Dongxia Hao

Porogen effects in synthesis of uniform micrometer-sized poly(divinylbenzene) microspheres with high surface areas

A new method of synthesizing uniform poly(divinylbenzene) (polyDVB) microspheres with high specific surface areas was designed by combining Shirasu porous glass (SPG) membrane emulsification, suspension polymerization, and post-crosslinking techniques. It was shown that the physicochemical properties of porogens have a great influence on the size distribution and porous features of microspheres. The low aqueous solubility of porogen facilitated preparation of uniform emulsions and microspheres, and high aqueous solubility led to polydispersed emulsions and poor microsphere yields. Such aqueous solubility effects can be tailored by adding a low molecular weight polystyrene (LPST) as costabilizer in porogen, thus improving the uniformity of microspheres. Moreover, different affinities of porogens for copolymers demonstrate various contributions to specific surface areas of microspheres in suspension polymerization especially post-crosslinking. Solvating porogen requires a much higher addition than nonsolvating porogen to obtain equal specific surface areas in polymerization, but has more potential to enhance the specific surface area in post-crosslinking. Two kinds of uniform microspheres were obtained with high specific surface areas, up to 706.6 m2/g by heptane and 937.5 m2/g by toluene.

Fabrication strategy for amphiphilic microcapsules with narrow size distribution by premix membrane emulsification

Amphiphilic co-polymer, which can maintain the stability of proteins and increase the protein loading efficiency, is considered as an exploring-worthy biodegrade polymer for drug delivery. However, amphiphilic microcapsules prepared by conventional methods, such like mechanical stirring and spray-drying methods, exhibit broad size distributions due to its hydrophilic sequences, leading to poor reproducibility. In this study, we employed poly(monomethoxypoly ethylene glycol-co-D,L-lactide) (mPEG-PLA, PELA), one of common amphiphilic polymers, as model to focus on investigating the process parameters and mechanisms to prepare PELA microcapsules with narrow size distribution and regular sphericity by combining premix membrane emulsification and double emulsion technique. The coarse double emulsion with broad size distribution was repeatedly pressed through Shirasu Porous Glass (SPG) membrane with relatively high pressure to form the fine emulsion with narrow size distribution. Then, the microcapsules with narrow size distribution can be obtained by solvent extraction method. It was found that it was more difficult to obtain PELA microcapsules with narrow size distribution and smooth surface due to its amphiphilic property, compared with the cases of PLA and PLGA. The smooth surface morphology was found to be related to several factors including internal water phase with less volume, slower stirring rate during solidification and using ethyl acetate as oil phase. It was also found that mass ratio of hydrophilic mPEG, stabilizer PVA concentration in external water phase and transmembrane pressure played important role on the distribution of microcapsules size. The suitable preparation conditions were determined as follows: for the membrane with pore size of 2.8 μm, the mass ratio of PLA/mPEG was 19:1, volume ratio of W(1)/O was 1:10 and O/W(2) was 1:5, PVA concentration (w/v) was 1.0%, magnetic stirring rate during solidification was 60 rpm and 300 kPa was chosen as transmembrane pressure. There was a linear relationship between the diameter of microcapsules and the pore size of the membranes. Finally, by manipulating the process parameters, PELA microcapsules with narrow size distributions (coefficient of variation was less than 15%), smooth morphology and various sizes, were obtained. Most importantly, the key factors affecting fabrication have been revealed and mechanisms were illustrated in detail, which would shed light on the research of amphiphilic polymer formulation.

Chitosan-based mucosal adjuvants: Sunrise on the ocean.

Abstract Mucosal vaccination, which is shown to elicit systemic and mucosal immune responses, serves as a non-invasive and convenient alternative to parenteral administration, with stronger capability in combatting diseases at the site of entry. The exploration of potent mucosal adjuvants is emerging as a significant area, based on the continued necessity to amplify the immune responses to a wide array of antigens that are poorly immunogenic at the mucosal sites. As one of the inspirations from the ocean, chitosan-based mucosal adjuvants have been developed with unique advantages, such as, ability of mucosal adhesion, distinct trait of opening the junctions to allow the paracellular transport of antigen, good tolerability and biocompatibility, which guaranteed the great potential in capitalizing on their application in human clinical trials. In this review, the state of art of chitosan and its derivatives as mucosal adjuvants, including thermo-sensitive chitosan system as mucosal adjuvant that were newly developed by authors group, was described, as well as the clinical application perspective. After a brief introduction of mucosal adjuvants, chitosan and its derivatives as robust immune potentiator were discussed in detail and depth, in regard to the metabolism, safety profile, mode of actions and preclinical and clinical applications, which may shed light on the massive clinical application of chitosan as mucosal adjuvant.

MULTI-SCALE STRUCTURES IN EMULSION AND MICROSPHERE COMPLEX SYSTEMS

Multi-scale structures involved in emulsion and microsphere complex systems are presented and discussed. The stability and spatio-temporal structures of emulsions, as well as nano-structures formed on the surface of microspheres after polymerization, are affected by the molecular emulsifier/stabilizer structures and the adsorbed emulsifier/stabilizer nano-structures on the oil/water interface. The broad size distribution and variation of surface features of droplets are responsible for variations of the adsorbed emulsifier/stabilizer structures and the stability of the emulsions. On the other hand, preparation of a uniformly sized emulsion and employment of a combined emulsifier/stabilizer system can preserve the stability of the emulsions and microspheres. The above phenomena should be modeled by a multi-scale method, in order to maintain the stability of individual emulsion systems and realize the desired nano-structures of microspheres by choosing adequate emulsifier/stabilizer and experimental parameters.

Comparison of covalent and physical immobilization of lipase in gigaporous polymeric microspheres.

Lipase (EC 3.1.1.3) is a versatile enzyme which has been widely used in ester-reaction industries. We have previously discovered that gigaporous polystyrene (PST) microspheres can be used as a novel immobilization carrier for lipase. In this work, a series of gigaporous microspheres with different densities of epoxy group including poly(glycidyl methacrylate) (PGMA) and poly(styrene-co-glycidyl methacrylate) [P(ST-GMA)] were evaluated as lipase immobilization carriers, which were also compared with gigaporous PST microspheres and the commercial immobilized lipase Novozym 435. Lipase immobilized in gigaporous PGMA microspheres showed the highest activity yield, reusability, and stability as well as the best affinity for the substrate. The characterizations of adsorption curves, the change of epoxy group amounts, and hydrophobic–hydrophilic properties of the microspheres were carried out to investigate the interaction between lipase molecules and carriers. It was found that covalent binding played a key role in improving the properties of lipase immobilized in gigaporous PGMA microspheres.

Multiscale evaluation of pore curvature effects on protein structure in nanopores

Protein structure in nanopores is an important determinant in porous substrate utilization in biotechnology and materials science. To date, accurate residue details of pore curvature induced protein binding and unfolding were still unknown. Here, a multiscale ensemble of chromatography, NMR hydrogen and deuterium (H/D) exchange, confocal scanning and molecular docking simulations was combined to obtain the protein adsorption information induced by pore size and curvature. Lysozyme and polystyrene microspheres within pores in the 14-120 nm range were utilized as models. With pore size increasing, the bound lysozyme presented a tendency of significantly decreased retention, less unfolding and fewer interacted sites. However, such a significant dependence between pore curvature and protein size only existed in a limited micro-pore range comparable to protein sizes. The mechanism behind the above events could be attributed to the diverse protein interaction area determined by pore curvature and size change, by models calculating the binding of lysozyme onto surfaces. Another surface of opposite curvature for nanoparticles was also calculated and compared, the rules were similar but with opposite direction and such a critical size also existed. These studies of proteins on curved interfaces may ultimately help to guide the design of novel porous materials and assist in the discrimination of the target protein from molecular banks.

Residue-level elucidation of the ligand-induced protein binding on phenyl-argarose microspheres by NMR hydrogen/deuterium exchange technique

Protein–ligand interactions on liquid–solid interfaces governed the design of functional biomaterials. However, accurate residue details of ligand induced protein binding and unfolding on an interface were still unknown by the current ensemble of protein structure characterizations. Here, a hydrogen/deuterium (H/D) approach coupled with analysis of NMR TOCSY spectra and the solvent accessible surface area (SASA) was designed to enable residue level understanding of lysozyme adsorbed at a phenyl-ligand modified surface. Results showed that the binding sites and unfolding of lysozyme molecules on phenyl-agarose microspheres demonstrated significant ligand-density dependence and protein-coverage dependence. Either increasing ligand density or decreasing adsorption coverage would lead to more binding sites and unfolding of the protein molecules. With the multipoint adsorption strengthening, the protein molecule changed from lying end-on to side-on. Finally, Molecular Dock simulation was utilized to evaluate the NMR determined binding sites based on energy ranking of the binding. It confirmed that this NMR approach represents a reliable route to in silico abundant residue-level structural information during protein interaction with biomaterials.

Towards A Deeper Understanding of the Interfacial Adsorption of Enzyme Molecules in Gigaporous Polymeric Microspheres

Compared with the one immobilized in the conventional mesoporous microspheres, the enzyme immobilized in gigaporous microspheres showed much higher activity and better stability. To gain a deeper understanding, we herein selected lipase as a prototype to comparatively analyze the adsorption behavior of lipase at interfaces in gigaporous and mesoporous polystyrene microspheres at very low lipase concentration, and further compared with the adsorption on a completely flat surface (a chip). Owing to the limited space of narrow pores, lipase molecules were inclined to be adsorbed as a monolayer in mesoporous microspheres. During this process, the interaction between lipase molecules and the interface was stronger, which could result in the structural change of lipase molecular and compromised specific activity. In addition to monolayer adsorption, more multilayer adsorption of enzyme molecules also occurred in gigaporous microspheres. Besides the adsorption state, the pore curvature also affected the lipase adsorption. Due to the multilayer adsorption, the excellent mass transfer properties for the substrate and the product in the large pores, and the small pore curvature, lipase immobilized in gigaporous microspheres showed better behaviors.

A novel rProtein A chromatographic media for enhancing cleaning-in-place performance

Protein A chromatography has been a popular method for purification of therapeutic monoclonal antibodies (mAb). Protein A chromatographic media using alkali-resistant rProtein A ligands from site-directed coupling method have been pursued for both high dynamic capacities and excellent stabilities. However, the mechanism of rProtein A leaking under cleaning-in-place (CIP) conditions is not very clear and difficulties have been commonly encountered when improving the medias chromatographic performance. We investigated the chromatographic performance of site-directed coupled rProtein A chromatographic media during CIP procedure. Trace amount of ligands leaked during the chromatographic medias incubation in 0.5 M NaOH was detected, explaining for the decline of chromatographic medias CIP performance. Decrease of rProtein As concentration in 0.5 M NaOH was consistent with chromatographic medias binding capacity. A novel rProtein A chromatographic media were prepared by site-directed coupling a newly-constructed alkali-resistant rProtein A to highly cross-linked agarose-based matrix. The media had a dynamic binding capacity of 63.2 mg hIgG/mL higher than 48.1 mg hIgG/mL of the commercial one, and the CIP performance was improved greatly with the remained dynamic binding capacity increased from 86% to 95% of the initial value after 40 CIP cycles.

Microscopic insight into role of protein flexibility during ion exchange chromatography by nuclear magnetic resonance and quartz crystal microbalance approaches.

Driven by the prevalent use of ion exchange chromatography (IEC) for polishing therapeutic proteins, many rules have been formulated to summarize the different dependencies between chromatographic data and various operational parameters of interest based on statically determined interactions. However, the effects of the unfolding of protein structures and conformational stability are not as well understood. This study focuses on how the flexibility of proteins perturbs retention behavior at the molecular scale using microscopic characterization approaches, including hydrogen-deuterium (H/D) exchange detected by NMR and a quartz crystal microbalance (QCM). The results showed that a series of chromatographic retention parameters depended significantly on the adiabatic compressibility and structural flexibility of the protein. That is, softer proteins with higher flexibility tended to have longer retention times and stronger affinities on SP Sepharose adsorbents. Tracing the underlying molecular mechanism using NMR and QCM indicated that an easily unfolded flexible protein with a more compact adsorption layer might contribute to the longer retention time on adsorbents. The use of NMR and QCM provided a previously unreported approach for elucidating the effect of protein structural flexibility on binding in IEC systems.