and the dose distribution within a 2D plane, respectively. Two irradiation scenarios were developed: (1) a 10 × 10 mm2 static field targeting the pelvis, and (2) a 5 × 5 mm2 90° arc targeting the brain. For static fields, the absolute difference between the planned dose and alanine measurement across all centres was 4.1 ± 4.3% (mean ± standard deviation), with an overall range of - 2.3 to 10.5%. For arc fields, the difference was - 1.2% ± 6.1%, with a range of - 13.1 to 7.7%. EBT3 dose measurements were greater than alanine by 2.0 ± 2.5% and 3.5 ± 6.0% (mean ± standard deviation) for the static and arc fields, respectively. 2D dose distributions showed discrepancies to the planned dose at the field edges. The audit demonstrates that further work on preclinical radiotherapy quality assurance processes is merited.

Membrane extraction with styrene-maleic acid copolymer results in insulin receptor autophosphorylation in the absence of ligand.

Extraction of integral membrane proteins with poly(styrene-co-maleic acid) provides a promising alternative to detergent extraction. A major advantage of extraction using copolymers rather than detergent is the retention of the lipid bilayer around the proteins. Here we report the first functional investigation of the mammalian insulin receptor which was extracted from cell membranes using poly(styrene-co-maleic acid). We found that the copolymer efficiently extracted the insulin receptor from 3T3L1 fibroblast membranes. Surprisingly, activation of the insulin receptor and proximal downstream signalling was detected upon copolymer extraction even in the absence of insulin stimulation. Insulin receptor and IRS1 phosphorylations were above levels measured in the control extracts made with detergents. However, more distal signalling events in the insulin signalling cascade, such as the phosphorylation of Akt were not observed. Following copolymer extraction, in vitro addition of insulin had no further effect on insulin receptor or IRS1 phosphorylation. Therefore, under our experimental conditions, the insulin receptor is not functionally responsive to insulin. This study is the first to investigate receptor tyrosine kinases extracted from mammalian cells using a styrene-maleic acid copolymer and highlights the importance of thorough functional characterisation when using this method of protein extraction.

Fluorescent styrene maleic acid copolymers to facilitate membrane protein studies in lipid nanodiscs.

Fluorescently-labelled variants of poly(styrene-co-maleic acid), SMA, have been synthesised by RAFT copolymerisation. We show that low ratios of vinyl fluorophores, analogous to styrene, can be successfully incorporated during polymerisation without detriment to nanodisc formation upon interaction with lipids. These novel copolymers are capable of encapuslating lipids and the model membrane protein, gramicidin, and hence have the potential to be applied in fluorescence-based biological studies. To demonstrate this, energy transfer is used to probe polymer-protein interactions in nanodiscs. The copolymers may also be used to monitor nanodisc self assembly by exploiting aggregation-caused-quenching (ACQ).

Ab initio reconstruction of small angle scattering data for membrane proteins in copolymer nanodiscs.

Background: Small angle scattering techniques are beginning to be more widely utilised for structural analysis of biological systems. However, applying these techniques to study membrane proteins still remains problematic, due to sample preparation requirements and analysis of the resulting data. The development of styrene-maleic acid co-polymers (SMA) to extract membrane proteins into nanodiscs for further study provides a suitable environment for structural analysis. Methods: We use small angle neutron scattering (SANS) with three different contrasts to determine structural information for two different polymer nanodisc-incorporated proteins, Outer membrane protein F (OmpF) and gramicidin. Ab initio modelling was applied to generate protein/lipid structures from the SANS data. Other complementary structural methodologies, such as DLS, CD and TEM were compared alongside this data with known protein crystal structures. Results: A single-phase model was constructed for gramicidin-containing nanodiscs, which showed dimer formation in the centre of the nanodisc. For OmpF-nanodiscs we were able to construct a multi-phase model, providing structural information on the protein/lipid and polymer components of the sample. Conclusions: Polymer-nanodiscs can provide a suitable platform to investigate certain membrane proteins using SANS, alongside other structural methodologies. However, differences between the published crystal structure and OmpF-nanodiscs were observed, suggesting the nanodisc structure could be altering the folding of the protein. General significance: Small angle scattering techniques can provide structural information on the protein and polymer nanodisc without requiring crystallisation of the protein. Additional complementary techniques, such as ab initio modelling, can generate alternative models both the protein and nanodisc system.

Development of Methodology to Investigate the Surface SMALPome of Mammalian Cells.

Extraction of membrane proteins from biological membranes has traditionally involved detergents. In the past decade, a new technique has been developed, which uses styrene maleic acid (SMA) copolymers to extract membrane proteins into nanodiscs without the requirement of detergents. SMA nanodiscs are compatible with analytical techniques, such as small-angle scattering, NMR spectroscopy, and DLS, and are therefore an attractive medium for membrane protein characterization. While mass spectrometry has also been reported as a technique compatible with copolymer extraction, most studies have focused on lipidomics, which involves solvent extraction of lipids from nanodiscs prior to mass-spectrometry analysis. In this study, mass spectrometry proteomics was used to investigate whether there are qualitative or quantitative differences in the mammalian plasma membrane proteins extracted with SMA compared to a detergent control. For this, cell surface proteins of 3T3L1 fibroblasts were biotinylated and extracted using either SMA or detergent. Following affinity pull-down of biotinylated proteins with NeutrAvidin beads, samples were analyzed by nanoLC-MS. Here, we report for the first time, a global proteomics protocol for detection of a mammalian cell "SMALPome", membrane proteins incorporated into SMA nanodiscs. Removal of SMA from samples prior to processing of samples for mass spectrometry was a crucial step in the protocol. The reported surface SMALPome of 3T3L1 fibroblasts consists of 205 integral membrane proteins. It is apparent that the detergent extraction method used is, in general, quantitatively more efficient at extracting proteins from the plasma membrane than SMA extraction. However, samples prepared following detergent extraction contained a greater proportion of proteins that were considered to be "non-specific" than in samples prepared from SMA extracts. Tantalizingly, it was also observed that proteins detected uniquely or highly preferentially in pull-downs from SMA extracts were primarily multi-spanning membrane proteins. These observations hint at qualitative differences between SMA and detergent extraction that are worthy of further investigation.

Reliability and prognostic value of radiomic features are highly dependent on choice of feature extraction platform.

OBJECTIVE: To investigate the effects of Image Biomarker Standardisation Initiative (IBSI) compliance, harmonisation of calculation settings and platform version on the statistical reliability of radiomic features and their corresponding ability to predict clinical outcome. METHODS: The statistical reliability of radiomic features was assessed retrospectively in three clinical datasets (patient numbers: 108 head and neck cancer, 37 small-cell lung cancer, 47 non-small-cell lung cancer). Features were calculated using four platforms (PyRadiomics, LIFEx, CERR and IBEX). PyRadiomics, LIFEx and CERR are IBSI-compliant, whereas IBEX is not. The effects of IBSI compliance, user-defined calculation settings and platform version were assessed by calculating intraclass correlation coefficients and confidence intervals. The influence of platform choice on the relationship between radiomic biomarkers and survival was evaluated using univariable cox regression in the largest dataset. RESULTS: The reliability of radiomic features calculated by the different software platforms was only excellent (ICC > 0.9) for 4/17 radiomic features when comparing all four platforms. Reliability improved to ICC > 0.9 for 15/17 radiomic features when analysis was restricted to the three IBSI-compliant platforms. Failure to harmonise calculation settings resulted in poor reliability, even across the IBSI-compliant platforms. Software platform version also had a marked effect on feature reliability in CERR and LIFEx. Features identified as having significant relationship to survival varied between platforms, as did the direction of hazard ratios. CONCLUSION: IBSI compliance, user-defined calculation settings and choice of platform version all influence the statistical reliability and corresponding performance of prognostic models in radiomics. KEY POINTS: • Reliability of radiomic features varies between feature calculation platforms and with choice of software version. • Image Biomarker Standardisation Initiative (IBSI) compliance improves reliability of radiomic features across platforms, but only when calculation settings are harmonised. • IBSI compliance, user-defined calculation settings and choice of platform version collectively affect the prognostic value of features.

Radiomics as a personalized medicine tool in lung cancer: Separating the hope from the hype.

Radiomics has become a popular image analysis method in the last few years. Its key hypothesis is that medical images harbor biological, prognostic and predictive information that is not revealed upon visual inspection. In contrast to previous work with a priori defined imaging biomarkers, radiomics instead calculates image features at scale and uses statistical methods to identify those most strongly associated to outcome. This builds on years of research into computer aided diagnosis and pattern recognition. While the potential of radiomics to aid personalized medicine is widely recognized, several technical limitations exist which hinder biomarker translation. Aspects of the radiomic workflow lack repeatability or reproducibility under particular circumstances, which is a key requirement for the translation of imaging biomarkers into clinical practice. One of the most commonly studied uses of radiomics is for personalized medicine applications in Non-Small Cell Lung Cancer (NSCLC). In this review, we summarize reported methodological limitations in CT based radiomic analyses together with suggested solutions. We then evaluate the current NSCLC radiomics literature to assess the risk associated with accepting the published conclusions with respect to these limitations. We review different complementary scoring systems and initiatives that can be used to critically appraise data from radiomics studies. Wider awareness should improve the quality of ongoing and future radiomics studies and advance their potential as clinically relevant biomarkers for personalized medicine in patients with NSCLC.

Preclinical dosimetry: exploring the use of small animal phantoms.

Preclinical radiotherapy studies using small animals are an indispensable step in the pathway from in vitro experiments to clinical implementation. As radiotherapy techniques advance in the clinic, it is important that preclinical models evolve to keep in line with these developments. The use of orthotopic tumour sites, the development of tissue-equivalent mice phantoms and the recent introduction of image-guided small animal radiation research platforms has enabled similar precision treatments to be delivered in the laboratory.These technological developments, however, are hindered by a lack of corresponding dosimetry standards and poor reporting of methodologies. Without robust and well documented preclinical radiotherapy quality assurance processes, it is not possible to ensure the accuracy and repeatability of dose measurements between laboratories. As a consequence current RT-based preclinical models are at risk of becoming irrelevant.In this review we explore current standardization initiatives, focusing in particular on recent developments in small animal irradiation equipment, 3D printing technology to create customisable tissue-equivalent dosimetry phantoms and combining these phantoms with commonly used detectors.

Sonochemical production and activation of responsive polymer microspheres.

This paper reports work aimed at extending previous studies of the sonochemical method for forming microspheres. It shows that a previously reported method for encapsulating and delivering hydrophilic species using a 'double emulsion' method can be used with chitosan or thiolated poly(methacrylic acid), PMAASH, based systems. One particular application involves targeted catalysis where gold nanoparticles are incorporated into chitosan microspheres and can be released to catalyse the borohydride reduction of 4-nitrophenol. Also reported is the use of ultrasound to 'trigger' the reduction reaction of 4-nitrophenol by rupturing nanoparticle-containing microspheres to release the catalyst. We also demonstrate that more sustainable and potentially lower environmental impact processes can be prepared by substituting commercial vegetable oil for the hydrocarbons used previously. We also report for the first time the use of responsive block copolymers to form microspheres. The copolymers consist of PMAASH blocks around a central, responsive block of poly(ethylene glycol), poly(4-vinylphenyl boronic acid) or poly(N-isopropylacrylamide) to give systems that potentially respond to pH, sugar concentrations or temperature.

Comparison of the effects of gamma or sonochemical irradiation of carbon nanotubes and the influence on the mechanical and dielectric properties of chitosan nanocomposites.

Chitosan-carbon nanotube (Chi-CNT) composite materials have been prepared with CNTs that were surface treated using either dilute acid combined with 20 kHz ultrasound or gamma-irradiation in air. The mechanical and dielectric properties have been measured and compared. Both modification methods gave nanocomposites with much improved tensile properties over native chitosan. The sonochemically treated samples were stronger with higher tensile strength but at the expense of lower elasticity and extensibility than found when γ-irradiation was used. Impedance spectra showed differences in the polymer chain transitions and in the conduction mechanisms within the nanocomposites. The results correlated well with previous work suggesting that the two modification techniques result in CNT surfaces with higher polarity. This enhances interfacial interactions with the chitosan matrix although the extent of functionalisation was greater in the sonochemical case. This work demonstrates that sonochemical modification under mild conditions is a useful method for modifying CNTs for inclusion in nanocomposite materials. However, the resulting material properties depend on the level of treatment so that the sonochemical conditions need to be carefully evaluated and controlled if the effects are to be optimised.

Synthesis, Radiolabelling and In Vitro Imaging of Multifunctional Nanoceramics.

Molecular imaging has become a powerful technique in preclinical and clinical research aiming towards the diagnosis of many diseases. In this work, we address the synthetic challenges in achieving lab-scale, batch-to-batch reproducible copper-64- and gallium-68-radiolabelled metal nanoparticles (MNPs) for cellular imaging purposes. Composite NPs incorporating magnetic iron oxide cores with luminescent quantum dots were simultaneously encapsulated within a thin silica shell, yielding water-dispersible, biocompatible and luminescent NPs. Scalable surface modification protocols to attach the radioisotopes 64Cu (t1/2=12.7 h) and 68Ga (t1/2=68 min) in high yields are reported, and are compatible with the time frame of radiolabelling. Confocal and fluorescence lifetime imaging studies confirm the uptake of the encapsulated imaging agents and their cytoplasmic localisation in prostate cancer (PC-3) cells. Cellular viability assays show that the biocompatibility of the system is improved when the fluorophores are encapsulated within a silica shell. The functional and biocompatible SiO2 matrix represents an ideal platform for the incorporation of 64Cu and 68Ga radioisotopes with high radiolabelling incorporation.

Residual Setup Errors Towards the Heart After Image Guidance Linked With Poorer Survival in Lung Cancer Patients: Do We Need Stricter IGRT Protocols?

PURPOSE: Image guided radiation therapy (IGRT) is widely used, but data directly relating set-up errors to patient outcome are scarce. This study investigates the relationship between residual IGRT shifts and overall patient survival and uses the observed relations to identify structures sensitive to radiation dose. METHODS AND MATERIALS: Residual shift data for 780 patients with non-small cell lung cancer were summarized for each patient over the course of treatment by determining the mean shifts, standard deviations, and the vector shift in the direction of the heart. These variables were related to overall survival, and significant variables were used to produce Kaplan-Meier plots of survival. The effect of shift directionality was studied by splitting the cohort into left, right, anterior, posterior, superior, and inferior groups and by analyzing the vector shift in the direction of the heart. The observed relationship was independently validated in an esophageal cancer cohort (n = 177). RESULTS: The shift data showed strong associations with survival. Left and right cohorts showed opposite directional shift effects, suggesting shifts toward the mediastinum have a negative effect on survival. Projection of the vector shift in the direction of the heart showed that patients with a residual shift toward the heart have significantly worse overall survival (P = .007, hazard ratio 1.091). The same effect was observed in the esophageal cancer cohort (P = .041, hazard ratio 1.164). CONCLUSIONS: Residual shift metrics derived from IGRT data can categorize patients with non-small cell lung cancer and those with esophageal cancer into populations with significantly different survival times on the basis of the size of the residual shift in the direction of the heart, thus providing evidence of the importance of using strict IGRT protocols to spare organs at risk and highlighting the heart as a dose-sensitive organ.

Results from a clinical trial evaluating the efficacy of real-time body surface visual feedback in reducing patient motion during lung cancer radiotherapy.

INTRODUCTION: Optical surface measurement devices are a maturing technology in radiotherapy. The challenge for such devices is to demonstrate how they can improve clinical care. We present results from a phase 1 clinical trial designed to test the hypothesis that if presented with live data from a novel optical measurement device, showing their deviation from an ideal radiotherapy treatment position, patients will be able to better control their motion and increase their geometrical conformance. METHOD AND MATERIALS: Fourteen lung cancer patients were enrolled in a prospective clinical study and asked to use a variety of visual feedback schema from a novel in-house developed optical surface measurement device. The magnitude and regularity of their body surface motion using the different schema was compared to that when free-breathing at three time-points throughout their radiotherapy treatment schedule. Additionally, 4D Cone Beam CT data, acquired simultaneously with the optical measurements, was used to test if improvements in external motion are reflected in changes in internal tumor motion. RESULTS: The primary endpoint of the trial, device tolerability assessed by the fraction of participants completing all study sessions, was 86%. Secondary endpoints showed that use of the visual feedback device was found to statistically significantly decrease body surface motion magnitude by an average of 17% over the study cohort, although not universally. Similarly body surface motion variability was decreased by 18% on average. Internal tumor motion magnitude was also found to be statistically significantly decreased by an average of 14% when using the feedback device. Reduction in external motion was predictive of reduced internal motion but no evidence of a simple correlation between changes in internal and external motion magnitude was found. CONCLUSIONS: Visual feedback of live motion is well tolerated by lung cancer patients and can reduce both body surface and tumor motion.

Influence of Poly(styrene- co-maleic acid) Copolymer Structure on the Properties and Self-Assembly of SMALP Nanodiscs.

Polymer stabilized nanodiscs are self-assembled structures composed of a polymer belt that wraps around a segment of lipid bilayer, and as such are capable of encapsulating membrane proteins directly from the cell membrane. To date, most studies on these nanodiscs have used poly(styrene- co-maleic acid) (SMA) with the term SMA-lipid particles (SMALPs) coined to describe them. In this study, we have determined the physical and thermodynamic properties of such nanodiscs made with two different SMA copolymers. These include a widely used and commercially available statistical poly(styrene- co-maleic acid) copolymer (coSMA) and a reversible addition-fragmentation chain transfer synthesized copolymer with narrow molecular weight distribution and alternating styrene and maleic acid groups with a polystyrene tail, (altSMA). We define phase diagrams for each polymer, and show that, regardless of polymer topological structure, self-assembly is driven by the free energy change associated with the polymers. We also show that nanodisc size is polymer dependent, but can be modified by varying polymer concentration. The thermal stability of each nanodisc type is similar, and both can effectively solubilize proteins from the E. coli membrane. These data show the potential for the development of different SMA polymers with controllable properties to produce nanodiscs that can be optimized for specific applications and will enable more optimized and widespread use of the SMA-based nanodiscs in membrane protein research.