Wenge Jiang

Yeast cells with an artificial mineral shell: protection and modification of living cells by biomimetic mineralization.

In all living organisms, whether very basic or highly complex, nature provides a multiplicity of materials, architectures, systems and functions. A number of unicellular organisms have an outer-surface proteinaceous membrane as a template for biomineralization. The resultant thin mineral layer is a functional covering. For example, the mineral shell can protect an egg from invasion from the exterior, and the diatom has an ornately patterned silicified shell that evolved as mechanical protection. But most cells in nature cannot make their own hard shells. Here we show a strategy to fashion an artificial shell for the yeast cell so that it has extensive protection. Individual Saccharomyces cerevisiae (S. cerevisiae) cells are coated with a uniform calcium mineral layer by first self-assembly of functional polymers (layer-bylayer technique, LbL) and then in situ mineralization under physiological conditions. The viability of the cells is maintained after the encapsulation. The enclosed cells become inert (stationary phase) and their lifetime can be extended. Furthermore, the mineral shell protects the cell under harsh conditions. The encapsulated S. cerevisiae can even survive the attack of the lytic enzyme zymolyase. The shell can also be used as a scaffold for chemical and biological functionalization. For example, S. cerevisiae becomes magnetic by the incorporation of Fe3O4 nanoparticles in the mineral layer. The present work demonstrates that the artificial shell has a great potential in the storage, protection, delivery, and modification of living cells. Furthermore, insights from systems biology combined with an understanding of the molecular mechanisms of functional shells will facilitate the tailoring of “super cells” through biomimetic mineralization. As S. cerevisiae shares a common life cycle and cell structure with higher eukaryotes, it is a popular and successful model system for understanding eukaryotic biology at the cellular and molecular levels. Like most natural cells, S. cerevisiae cannot induce spontaneous mineralization on its surface. Figure 1a shows a typical scanning electron microscopy (SEM) image of S. cerevisiae. Although the precipitation of calcium phosphate is induced in a supersaturated calcium phosphate solution (concentrations of calcium and phosphate ions are 5.0 mm, ionic strength 0.12m, and pH 6.8), most reactions do not occur on the cell surface. The surface of S. cerevisiae is unchanged during the precipitation and the mineral forms separately (Figure 1b). Although some mineralization can occur on the cell surface, the deposited minerals are not uniform and they cannot form an ideal shell around the cell (Figure 1c). This phenomenon can be explained by the chemical structure of the cell wall of S. cerevisiae, which consists mainly of polysaccharides made up of glucose, mannose, and N-acetylglucosamine. Such a structure hardly induces the templated crystallization of calcium phosphates, which is caused by the relatively low density of electronic charge. Previous studies have already pointed out that the electronic interaction is a key factor in biomineralization. It is generally agreed that proteins that are most active in the mediation of biologically directed mineralization contain regions rich in carboxylates or other charged functional groups. The mineralization skill of living cells can be improved by introducing functional factors onto the cell surface. LbL has been applied as a general approach for the fabrication of multicomponent films on solid supports. We used two polyelectrolytes with opposite charges, poly(diallyldimethylammonium chloride) (PDADMAC) and poly(acrylic sodium) (PAA) are used. PAA has a high density of carboxylate groups, which provide the active nucleation sites for calcium minerals. When the adsorbed PAA molecules are on the outermost layer of the yeast cell, the physicochemical properties of the cells are altered significantly. The carboxylate groups migrate toward the water–polymer interface and bind Ca ions. Upon contact with calcification solution the reorganized surface induces the heterogeneous nucleation of calcium minerals. Besides, it is emphasized the LbL coating does not kill the yeast cells after the immobilization. In situ precipitation of calcium phosphates on the LbLtreated S. cerevisiae cell surface dominates the mineral formation, and the cells can be fully enclosed by the mineral phase (Figure 1d). SEM shows that the surface of S. cerevisiae typically becomes rough and porous and is covered by numerous flakelike nanocrystals. A large-scale SEM view (Figure 1e) shows that practically all of the treated cells have mineral surfaces. The formation of the mineral shell around S. cerevisiae cells is confirmed by transmission electron [*] B. Wang, P. Liu, W. Jiang, Dr. H. Pan, Dr. X. Xu, Prof. R. Tang Center for Biomaterials and Biopathways Department of Chemistry, Zhejiang University Hangzhou, 310027 (China) Fax: (+86)571-8795-3736 E-mail: rtang@zju.edu.cn

Atomic force microscopy reveals hydroxyapatite-citrate interfacial structure at the atomic level.

An approach to organic-inorganic interfacial structure at the atomic level is a great challenge in the studies of biomineralization. We demonstrate that atomic force microscopy (AFM) is powerful tool to discover the biomineral interface in detail. By using a model system of (100) hydroxyapatite (HAP) face and citrate, it reveals experimentally that only a side carboxylate and a surface calcium ion are involved in the binding effect during the citrate adsorption, which is against the previous understandings by using Langmuir adsorption and computer simulation. Furthermore, the adsorbed citrate molecules can use their free carboxylate and hydroxyl groups to be self-assembled on the HAP surface. AFM examination also finds that the presence of citrate molecules on the HAP crystal faces can enhance the adhesion force of the HAP surface. We suggest that the established AFM method can be used for a precise and direct understanding of biointerfaces at the atomic level.

Biomimetically Triggered Inorganic Crystal Transformation by Biomolecules: A New Understanding of Biomineralization

Phase transformation is an important strategy in biomineralization. However, the role of biomolecules in the mineral transition is poorly understood despite the fact that the biomineralization society greatly highlights the organic controls in the formation of the inorganic phase. Here, we report an induced biomimetic phase transformation from brushite (a widely used calcium phosphate precursor in biological cement) to hydroxyapatite (main inorganic composition of skeletal mineral) by citrate (a rich organic component in bone tissue). The transformation in the absence of the organic additive cannot be spontaneously initiated in an aqueous solution with a pH of 8.45 (no phase transition is detected in 4 days), which is explained by a high interfacial energy barrier between brushite-solution and hydroxyapatite-solution interfaces. Citrate can oppositely regulate these two interfaces, which decreases and increases the stabilities of brushite and hydroxyapatite surfaces in the solution, respectively. Thus, the interfacial energy barrier can be greatly reduced in the presence of citrate and the reaction is triggered; e.g., at 1 mM citrate, the total transformation from brushite to hydroxyapatite can be completed within 3 days. The relationship between the transition kinetics and citrate concentration is also studied. The work reveals how the organic components direct solid-solid phase transformation, which can be understood by an energetic control of the interfacial barrier. It is emphasized that the terms of interfacial energy must be taken into account in the studies of phase transformation. We suggest that this biomimetic approach may provide an in-depth understanding of biomineralization.

Unique roles of acidic amino acids in phase transformation of calcium phosphates.

Although phase transformation is suggested as a key step in biomineralization, the chemical scenario about how organic molecules mediate inorganic phase transformations is still unclear. The inhibitory effect of amino acids on hydroxyapatite (HAP, the main inorganic component of biological hard tissues such as bone and enamel) formation was concluded by the previous biomimetic modeling based upon direct solution crystallization. Here we demonstrate that acidic amino acids, Asp and Glu, could promote HAP crystallization from its precursor crystal, brushite (DCPD). However, such a promotion effect could not be observed when the nonacidic amino acids were applied in the transformation-based HAP formation. We found that the specific modification of acidic amino acid on crystal-solution interfaces played a key role in the phase transition. The distinct properties between DCPD and HAP in the solution resulted in an interfacial energy barrier to suppress the spontaneous formation of HAP phase on DCPD phase. Different from the other amino acids, the carboxylate-rich amino acids, Asp and Glu, could modify the interfacial characteristics of these two calcium phosphate crystals to make them similar to each other. The experiments confirmed that the involvement of Asp or Glu reduced the interfacial energy barrier between DCPD and HAP, leading to a trigger effect on the phase transformation. An in-depth understanding about the unique roles of acidic amino acids may contribute to understanding phase transformation controls druing biomineralization.

Self‐Assembled Organic–Inorganic Hybrid Elastic Crystal via Biomimetic Mineralization

It is generally accepted that biomaterials have unique physicochemical properties. [ 1 ] Inspired by biological systems, scientists have been studying biomimetic methods to fabricate functional materials. [ 2 ] Almost all biomaterials possess a common multi-component feature. [ 1 , 3 ] These composites frequently have ordered organic–inorganic hybrid structures and their properties are distinct from the individual components. For example, in a multilayered complex of inorganic aragonite tablets and organic substrate, the fracture toughness of nacre is signifi cantly improved to three thousand times greater than that of synthetic aragonite. [ 4 ] Another striking example is biological bone. In bone, the hydroxyapatite (HAP) phase crystallizes in the nanoscaled channels formed by the staggered alignment of the protein matrix. The typical HAP crystals in bone are plate-shaped with extremely thin thickness (1.5–2 nm), which is the smallest known dimension of the biologically formed crystals. [ 5 ] In nature the organic and inorganic components intimately associate into well-organized hybrid structures to ensure optimal strength and fl exural stress. [ 6 , 7 ] Therefore, in biomimetic designs and fabrications the formation of such ordered nanostructures is a key challenge. The formation of inorganic crystals in living organisms is regulated by the organic matrix. Generally, different organic templates and additives lead to variety in the morphology, size, orientation, and assembly of the inorganic crystal by mediating its nucleation and growth. [ 8 , 9 ] Although many organic– inorganic nanocomposites have been reported, [ 10 ] the selfformation of ultrathin organic–inorganic substructures is still diffi cult to achieve by using a simple bottom-up approach. But the self-formed ordered and intimate combination of organic additives and inorganic crystals at the nanoscale is a crucial requirement for bioactive composites. [ 11 ] Here we prepare an organic–inorganic hybrid crystal by the self-assembly of calcium phosphate, surfactant, and protein. This hybrid crystal is composed of uniform and alternate organic–inorganic layers at the nanoscale. Both the inorganic crystalline phase and

Switchable Chiral Selection of Aspartic Acids by Dynamic States of Brushite

We herein show the chiral recognition and separation of aspartic acid (Asp) enantiomers by achiral brushite due to the asymmetries of their dynamical steps in its nonequilibrium states. Growing brushite has a higher adsorption affinity to d-Asp, while l-Asp is predominant on the dissolving brushite surface. Microstructural characterization reveals that chiral selection is mainly attributed to brushite [101] steps, which exhibit two different configurations during crystal growth and dissolution, respectively, with each preferring a distinct enantiomer due to this asymmetry. Because these transition step configurations have different stabilities, they subsequently result in asymmetric adsorption. By varying free energy barriers through solution thermodynamic driving force (i.e., supersaturation), the dominant nonequilibrium intermediate states can be switched and chiral selection regulated. This finding highlights that the dynamic steps can be vital for chiral selection, which may provide a potential pathway for chirality generation through the dynamic nature.