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.Keywords: doxorubicin; gating; pH-responsive nanoparticles; squeezing effect; swollen micelles;

•A high-speed thermo- and pH-responsive matrix was prepared in two steps.•Proteins separation was achieved by combining a step pH and temperature gradient.•A clear separation mechanism was proposed.•It was able to separate proteins at the mobile phase velocity up to 2528 cm h−1.Dual thermo- and pH-responsive chromatography has been proposed using poly(N-isopropylacrylamide-co-butyl methacrylate-co-N,N-dimethylaminopropyl acrylamide) (P(NIPAM-co-BMA-co-DMAPAAM)) brushes grafted gigaporous polystyrene microspheres (GPM) as matrix. Atom transfer radical polymerization (ATRP) initiator was first coupled onto GPM through Friedel–Crafts acylation with 2-bromoisobutyryl bromide. The dual-responsive polymer brushes were then grafted onto GPM via surface-initiated ATRP. The surface composition, gigaporous structure, protein adsorption and dual-responsive chromatographic properties of the matrix (GPM-P(NIPAM-co-BMA-co-DMAPAAM) were characterized in detail. Results showed that GPM were successfully grafted with thermoresponsive cationic polymer brushes and that the gigaporous structure was well maintained. A column packed with GPM-P(NIPAM-co-BMA-co-DMAPAAM presented low backpressure, good permeability and appreciable thermo-responsibility. By changing pH of the mobile phase and temperature of the column in turn, the column can separate three model proteins at the mobile phase velocity up to 2528 cm h−1. A separation mechanism of this matrix was also proposed. All results indicate that the dual thermo- and pH-responsive chromatography matrix has great potentials in ‘green’ high-speed protein chromatography.

•A high-speed thermoresponsive bioseparation medium was prepared in two steps.•Non-specific adsorption of proteins on thermoresponsive medium was greatly reduced.•Separation of proteins was achieved by only adjusting column temperature.•It was able to separate proteins at the mobile phase velocity up to 2167 cm h−1.A high-speed thermoresponsive medium was developed by grafting poly(N-isopropylacrylamide-co-butyl methacrylate) (P(NIPAM-co-BMA)) brushes onto gigaporous polystyrene (PS) microspheres via surface-initiated atom transfer radical polymerization (ATRP) technique, which has strong mechanical strength, good chemical stability and high mass transfer rate for biomacromolecules. The gigaporous structure, surface chemical composition, static protein adsorption, and thermoresponsive chromatographic properties of prepared medium (PS–P(NIPAM-co-BMA)) were characterized in detail. Results showed that the PS microspheres were successfully grafted with P(NIPAM-co-BMA) brushes and that the gigaporous structure was robustly maintained. After grafting, the nonspecific adsorption of proteins on PS microspheres was greatly reduced. A column packed with PS–P(NIPAM-co-BMA) exhibited low backpressure and significant thermo-responsibility. By simply changing the column temperature, it was able to separate three model proteins at the mobile phase velocity up to 2167 cm h−1. In conclusion, the thermoresponsive polymer brushes grafted gigaporous PS microspheres prepared by ATRP are very promising in ‘green’ high-speed preparative protein chromatography.

Gigaporous polystyrene (PS) microspheres were hydrophilized by in situ polymerization to give a stable cross-linked poly(vinyl alcohol) (PVA) hydrogel coating, which can shield proteins from the hydrophobic PS surface underneath. The amination of microspheres (PS-NH2) was first carried out through acetylization, oximation and reduction, and then 4,4′-azobis (4-cyanovaleric acid) (ACV), a polymerization initiator, was covalently immobilized on PS-NH2 through amide bond formation, and the cross-linked poly(vinyl acetate) (PVAc) was prepared by radical polymerization at the surfaces of ACV-immobilized PS microspheres (PS-ACV). Finally, the cross-linked PVA hydrogel coated gigaporous PS microspheres (PS-PVA) was easily achieved through alcoholysis of PVAc. Results suggested that the PS microspheres were effectively coated with cross-linked PVA hydrogel, where the gigaporrous structure remained under optimal conditions. After hydrophilic modification (PS-PVA), the protein-resistant ability of microspheres was greatly improved. The hydroxyl-rich PS-PVA surface can be easily derivatized by classical chemical methods. Performance advantages of the PS-PVA column in flow experiment include good permeability, low backpressure, and mechanical stability. These results indicated that PS-PVA should be promising in rapid protein chromatography.Keywords: coating; cross-linked poly(vinyl alcohol) hydrogel; gigaporous; polystyrene particles; protein adsorption

A magnetic nano-sized carrier for 10-hydroxycamptothecin (HCPT) was prepared by using Fe3O4 nanoparticles as cores and chitosan (CS) as a polymeric shell by a novel reverse ultrasonic emulsification method. Poly(ethylene glycol) (PEG) chains were then coupled onto the magnetic particles (CS-Fe3O4) to improve their biocompatibility (PEG-CS-Fe3O4). HCPT was loaded onto PEG-CS-Fe3O4 by a subtle precipitation method. Under optimum conditions, the CS-Fe3O4 was close to spherical in shape with an average size of 174 nm and a high saturated magnetization. After coupling PEG chains, the unspecific adsorption of bovine serum albumin (BSA) on PEG-CS-Fe3O4 decreased significantly. The drug loading content and loading efficiency were 9.8–11.8% and 49–59% for magnetic composite nanoparticles, respectively. HCPT-loaded magnetic composite nanoparticles showed sustained release profiles up to 48 h, and the cumulative release amount of HCPT from nanoparticles at 45 °C increased significantly compared to that at 37 °C. Cytotoxicity assay suggests that CS-Fe3O4 does not exhibit noteworthy cytotoxicity against HepG2 cells, but the antitumor activities of HCPT-loaded magnetic composite nanoparticles against HepG2 cells increased significantly in comparison with that of pristine HCPT powder. These results reveal the promising potential of PEG-CS-Fe3O4 as a stable magnetic targeting drug carrier in cancer therapy.Graphical abstractHighlights► A nano-sized carrier with a high saturated magnetization was prepared by a novel reverse ultrasonic emulsification method. ► The unspecific adsorption of protein on PEG-CS-Fe3O4 decreased significantly after coupling PEG. ► Water-insoluble HCPT was loaded onto PEG-CS-Fe3O4 by a subtle precipitation method. ► The HCPT-loaded PEG-CS-Fe3O4 exhibited a high cytotoxicity against HepG2 cells.

Agarose coated gigaporous polystyrene microspheres were evaluated as a novel matrix for immobilized-metal affinity chromatography (IMAC). With four steps, nickel ions were successfully immobilized on the microspheres. The gigaporous structure and chromatographic properties of IMAC medium were characterized. A column packed with the matrix showed low column backpressure and high column efficiency at high flow velocity. Furthermore, this matrix was used for purifying superoxide dismutase (SOD), which was expressed in Escherichia coli (E. coli) in submerged fermentation, on an Äkta purifier 100 system under different flow velocities. The purity of the SOD from this one-step purification was 79% and the recovery yield was about 89.6% under the superficial flow velocity of 3251 cm/h. In conclusion, all the results suggested that the gigaporous matrix has considerable advantages for high-speed immobilized-metal affinity chromatography.

•Chloromethylated polystyrene microspheres were found having inherent fluorescence.•No photo-bleaching or leaking issues were observed owing to the stable structure of the microspheres.•The stable bright fluorescence of the microspheres in dry powder state makes them suitable for coating application.•The fluorescence of microspheres can be selectively quenched by Fe3+ in solution or in cytoplasm.Commercially available polystyrene (PS) fluorescent microspheres are widely used in biological field for tracing, in vivo imaging and calibration of flow cytometry, among other applications. However, these particles do suffer from some drawbacks such as the leakage and photobleaching of organic dyes within them. In the present study, inherently fluorescent properties of PS based microspheres have been explored for the first time. Here we find that a simple chloromethylation reaction endows the polystyrene particles with inherent fluorescence without any subsequent conjugation of an external fluorophore. A possible mechanism for fluorescence is elucidated by synthesizing and investigating p-ethylbenzyl chloride, a compound with similar structure. Significantly, no photobleaching or leaking issues were observed owing to the stable structure of the microspheres. Chloromethylated PS (CMPS) microspheres can keep their perpetual blue fluorescence even in dry powder state making them attractive as a potential coating material. Furthermore, the chloromethyl groups on CMPS microspheres make them very convenient for further functionalization. Poly(ethylene glycol) (PEG) grafted microspheres showed good biocompatibility and negligible cytotoxicity, and could be used to image intracellular Fe3+ due to the selective fluorescence quenching effect of aqueous Fe3+ in cytoplasm.