Zhihua An

Crystal Growth Inhibitors for the Prevention of l-Cystine Kidney Stones Through Molecular Design

Taking the Cystine Kidney stones that form from l-cystine are much less common than those forming from calcium oxalate monohydrate, but are more likely to cause chronic kidney disease. Rimer et al. (p. 337; see the cover; see the Perspective by Coe and Asplin) designed two structural mimics for l-cystine. Atomic force microscopy showed that at low concentrations, the mimics could change the l-cystine crystal habit and inhibit overall crystal growth. These structural mimics may thus offer hope for treating cystinuria. Structural mimics for l-cystine may provide drug treatments for certain types of kidney stones. Crystallization of l-cystine is a critical step in the pathogenesis of cystine kidney stones. Treatments for this disease are somewhat effective but often lead to adverse side effects. Real-time in situ atomic force microscopy (AFM) reveals that l-cystine dimethylester (L-CDME) and l-cystine methylester (L-CME) dramatically reduce the growth velocity of the six symmetry-equivalent {100} steps because of specific binding at the crystal surface, which frustrates the attachment of l-cystine molecules. L-CDME and L-CME produce l-cystine crystals with different habits that reveal distinct binding modes at the crystal surfaces. The AFM observations are mirrored by reduced crystal yield and crystal size in the presence of L-CDME and L-CME, collectively suggesting a new pathway to the prevention of l-cystine stones by rational design of crystal growth inhibitors.

Molecular assembly of biomimetic microcapsules

Our recent work on the fabrication of microcapsules comprised of human serum albumin (HSA) and -α-dimyristoylphosphatitic acid (DMPA) by means of stepwise adsorption of HSA and DMPA on fluid droplet surfaces or charged colloids and subsequent dissolution of the cores is reviewed. The lipid self-assembles as a bilayer on the protein surface and the completed microcapsule serves as a biomimetic membrane model. The DMPA/HSA microcapsules have good biocompatibility and the potential for the insertion of recognition units in the lipid bilayers. The structure and properties of the lipid/protein microcapsules are described and their potential for applications in sustained drug release are introduced.

Polyelectrolyte microcapsule interactions with cells in two- and three-dimensional culture

Microcapsules fabricated by layer-by-layer self-assembly have unique physicochemical properties that make them attractive for drug delivery applications. This study chiefly investigated the biocompatibility of one of the most stable types of microcapsules, those composed of poly-(sodium 4-styrene sulfonate) [PSS] and poly-(allylamine hydrochloride) [PAH], with cells cultured on two-dimensional (2D) substrates and in three-dimensional (3D) matrices. C6 glioma and 3T3 fibroblast cell morphology was observed after 24h of co-culture with PSS/PAH microcapsules on a 2D substrate. Cells were also cultured with four other types of microcapsules, each composed of at least one naturally occurring polyelectrolyte. At microcapsule to cell ratios up to 100:1, it was found that PSS/PAH microcapsules do not affect number of viable cells more substantially than do the other microcapsules investigated. However, differences in number of viable cells were found as a function of microcapsule composition, and our results suggest particular biochemical interactions between cells and internalized microcapsules, rather than mechanical effects, are responsible for these differences. We then investigated the effects of PSS/PAH microcapsules on cells embedded in 3D collagen matrices, which more closely approximate the tumor environments in which microcapsules may be useful drug delivery agents. Matrix structure, cell invasion, and volumetric spheroid growth were investigated, and we show that these microcapsules have a negligible effect on cell invasion and tumor spheroid growth even at high concentration. Taken together, this work suggests that PSS/PAH microcapsules have sufficiently high biocompatibility with at least some cell lines for use as proof of principle drug delivery agents in in vitro studies.

Suberoylanilide hydroxamic acid limits migration and invasion of glioma cells in two and three dimensional culture

High grade gliomas are aggressive cancers that are not well addressed by current chemotherapies, in large measure because these drugs do not curtail the diffuse invasion of glioma cells into brain tissue surrounding the tumor. Here, we investigate the effects of suberoylanilide hydroxamic acid (SAHA) on glioma cells in 2D and 3D in vitro assays, as SAHA has previously been shown to significantly increase apoptosis, decrease proliferation, and interfere with migration in other cell lines. We find that SAHA has significant independent effects on proliferation, migration, and invasion. These effects are seen in both 2D and 3D culture. In 3D culture, with glioma spheroids embedded in collagen I matrices, SAHA independently limits both glioma invasion and the reorganization of the tumor surroundings that usually proceeds such invasion. The decreased matrix reorganization and invasion is not accompanied by decreased production or activity of matrix-metalloproteases but instead may be related to increased cell-cell adhesion.

Illusory spirals and loops in crystal growth

Significance Molecular mechanisms of crystal growth from solution remain ill-defined. Scanning probe microscopies have begun to illustrate what was before insightful theory. The in situ observations described here for hexagonal l-cystine crystals, which are known to form kidney stones, demonstrate that crystals with certain symmetries can exhibit unusual structural and growth behaviors that produce unexpected and deceptive morphological features. Such features can appear to violate a classic theory of crystal growth enshrined more than 60 y ago and could lead to incorrect conclusions about growth mechanisms. The theory of dislocation-controlled crystal growth identifies a continuous spiral step with an emergent lattice displacement on a crystal surface; a mechanistic corollary is that closely spaced, oppositely winding spirals merge to form concentric loops. In situ atomic force microscopy of step propagation on pathological l-cystine crystals did indeed show spirals and islands with step heights of one lattice displacement. We show by analysis of the rates of growth of smaller steps only one molecule high that the major morphological spirals and loops are actually consequences of the bunching of the smaller steps. The morphology of the bunched steps actually inverts the predictions of the theory: Spirals arise from pairs of dislocations, loops from single dislocations. Only through numerical simulation of the growth is it revealed how normal growth of anisotropic layers of molecules within the highly symmetrical crystals can conspire to create features in apparent violation of the classic theory.

Stabilized complex film formed by co-adsorption of β-lactoglobulin and phospholipids at liquid/liquid interface

Abstract A complex film has been formed at a drop surface by the co-adsorption of β-lactoglobulin with the l -α-dipalmitoylphosphatidylcholine (DPPC), l -α-dipalmitoylphosphatidyl-ethanolamine (DPPE), and l -α-dipalmitoylphosphatidic acid sodium salt (DPPA), respectively, at the water/chloroform interface. By using pendent drop technique we studied the headgroup effect of lipids on the kinetics of co-adsorption layers. It was found experimentally that a folded drop surface was formed during co-adsorption and the headgroup of lipids affected on the formation rate of the folded drop surface. Such a skinlike film demonstrates that there exits an strong interaction between β-lactoglobulin and each lipid. By depositing one lipid mono- and bilayers onto a solid surface morphology of the mixed lipid/β-lactoglobulin layers has been investigated by atomic force microscopy (AFM).

Synthesis of cerium oxide particles via polyelectrolyte controlled nonclassical crystallization for catalytic application

A polyelectrolyte controlled nonclassical crystallization method has been used to synthesize cerium oxide particles with tailored morphology. The results reveal that the as-prepared particles exhibit high specific surface area and enhanced oxygen storage capacity.

Attachment of Calcium Oxalate Monohydrate Crystals on Patterned Surfaces of Proteins and Lipid Bilayers

The attachment of calcium oxalate monohydrate (COM) crystals to renal tubules is thought to be one of the critical steps of kidney stone formation. Patterns of phosphatidylserine (DPPS) bilayers and osteopontin (OPN) were fabricated on silica substrates through the combination of a microcontact printing technique and fusion of lipid vesicles to create spatially organized surfaces of lipids and proteins that may mimic renal tubule surfaces while allowing direct visualization of the competition for COM attachment to compositionally different regions. In the case of DPPS-OPN patterns, micrometer-sized COM crystals dispersed in saturated aqueous calcium oxalate solutions attached preferentially to the OPN regions, in agreement with other in vitro studies that have suggested a binding affinity of OPN to COM crystal surfaces. COM crystals attached with nearly equal coverage to OPN and DPPS surfaces alone, suggesting that the preferential segregation of COM crystals to the OPN regions on the patterned surfaces reflects reversible attachment of micrometer-sized COM crystals capable of Brownian motion. These attached microcrystals then grow larger over time during immersion in the supersaturated calcium oxalate solutions. Free OPN, a major constituent in urine, adsorbs on COM crystals and suppresses attachment to DPPS, suggesting a link between OPN and reduced attachment of COM crystals to renal epithelium. This patterning protocol can be expanded to other urinary molecules, providing a convenient approach for understanding the effects of biomolecules on COM crystal attachment and the pathogenesis of kidney stones.