Multifunctional Poly[<i>N</i>‑(2-hydroxypropyl)methacrylamide] Copolymers via Postpolymerization Modification and Sequential Thiol–Ene Chemistry

Poly­[<i>N</i>-(2-hydroxypropyl)­methacrylamide] is a promising candidate material for biomedical applications. However, synthesis of functional pHPMA via compolymerization results can lead to variations in monomer composition, molar mass, and dispersity making comparison difficult. Postpolymerization modification routes, most commonly aminolysis of poly­[active ester methacrylates], have alleviated some of these problems, but ester hydrolysis can lead to other problems. Here we report the synthesis of multifunctional pHPMA via a simple two-step derivatization of pHPMA homopolymer using readily available standard reagents and atom-efficient procedures. First, treatment with allyl isocyanate yields the corresponding carbamate with predictable incorporation of side-chain functionality. Allyl-pHPMA can then be derivatized further via radical thiol–ene reactions to generate pHPMA with multiple diverse functionalities but without adverse effects on the molecular weight and dispersity of the polymer. The applicability of the method to production of biologically relevant materials is demonstrated by cytocompatibility and cell labeling experiments with easily prepared ligand-functionalized pHPMA in the HCT 116 model cell line

Hollow Colloidosomes Prepared Using Accelerated Solvent Evaporation

We demonstrate a new, scalable, simple, and generally applicable two-step method to prepare hollow colloidosomes. First, a high volume fraction oil-in-water emulsion was prepared. The oil phase consisted of CH₂Cl₂ containing a hydrophobic structural polymer, such as polycaprolactone (PCL) or polystyrene (PS), which was fed into the water phase. The water phase contained poly­(vinylalcohol), poly­(<i>N</i>-isopropylacrylamide), or a range of cationic graft copolymer surfactants. The emulsion was rotary evaporated to rapidly remove CH₂Cl₂. This caused precipitation of PCL or PS particles which became kinetically trapped at the periphery of the droplets and formed the shell of the hollow colloidosomes. Interestingly, the PCL colloidosomes were birefringent. The colloidosome yield increased and the polydispersity decreased when the preparation scale was increased. One example colloidosome system consisted of hollow PCL colloidosomes stabilized by PVA. This system should have potential biomaterial applications due to the known biocompatibility of PCL and PVA

Thermally Triggered Assembly of Cationic Graft Copolymers Containing 2-(2-Methoxyethoxy)ethyl Methacrylate Side Chains

Thermoresponsive copolymers continue to attract a great deal of interest in the literature. In particular, those based on ethylene oxide-containing methacrylates have excellent potential for biomaterial applications. Recently, some of us reported a study of thermoresponsive cationic graft copolymers containing poly(<i>N</i>-isopropylacrylamide), PNIPAm, (Liu et al., <i>Langmuir</i>, <b>24</b>, 7099). Here, we report an improved version of this new family of copolymers. In the present study, we replaced the PNIPAm side chains with poly(2-(2-methyoxyethoxy)ethylmethacrylate), PMeO₂MA. These new, nonacrylamide containing, cationic graft copolymers were prepared using atom transfer radical polymerization (ATRP) and a macroinitiator. They contained poly(trimethylamonium)-aminoethyl methacrylate and PMeO₂MA, i.e., PTMA⁺_<i>x</i>-<i>g</i>-(PMeO₂MA_<i>n</i>)_<i>y</i>. They were investigated using variable-temperature turbidity, photon correlation spectroscopy (PCS), electrophoretic mobility, and ¹H NMR measurements. For one system, four critical temperatures were measured and used to propose a mechanism for the thermally triggered changes that occur in solution. All of the copolymers existed as unimolecular micelles at 20 °C. They underwent reversible aggregation with heating. The extent of aggregation was controlled by the length of the side chains. TEM showed evidence of micellar aggregates. The thermally responsive behaviors of our new copolymers are compared to those for the cationic PNIPAm graft copolymers reported by Liu et al. Our new cationic copolymers retained their positive charge at all temperatures studied, have high zeta potentials at 37 °C, and are good candidates for conferring thermoresponsiveness to negatively charged biomaterial surfaces

Assay characterisation using Z-factors, Signal Window (SW) and Coefficient of variation (CV) for NSCs. Z-factors (A–C), Signal window (D–F) and Coefficient of variation (G–I).

<p>Acceptance criteria Z-factor>0.4, SW>2 and CV<20% were colour coded so that values above the green lines meet quality criteria whereas values above the red line fail. Dotted line at 10000 cells represents chosen seeding density for spheroid cytotoxicity screening.</p

Dose-response curves for UW228-3 and NSCs spheroids exposed to increasing concentrations of etoposide.

<p>Normalized viability is expressed as volume, resazurin reduction, acid phosphatase activity and cell number. Data is pooled from at least three separate experiments.</p

Confidence intervals for etoposide IC50 determinations for resazurin and Volume in neural stem cells.

<p>The 95% confidence intervals (CI) for each experiment were plotted against the geometric mean (black dotted line) and 95% CIs (green dotted lines) for all individual experiments for resazurin and Volume determinations.</p

Phase-contrast microscope images of NSC exposed to increasing concentrations of etoposide.

<p>A–C spheroids before PBS wash. A′–C′- the same spheroids after PBS wash. Control is grown in plain media, concentrations of etoposide on drug treated spheroids shown in µM, scale bar applies to all panels.</p

Phase-contrast microscope image of UW228-3 spheroids exposed to increasing concentrations of etoposide.

<p>Panels A–C show intact UW228-3 spheroids with a halo of debris and dead cells at high drug doses impeding image analysis. Panels A′–C′ capture the same UW228-3 spheroids after PBS rinse. Controls were cultured in plain media, concentration of etoposide is given in µM and scale bar applies for all images.</p

Assay characterisation using Z-factors, Signal Window (SW) and Coefficient of variation (CV) for UW228-3 cells.

<p>Z-factors (A–C), Signal window (D–F) and Coefficient of variation (G–I). Acceptance criteria Z-factor>0.4, SW>2 and CV<20% were colour coded so that values above the green lines meet quality criteria whereas values above the red line fail. Dotted line at 5000 cells represents chosen seeding density for spheroid cytotoxicity screening.</p

Spheroid volume increase of NSC and UW228-3 cells as a function of initial seeding number.

<p>Volume increase % = 100*(V_day7−V_day1)/V_day1. NSCs grew more in a week than UW cells, reaching maximum growth increase of 600% for seeding 1000 cells per spheroid, whereas the maximum growth increase for tumour cells was around 170% for 2000–5000 cells/spheroid.</p