Dr Robert Chapman

Peristalsis in the digestive tract is crucial to maintain physiological functions. It remains challenging to mimic the peristaltic microenvironment in gastrointestinal organoid culture. Here, we present a method to model the peristalsis for human colon tumor organoids on a microfluidic chip. The chip contains hundreds of lateral microwells and a surrounding pressure channel. Human colon tumor organoids growing in the microwell were cyclically contracted by pressure channel, mimicking the in vivo mechano-stimulus by intestinal muscles. The chip allows the control of peristalsis amplitude and rhythm and the high throughput culture of organoids simultaneously. By applying 8% amplitude with 8 ~ 10 times min-1, we observed the enhanced expression of Lgr5 and Ki67. Moreover, ellipticine-loaded polymeric micelles showed reduced uptake in the organoids under peristalsis and resulted in compromised anti-tumor efficacy. The results indicate the importance of mechanical stimuli mimicking the physiological environment when using in vitro models to evaluate nanoparticles. This work provides a method for attaining more reliable and representative organoids models in nanomedicine.

We show that photoinitiated electron/energy transfer-reversible addition-fragmentation chain transfer (PET-RAFT) enables vastly superior control over the polymerization of multiblock star copolymers compared to conventional techniques. Monomodal distributions with dispersities <1.3 could be achieved after the 10th block despite pushing the polymerization to >95% conversion in each block extension. The improvement in control is likely due to the reabsorption of the free radical at the propagating chain end by the excited catalyst, which would otherwise lead to a termination product. Simple modeling shows the dramatic effect that this has in the case of star polymerizations. Because PET-RAFT is also tolerant to oxygen, we were able to automate the synthesis of up to heptablock stars at short block lengths, providing a useful technique for screening the effect of polymer composition on the solution structure.

Trehalose is widely assumed to be the most effective sugar for protein stabilization, but exactly how unique the structure is and the mechanism by which it works are still debated. Herein, we use a polyion complex micelle approach to control the position of trehalose relative to the surface of glucose oxidase within cross-linked and non-cross-linked single-enzyme nanoparticles (SENs). The distribution and density of trehalose molecules in the shell can be tuned by changing the structure of the underlying polymer, poly(N-[3-(dimethylamino)propyl] acrylamide (PDMAPA). SENs in which the trehalose is replaced with sucrose and acrylamide are prepared as well for comparison. Isothermal titration calorimetry, dynamic light scattering, and asymmetric flow field-flow fraction in combination with multiangle light scattering reveal that two to six polymers bind to the enzyme. Binding either trehalose or sucrose close to the enzyme surface has very little effect on the thermal stability of the enzyme. By contrast, encapsulation of the enzyme within a cross-linked polymer shell significantly enhances its thermal stability and increases the unfolding temperature from 70.3 °C to 84.8 °C. Further improvements (up to 92.8 °C) can be seen when trehalose is built into this shell. Our data indicate that the structural confinement of the enzyme is a far more important driver in its thermal stability than the location of any sugar.

Many diseases are associated with the dysregulated activity of enzymes, such as matrix metalloproteinases (MMPs). This dysregulation can be leveraged in drug delivery to achieve disease- or site-specific cargo release. Self-assembled polymeric nanoparticles are versatile drug carrier materials due to the accessible diversity of polymer chemistry. However, efficient loading of sensitive cargo, such as proteins, and introducing functional enzyme-responsive behaviour remain challenging. Herein, peptide-crosslinked, temperature-sensitive nanogels for protein delivery were designed to respond to MMP-7, which is overexpressed in many pathologies including cancer and inflammatory diseases. The incorporation ofN-cyclopropylacrylamide (NCPAM) intoN-isopropylacrylamide (NIPAM)-based copolymers enabled us to tune the polymer lower critical solution temperature from 33 to 44 °C, allowing the encapsulation of protein cargo and nanogel-crosslinking at slightly elevated temperatures. This approach resulted in nanogels that were held together by MMP-sensitive peptides for enzyme-specific protein delivery. We employed a combination of cryogenic transmission electron microscopy (cryo-TEM), dynamic light scattering (DLS), small angle neutron scattering (SANS), and fluorescence correlation spectroscopy (FCS) to precisely decipher the morphology, self-assembly mechanism, enzyme-responsiveness, and model protein loading/release properties of our nanogel platform. Simple variation of the peptide linker sequence and combining multiple different crosslinkers will enable us to adjust our platform to target specific diseases in the future.

We present the use of an oxygen tolerant controlled radical polymerization (photoinduced electron/energy transfer-reversible addition fragmentation chain transfer polymerization, PET-RAFT) as a simple method for preparing controlled radical polymers in an undergraduate laboratory. Unlike conventional techniques, PET-RAFT polymerizations require no deoxygenation, heating, or special equipment. This removes hazards and complexity from the experimental setup, as well as sources of error and variability in the polymers produced. In this program, students used PET-RAFT to synthesize poly(N-isopropylacrylamide) coated gold nanoparticles and studied the effect of the polymer on the size, optical properties, and aggregation state of the nanoparticles in response to temperature. In parallel, students used PET-RAFT to study the chemistry of radical copolymerization by measuring the reactivity ratios of a range of vinylic monomers. Both experiments would be very difficult to perform in an undergraduate laboratory by conventional controlled radical polymerization techniques. The PET-RAFT technique we present can be used to prepare well-defined polymers easily for any number of applications beyond this, particularly in materials, biology, engineering, and physics laboratories that are not set up for complicated polymer synthesis.

Single-enzyme nanoparticles (SENs), which encapsulate individual enzymes in a thin polymer network, offer exciting possibilities for stabilizing enzymes and tuning their activity, but most approaches to date rely on covalent modification of the enzyme. We introduce here a new approach to their synthesis in which a pre-prepared polymer synthesized by reversible addition-fragmentation chain-transfer polymerization is first tethered to the surface via electrostatic interactions. Isothermal titration calorimetry and asymmetric flow field-flow fraction (AF4) in combination with multiangle light scattering (MALS) reveal weak binding of 2-5 chains/enzymes. The strength of binding can be tuned based on the charge density of the bound polymer. AF4-MALS and small-angle X-ray scattering confirm the formation of a thin cross-linked shell around the enzyme following chain extension of this polymer in the presence of a bis-functional monomer. The mild conditions of this method of SEN formation, which avoids any covalent modification of the enzyme, result in no loss in activity on our model enzyme (glucose oxidase) and 4-fold increase in thermal stability. It offers much greater control over the chemistry of the SEN, which we demonstrate by incorporation of trehalose between the enzyme and cross-linked shell.

Nanogels are attractive delivery vehicles for small hydrophilic cargo, such as peptides, but there is a limited understanding of how the structure of both the nanogel and cargo affect the drug loading and release properties, particularly in biological environments. We have used Förster resonance energy transfer (FRET) to study the loading and release behaviour of a series of hydrophilic charged peptides (SNKAY, SNKKY and SNDDY) in a set of pH-responsive methacrylic acid (pMAA) core crosslinked nanogels that were prepared through miniemulsion polymerisation from a PEGMEMA-DMAEMA-tBuMA terblock copoylmer. Our nanogels show an extremely high loading capacity of the positively charged peptides (400-800 wt% in the best cases), absorbing them from solution at pH 7.4 without any need for purification. At pH values below 6, the peptide is rapidly expelled from the nanogel due to the collapse of the core and protonation of the positively charged inner shell. By combining FRET with fluorescence lifetime imaging microscopy (FLIM), we were able to monitor this in vitro and found that most of the drug is released within the first 10 min after cell uptake.

Variable defects such as vacancies and grain boundaries are unavoidable in the synthesis of graphene, but play a central role in the activation of oxidation. Here, we apply reactive molecular dynamics simulations to reveal the underpinning mechanisms of oxidation in graphene with or without defects at the atomic scale. There exist four oxidation modes generating CO2 or CO in different stages, beginning from a single-atom vacancy, and proceeding until the ordered structure broken down into carbon oxide chains. The oxidation process of the graphene sheets experiences four typical stages, in which alternately symmetrical escape phenomenon is observed. Importantly, disordered rings can self-restructure during the oxidation of grain boundaries. Of all defects, the oxidation of vacancy has the lowest energy barrier and is therefore the easiest point of nucleation. This study demonstrates the crucial role of defects in determining the oxidation kinetics, and provides theoretical guidance for the oxidation prevention of graphene and the production of functionalized graphene.

Structure¿function relationships for multivalent polymer scaffolds are highly complex due to the wide diversity of architectures offered by such macromolecules. Evaluation of this landscape has traditionally been accomplished case-by-case due to the experimental difficulty associated with making these complex conjugates. Here, we introduce a simple dual-wavelength, two-step polymerize and click approach for making combinatorial conjugate libraries. It proceeds by incorporation of a polymerization friendly cyclopropenone-masked dibenzocyclooctyne into the side chain of linear polymers or the a-chain end of star polymers. Polymerizations are performed under visible light using an oxygen tolerant porphyrin-catalyzed photoinduced electron/energy transfer-reversible addition¿fragmentation chain-transfer (PET-RAFT) process, after which the deprotection and click reaction is triggered by UV light. Using this approach, we are able to precisely control the valency and position of ligands on a polymer scaffold in a manner conducive to high throughput synthesis.

Combinatorial and high throughput (HTP) methodologies have long been used by the pharmaceutical industry to accelerate the rate of drug discovery. HTP techniques can also be applied in polymer chemistry to more efficiently elucidate structure-property relationships, to increase the speed of new material development, and to rapidly optimize polymerization conditions. Controlled living/radical polymerization (CLRP) is widely employed in the preparation of potential materials for bioapplications being suitable for a large variety of polymeric materials with various architectures. The versatility of CLRP makes it an ideal candidate for combinatorial and HTP approaches to research, and recently, the development of oxygen tolerant CLRP techniques has greatly simplified the methodology. In this Perspective, we provide an overview of conventional CLRP, including automated parallel synthesizers, as well as oxygen tolerant CLRP applications for HTP polymer research.

LB76 is a cyclic peptide that shows great promise as a selective heat shock protein 90 (Hsp90) inhibitor. However despite strong binding to and inhibition of Hsp90 in cell lysate its polar structure prevents it from crossing the cell membrane. We have developed a pH responsive polymer nanoparticle that effectively encapsulates LB76 from solution without need for purification. The nanoparticle releases the molecule upon crossing the cell membrane. Treatment of human colon cancer HCT116 cells with nanoparticles laden with LB76 produces the typical phenotype associated with Hsp90 inhibition, providing evidence of a therapeutically active payload.

Polyion complex (PIC) micelles are frequently used as a means to deliver biologics such as proteins. While it is known that the polymer structure can affect the stability of these micelles, few studies have investigated their stability in biological settings. In this paper, we have prepared a library of poly[poly(ethylene glycol) methyl ether acrylate]-block-poly(2-carboxyethyl acrylate)s (PPEGMEA-b-PCEA) and poly[poly(ethylene glycol) methyl ether acrylate]-block-poly(acrylic acid)s (PPEGMEA-b-PAA), each with varying chain length of the charged polymer block. In addition terpolymers were prepared that included hydrophobic n-butyl acrylate (BA) units randomly incorporated along the charged block and as triblock terpolymers (PPEGMEA-b-(PCEA-co-PBA), PPEGMEA-b-PCEA-b-PBA or PPEGMEA-b-PBA-b-PCEA). These polymers were condensed with lysozyme to generate PIC micelles of various compositions. While stable in the intracellular space, the diblock PIC micelles readily disassembled inside the cells of spheroid cancer models as evidenced by light-sheet fluorescence microscopy (LSFM) and Förster resonance energy transfer (FRET) studies. Terpolymer PIC micelles were more resistant to disassembly, but only when the hydrophobic BA groups were randomly distributed within the pCEA blocks and not when incorporated as a separate block. These results show that simple PIC micelles will rapidly deliver biological cargo after cell internalization and their stability can be tuned by the introduction of hydrophobic units within the charged block.

Herein we report an approach to encapsulate enzymes within polymeric nanocapsules dispersed in an organic solvent via inverse miniemulsion periphery RAFT polymerization (IMEPP). Glucose oxidase (GOx), which has various applications but is unstable at elevated temperature and in organic solvents, was chosen as a model enzyme. In this study, we have explored the use of photoinitiation under visible (blue) light instead of thermal initiation to avoid enzyme denaturation by heating. GOx was successfully encapsulated within polymeric nanocapsules (~200 nm) and showed high activity (71-100% relative to free GOx in PBS) dispersed in toluene/t-BuOH. The nanocapsules were thus able to protect GOx and enable it to function in an organic solvent mixture where native GOx would otherwise undergo denaturation. This approach of enzyme encapsulation is significant as it may lead to increased industrial applications of enzyme biocatalysis, expanding the use of enzymes as nontoxic and environmentally friendly biocompatible catalysts.

We investigate a high throughput approach to polymer synthesis by employing photoinduced electron/energy transfer-reversible addition-fragmentation chain transfer (PET-RAFT) polymerization. Polymerization of a broad range of monomers, including acrylates, methacrylates, acrylamides, and styrenic monomers, was achieved directly in a multiwell plate by employing 5,10,15,20-tetraphenylporphine zinc (ZnTPP) as a photocatalyst under yellow LED light. Various parameters such as monomer concentration and degree of polymerization were investigated with respect to their effect on polymerization rate and the degree of control over the molecular weight and molecular weight distribution. Finally, the synthesis of well-defined multiblock copolymers (up to a hexablock copolymer) was shown to be achievable entirely within a multiwell plate without any intermediate purification. The versatility and ease of this oxygen tolerant polymerization in high throughput formats make it an excellent technique for the generation of polymer arrays.

A benchtop approach is developed for the synthesis of various polymeric architectures using an aqueous Reversible Addition-Fragmentation chain Transfer (RAFT) photopolymerization technique. Under visible green light irradiation (¿ = 530 nm), eosin Y (EY) in the presence of ascorbic acid (AscA) as a reducing agent can initiate RAFT polymerization of a range of monomers (acrylamide, acrylate and methacrylate families) in water. More importantly, this process proceeds rapidly without the need for traditional deoxygenation and thus allows RAFT polymerizations to be performed in ultralow volumes (20 µL). This photopolymerization approach can be applied on a 96-well microtiter plate for the synthesis of a range of homopolymer and diblock copolymers. Furthermore, more complex polymeric architectures such as star polymers (arm first) and polymeric nanoparticles (via a polymerization-induced self-assembly (PISA) approach) were successfully synthesized in low volumes and without prior deoxygenation.

The preparation of polyolefins using controlled radical polymerisation (CRP) has long been a goal of polymer chemistry, but is hampered by the instability of olefin radicals. Herein we propose a simple strategy for the preparation of well-defined polyolefins such as polypropylene, by post-modification of poly(acrylic acid) with dialkylzinc reagents. The starting polymers can be readily synthesised by existing CRP techniques to almost any desired length and architecture. After activation of the carboxylic acid side chain and reaction with the dialkylzinc, a new C-C bond is formed between the alkyl group and the backbone carbon, and the carboxylic acid functionality is lost as CO2. We used this strategy to prepare well-defined polyolefins with methyl, ethyl, propyl and butyl side chains. As the dialkylzinc reagents are unreactive towards esters we were also able to use this approach to synthesise and self-assemble a range of PEGMEA-olefin block copolymers.

Natural variations in pH levels of tissues in the body make it an attractive stimuli to trigger drug release from a delivery vehicle. A number of such carriers have been developed but achieving high drug loading combined with low leakage at physiological pH and tunable controlled release at the site of action is an ongoing challenge. Here we report a novel strategy for the synthesis of entirely hydrophilic stimuli-responsive nanocarriers with high passive loading efficiency of doxorubicin (DOX), which show good stability at pH 7 and rapid tunable drug release at intracellular pH. The particles (Dh = 120-150 nm), are prepared by cross-linking the core of swollen micelles of the triblock copolymer poly[poly(ethylene glycol) methyl ether methacrylate-b-N,N'-di(methylamino)ethyl methacrylate-b-tert-butyl methacrylate] (poly(PEGMEM A)-b- PDMAEMA-b-PtBMA)). After subsequent deprotection of the tert-butyl groups a hydrophilic poly(methacrylic acid) (PMAA) core is revealed. Due to the negative charge in the acidic core the particles absorb 100% of the DOX from solution at pH 7 at up to 50 wt % DOX/polymer, making them extremely simple to load. Unlike other systems, the DMAEMA "gating" shell ensures low drug leakage at pH 7, whereas physical shrinkage of the MAA core allows rapid release below pH 6. The particles deliver DOX with high efficiency to human pancreatic cancer AsPC-1 cell lines, even lowering the IC50 of DOX. As the particles are stable as a dry powder and can be loaded with any mixture of positively charged drugs without complex synthetic or purification steps, we propose they will find use in a range of delivery applications.

The synthesis of well-defined polymers in a low-volume, combinatorial fashion has long been a goal in polymer chemistry. Here, we report the preparation of a wide range of highly controlled homo and block co-polymers by Enz-RAFT (enzyme-assisted reversible addition-fragmentation chain transfer) polymerization in microtiter plates in the open atmosphere. The addition of 1 µm glucose oxidase (GOx) to water/solvent mixtures enables polymerization reactions to proceed in extremely low volumes (40 µL) and low radical concentrations. This procedure provides excellent control and high conversions across a range of monomer families and molecular weights, thus avoiding the need to purify for screening applications. This simple technique enables combinatorial polymer synthesis in microtiter plates on the benchtop without the need of highly specialized synthesizers and at much lower volumes than is currently possible by any other technique.

Secretory phospholipase A2 group IIA (sPLA2-IIA) was examined as a point of care marker for determining disease activity in rheumatoid (RA) and psoriatic (PsA) arthritis. Serum concentration and activity of sPLA2-IIA were measured using in-house antibodies and a novel point of care lateral flow device assay in patients diagnosed with varying severities of RA (n = 30) and PsA (n = 25) and found to correlate strongly with C-reactive protein (CRP). Levels of all markers were elevated in patients with active RA over those with inactive RA as well as both active and inactive PsA, indicating that sPLA2-IIA can be used as an analogue to CRP for RA diagnosis at point of care.

Acute pancreatitis is a relatively common and potentially fatal condition, but the presenting symptoms are non-specific and diagnosis relies largely on the measurement of amylase activity by the hospital clinical laboratory. In this work we develop a point of care test for pancreatitis measuring concentration of secretory phospholipase A2 group IB (sPLA2-IB). Novel antibodies for sPLA2-IB were raised and used to design an ELISA and a lateral flow device (LFD) for the point of care measurement of sPLA2-IB concentration, which was compared to pancreatic amylase activity, lipase activity, and sPLA2-IB activity in 153 serum samples. 98 of these samples were obtained from the pathology unit of a major hospital and classified retrospectively according to presence or absence of pancreatitis, and the remaining 55 were obtained from commercial sources to serve as high lipase (n = 20), CA19-9 positive (n = 15), and healthy (n = 20) controls. sPLA2-IB concentration correlated well with the serum activity of both amylase and lipase, and performed at least as well as either markers in the differentiation of pancreatitis from controls.