“Polysoaps” via RAFT Copolymerization to Form Well-Defined Micelles for Water Remediation and Targeted Drug Delivery Applications

Amphiphilic copolymers have become increasingly important for environmental and biological applications due to their behavioral characteristics in aqueous solution. For example, structurally-tailored statistical amphiphilic copolymers or “polysoaps” can self-assemble into micelles or other architectures in water at various concentrations. Polysoaps may be differentiated from small molecule surfactant micelles in their capability to self-assemble into unimolecular associates (unimolecular micelles) with no dependence on concentration. Such micelles offer enormous potential for dispersion of hydrophobic species in water at high dilution. Importantly, each polymer chain forms its own micelle and upon dilution, these micelles remain intact and capable of dispersing hydrocarbon material in their core domain. This dissertation focuses on determining the parameters that contribute to the unique unimeric micelle properties of polysoaps. We utilize RAFT copolymerization to prepare well-controlled copolymers with a variety of functional groups, molecular weights, and hydrophobic mole fractions. With this research platform, we achieve a better understanding of how single polymer chains form unimeric assemblies capable of sequestering hydrocarbons in water. With sufficiently high hydrophobic content along a water-soluble backbone, micelles may form, some with unimeric structures. The unimeric micelles have been shown to have higher uptake efficiencies of hydrocarbon into their core domains as compared to multimeric, polymer-based and small molecule, surfactant-based micelle. This work demonstrates the potential utility of polysoaps for contemporary applications including oil spill remediation, water treatment, and targeted drug delivery

Label-Free Characterization of Organic Nanocarriers Reveals Persistent Single Molecule Cores For Hydrocarbon Sequestration

Self-assembled molecular nanostructures embody an enormous potential for new technologies, therapeutics, and understanding of molecular biofunctions. Their structure and function are dependent on local environments, necessitating in-situ/operando investigations for the biggest leaps in discovery and design. However, the most advanced of such investigations involve laborious labeling methods that can disrupt behavior or are not fast enough to capture stimuli-responsive phenomena. We utilize X-rays resonant with molecular bonds to demonstrate an in-situ nanoprobe that eliminates the need for labels and enables data collection times within seconds. Our analytical spectral model quantifies the structure, molecular composition, and dynamics of a copolymer micelle drug delivery platform using resonant soft X-rays. We additionally apply this technique to a hydrocarbon sequestrating polysoap micelle and discover that the critical organic-capturing domain does not coalesce upon aggregation but retains distinct single-molecule cores. This characteristic promotes its efficiency of hydrocarbon sequestration for applications like oil spill remediation and drug delivery. Such a technique enables operando, chemically sensitive investigations of any aqueous molecular nanostructure, label-free

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

The Effects of Pi-Pi Stacking on the Controlled Radical Polymerization of Vinyl Aromatics

The atom transfer radical polymerization (ATRP) of styrene (St) was conducted in the presence of varying equivalence (eq) of hexafluorobenzene (HFB) and octafluorotoluene (OFT) to probe the effects of pi-pi stacking on the rate of the polymerization and on the tacticity of the resulting polystyrene (PSt). The extent of the pi-pi stacking interaction between HFB/OFT and the terminal polystyrenic phenyl group was also investigated as a function of solvent, both non-aromatic solvents (THF and hexanes) and aromatic solvents (benzene and toluene). In all cases the presence of HFB or OFT resulted in a decrease in monomer conversion indicating a reduction in the rate of the polymerization with greater retardation of the rate with increase eq of HFB or OFT (0.5 eq to 1 eq HFB/OFT compared to St). Additionally, when aromatic solvents were used instead of non-aromatic solvents the effect of the HFB/OFT on the rate was minimized, consistent with the aromatic solvent competitively interacting with the HFB/OFT. The effects of temperature and ligand strength on the ATRP of St in the presence of HFB were also probed. It was found that when using N,N,N’,N’,N’’-pentamethyldiethylenetriamine (PMDETA) as the ligand the effects of HFB at 38o were the same as at 86oC. When tris[2-(dimethylamino)ethyl]-amine (Me6TREN) was used as the ligand at 38o there was a decrease in monomer conversion similar to the analogous PMDETA reaction. When the polymerization was conducted at 86oC there was no effect on the monomer conversion with HFB present compared to when HFB was absent. To investigate the pi-pi stacking effect even further, the reverse pi-pi stacking system was observed by conducting the ATRP of pentafluorostyrene (PFSt) in the presence of varying eq of benzene and toluene, which in both cases resulted in an increase in monomer conversion compared to when benzene or toluene were absent; in summary the rate of the ATRP of PFSt increases when benzene or toluene waas present in the reaction. The pi-pi stacking interaction between the HFB/OFT and the dormant alkyl bromide of the polymer chain was verified by 1H-NMR with 1-bromoethylbenzene as the alkyl bromide. Also verified by 1H-NMR was the interaction between HFB/OFT and St and the interaction between PFSt and benzene. In all 1H-NMR spectra a perturbation in the aromatic and/or vinyl peaks was observed when the pi-pi stacking agent was present compared to when it was absent. The tacticity of the PSt formed in the presence of 1 eq of HFB was compared to the PSt formed in the absence of HFB by observing the C1 signal in their 13C-NMR spectra, but no change in shape or chemical shift of the signal was observed indicating that there was no change in tacticity

Structurally Controlled Polysoaps \u3ci\u3evia\u3c/i\u3e RAFT Copolymerization of AMPS and \u3ci\u3en\u3c/i\u3e-Dodecyl Acrylamide for Envio

A series of micelle-forming, amphiphilic copolymers or “polysoaps” with potential as dispersants for oil spill remediation has been synthesized via statistical RAFT copolymerization of specific molar ratios of 2-acrylamido-2-methylpropane sulfonic acid (AMPS) and n-dodecyl acrylamide (DDAM). Control over composition, degree of polymerization, polydispersity, and reaction kinetics was attained by conducting the copolymerizations at 60 °C in dimethylformamide with AIBN and CEP as initiator and chain-transfer agent, respectively. Molecular weight, hydrodynamic dimensions, composition- and concentration-dependent associative properties, and hydrophobic domain characteristics in water for copolymers with 10, 20, 30, 40, 50, and 60 mol% feed of DDAM have been studied utilizing NMR, SEC/MALLS, DLS, SLS, surface tensiometry, and fluorescence spectroscopy. It was found that the lower hydrophobic content (10%, 20%, and 30%) polysoaps form multimeric associations as indicated by increasing hydrodynamic dimensions as concentration is increased. On the other hand, the higher hydrophobic content (40%, 50%, and 60%) polysoaps form unimolecular micelles with consistent sizes and with distinct hydrophobic cores over the entire concentration range probed. UV/Vis absorbance experiments provided additional insight into the association and sequestration properties of the polysoaps. The higher hydrophobic content polysoaps show increased capabilities for dissolution of pyrene as compared to SDS above its CMC. Finally, the cytotoxicity of these polysoaps was determined utilizing KB cell lines. The polysoaps of this study exhibited up to 60× less cytotoxicity than SDS as measured by IC50 values. It was also found that as the molecular weight of the polysoap increased, the cytotoxicity decreased. The results of this study point to potential of unimolecular micelles for oil spill remediation, allowing sequestration and subsequent hydrocarbon break-down by endogenous bacteria in the marine environment

Structurally Controlled Polysoaps via RAFT Copolymerization of AMPS and n-Dodecyl Acrylamide for Environmental Remediation

A series of micelle-forming, amphiphilic copolymers or polysoaps with potential as dispersants for oil spill remediation has been synthesized via statistical RAFT copolymerization of specific molar ratios of 2-acrylamido-2-methylpropane sulfonic acid (AMPS) and n-dodecyl acrylamide (DDAM). Control over composition, degree of polymerization, polydispersity, and reaction kinetics was attained by conducting the copolymerizations at 60 degrees C in dimethylformamide with AIBN and CEP as initiator and chain-transfer agent, respectively. Molecular weight, hydrodynamic dimensions, composition- and concentration-dependent associative properties, and hydrophobic domain characteristics in water for copolymers with 10, 20, 30, 40, 50, and 60 mol% feed of DDAM have been studied utilizing NMR, SEC/MALLS, DLS, SLS, surface tensiometry, and fluorescence spectroscopy. It was found that the lower hydrophobic content (10%, 20%, and 30%) polysoaps form multimeric associations as indicated by increasing hydrodynamic dimensions as concentration is increased. On the other hand, the higher hydrophobic content (40%, 50%, and 60%) polysoaps form unimolecular micelles with consistent sizes and with distinct hydrophobic cores over the entire concentration range probed. UV/Vis absorbance experiments provided additional insight into the association and sequestration properties of the polysoaps. The higher hydrophobic content polysoaps show increased capabilities for dissolution of pyrene as compared to SDS above its CMC. Finally, the cytotoxicity of these polysoaps was determined utilizing KB cell lines. The polysoaps of this study exhibited up to 60x less cytotoxicity than SDS as measured by IC50 values. It was also found that as the molecular weight of the polysoap increased, the cytotoxicity decreased. The results of this study point to potential of unimolecular micelles for oil spill remediation, allowing sequestration and subsequent hydrocarbon break-down by endogenous bacteria in the marine environment

Label-Free Characterization of Aqueous Micelle Nanostructures Via Novel Liquid In-Situ Resonant Soft X-Ray Scattering (RSoXS)

Micelles are fundamental to nanocarrier applications from drug delivery to environmental remediation. Their structure and dynamics are critical to their properties and functions but are challenging to measure. Here, we demonstrate a novel technique capable of such measurements based on resonant soft X-ray scattering (RSoXS). It uniquely probes organic materials using their intrinsic chemical bonds rather than laborious and disruptive labeling techniques. Our customized microfluidic cell enables RSoXS to be performed in liquid environments, allowing structure and dynamics to be measured in-situ. Using this technique in a multimodal approach, we investigated an amphiphilic polyelectrolyte copolymer micelle of three different molecular weights (Mw). Despite no measurable critical micelle concentration, structural analyses point towards multimeric structures for most Mw’s. The sizes of the micelle substructures are independent of both concentration and Mw. Combining these results with a Mw-invariant surface charge and zeta potential strengthens the link between nanoparticle size and ionic charge in solution that governs polysoap micelle structure. Such control would be critical for nanocarrier applications such as drug delivery and water remediation

Amphoteric, Sulfonamide-Functionalized Polysoaps : CO-Induced Phase Separation for Water Remediation

Amphoteric polysoaps have been prepared via statistical RAFT copolymerization using either methacryloyl sulfacetamide (mSAC) or methacryloyl sulfmethazine (mSMZ) and 4-hexylphenyl methylacrylamide (4HPhMA). These copolymers form pH- and CO2-responsive polymeric micelles capable of sequestering hydrophobic molecules in water. The composition and structure of the respective copolymers can be changed to tailor the onset and extent of CO2-dependent phase behavior. When CO2 is introduced into the system, resulting in carbonic acid formation, the pH drops below the pKa of the sulfonamide units along the copolymer backbone, and phase separation occurs. Purging with N2 results in an increase in pH and redissolution of the polysoap; this process can be repeated multiple times. The mSMZ polysoaps, which show complete phase transitions using this reversible process, were especially efficient in removing the model contaminants pyrene and 9-anthracenemethanol from water. The feasibility of recovering and reusing these copolymers is demonstrated, pointing to the potential utility of such CO2-responsive systems in water treatment and related environmental remediation applications