Defect Induced Charge Redistribution and Enhanced Adsorption of Lysozyme on Hydroxyapatite for Efficient Antibacterial Activity.
Wang Ming,Zhu Yingchun
Langmuir : the ACS journal of surfaces and colloids
Defects in hydroxyapatite (HA) have attracted increasing research interest due to their significant functions to increase the bioactivity and antibacterial ability of hard-tissue implants. However, little is known about the natural property and functional mechanism of the defects in HA. Herein, we reported on the defect property concerned with the coordination state and charge distribution in Al doped HA, as well as the consequent interface and protein capture ability for improved antibacterial activity. Systemic investigations suggested that Al replacing Ca in HA induced coordination defect with decreased coordination number and bond distance, caused charge transfer and redistribution of surrounding O atom and resulted in an increase in negative charge of coordinated O atoms. These O atoms coordinated with Al further served as docking sites for lysozyme molecules via electrostatic and H-bonding interaction. The capacity of lysozyme adsorption for Al-HA increased approximately 10-fold more than that of HA, which significantly increased the antibacterial activity through lysozyme-catalyzed splitting of cell wall of bacteria. Moreover, in vitro studies indicated that Al-HA materials showed good cytocompatibility. These findings not only provided new insights into the important effect of defects on the performances of HA biomaterials by modulation of the coordination state, charge distribution, and chemical activity, but also proposed a promising method for efficient antibacterial activity of HA biomaterials.
10.1021/acs.langmuir.1c01666
Adsorption of human lysozyme onto hydroxyapatite. Identification of its adsorbing site using site-directed mutagenesis.
Aizawa T,Koganesawa N,Kamakura A,Masaki K,Matsuura A,Nagadome H,Terada Y,Kawano K,Nitta K
FEBS letters
To elucidate hydroxyapatite-protein interaction, mutant human lysozymes in which the surface charge was modified by site-directed mutagenesis were used. Five mutant human lysozymes (K1A, K13A, K33A, R10A, R14A) were expressed in yeast. The chromatographic behavior of these lysozymes was studied with a HPLC hydroxyapatite column. Elution molarities of K1A and R14A mutants were greatly lowered. While Lys-13 and Arg-10 are located around Lys-1 and Arg-14, K13A and R10A mutants bound onto hydroxyapatite stronger than K1A and R14A mutants. In combination with an X-ray crystal structure of human lysozyme, it is concluded that the adsorbing site of human lysozyme is at the back of the active site and that Arg-14, Lys-1, Arg-10 and Lys-13 play important roles in binding.
10.1016/s0014-5793(97)01621-9
Identification of the adsorbing site of lysozyme onto the hydroxyapatite surface using hydrogen exchange and 1H NMR.
Nagadome H,Kawano K,Terada Y
FEBS letters
The lysozyme-hydroxyapatite interaction was studied by measuring individual hydrogen-deuterium (H-D) exchange rates of amide protons. The H-D exchange reaction was initiated by transferring the lysozyme adsorbed on hydroxyapatite powder from H2O into D2O. After various H-D exchange time periods (pH 7.0, 25 degrees C), the complex was dissociated and the remaining hydrogen label was determined by 2D NMR analysis. The H-D exchange rate of amide protons of residues 9, 11, 13, and 83 was slowed in the hydroxyapatite-lysozyme complex compared with free lysozyme. Residues 9, 11 and 13 positioned at the back of the active site would be the location of the binding site.
10.1016/0014-5793(93)81506-u
Hydroxyapatite particles as drug carriers for proteins.
Tomoda Keishiro,Ariizumi Hidehiko,Nakaji Takatomo,Makino Kimiko
Colloids and surfaces. B, Biointerfaces
Recently, much attention has been paid to hydroxyapatite (HA) particles as protein drug carriers. HA is biological substance like tooth and bone, and HA has biodegradable and biocompatible property. Also HA has high affinity for versatile substances, such as proteins. The aim of this study was to prepare HA particles and to evaluate the effects of various experimental conditions on particles properties (i.e. shape and amount of protein adsorption to HA). HA particles were prepared by three methods including solvent diffusion methods, the method using sintering porous HA microgranules, and homogeneous precipitation method. Complicated spiky crystals were prepared by solvent diffusion method, whereas spherical amorphous agglomerates were prepared by sintering of porous HA granules. Spherical particles were obtained by homogeneous precipitation method and were well dispersed. The spherical particles were composed of minute spiky crystals. The temperature and time duration of the producing process and the amount of additives, such as urea and EDTANa(2), strongly affected the particle shape and size. When the production process is kept under low temperature, tiny crystals were not mutually sintered and also their aggregation was not deposited. Also, inhomogeneous crystal growth occurred when the reaction time was long. Addition of enough amount of EDTANa(2) to reaction solution made the small and spherical HA particles. Bovine serum albumin (BSA) or lysozyme hydrochloride (LSZ)-adsorbed HA particles were prepared. The affinity of HA particles and release profile of proteins from the particles were evaluated. X-ray powder diffraction measurements suggested that the surface area ratio of a plane against total surface area of HA crystal became smaller by decreasing EDTANa(2) concentration. The surface of plane a is positively charged but that of plane c is negatively charged. HA particle size decreased as EDTANa(2) concentration increased. The amounts of BSA and LSZ adsorbing onto HA particle surface were different, since BSA was negatively charged and LSZ was positively charged in the solution. Therefore BSA seems to adsorb onto plane c, whereas LSZ adsorb onto plane a. The differences of the ratio of surface a plane against the total surface area of HA particles and particles size influenced the amount of protein adsorption. The sustained release of BSA and LSZ from HA particles were observed for two weeks.
10.1016/j.colsurfb.2009.10.039
Endowing Orthopedic Implants' Antibacterial, Antioxidation, and Osteogenesis Properties Through a Composite Coating of Nano-Hydroxyapatite, Tannic Acid, and Lysozyme.
Wang Guofeng,Zhu Yaxin,Zan Xingjie,Li Meng
Frontiers in bioengineering and biotechnology
There is a substantial global market for orthopedic implants, but these implants still face the problem of a high failure rate in the short and long term after implantation due to the complex physiological conditions in the body. The use of multifunctional coatings on orthopedic implants has been proposed as an effective way to overcome a range of difficulties. Here, a multifunctional (TA@HA/Lys) coating composed of tannic acid (TA), hydroxyapatite (HA), and lysozyme (Lys) was fabricated in a layer-by-layer (LBL) manner, where TA deposited onto HA firmly stuck Lys and HA together. The deposition of TA onto HA, the growth of (TA@HA/Lys), and multiple related biofunctionalities were thoroughly investigated. Our data demonstrated that such a hybrid coating displayed antibacterial and antioxidant effects, and also facilitated the rapid attachment of cells [both mouse embryo osteoblast precursor cells (MC3T3-E1) and dental pulp stem cells (DPSCs)] in the early stage and their proliferation over a long period. This accelerated osteogenesis and promoted bone formation . We believe that our findings and the developed strategy here could pave the way for multifunctional coatings not only on orthopedic implants, but also for additional applications in catalysts, sensors, tissue engineering, etc.
10.3389/fbioe.2021.718255
Decomposition of proteins by photocatalytic Ti(IV)-doped calcium hydroxyapatite particles.
Kandori Kazuhiko,Oketani Makoto,Wakamura Masato
Colloids and surfaces. B, Biointerfaces
The decomposition of protein molecules on the surface of Ti(IV)-doped calcium hydroxyapatite (TiHap) particles with a Ti/(Ca+Ti) atomic ratio among 0-0.20 under UV irradiation of 365 nm in wavelength was disclosed. The acidic bovine serum albumin (BSA), neutral myoglobin (MGB) and basic lysozyme (LSZ) were employed as a model of pathogenic proteins. The photocatalytic activities of TiHap particles were estimated from the decomposition of each protein under 1 mW/cm(2) UV irradiation dispersed in 10 mL quartz tube. The concentrations of each protein in the supernatant after centrifugation during the UV irradiation were determined both by a HPLC and a SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis methods. No change in BSA concentration ([BSA]) by UV irradiation was observed for all the unheated original TiHap particles with low photocatalytic activity. The similar results were observed for the systems employed heat treated particles endowed a high photocatalytic activity by heat treatment at 650°C for 1h. These results indicated that the decomposition of BSA molecules is hard to take place. The heme structured MGB molecules are decomposed by UV irradiation irrespective of the presence of TiHap particles. In the case of heat treated particles, MGB molecules were further decomposed by the UV irradiation. The strongest photocatalytic activity was observed for the decomposition systems of LSZ by using heat treated particles. In this system, all the TiHap particles completely decomposed LSZ molecules after started the UV irradiation. It was concluded that the ability of decomposition of proteins is strongly related to the molecular weight and rigidity of proteins molecules. The LSZ molecule with low molecular weight and rigid structure was easily decomposed on the surface of heat treated TiHap particles under UV irradiation.
10.1016/j.colsurfb.2012.09.022
Biomimetic mineralization of nano-sized, needle-like hydroxyapatite with ultrahigh capacity for lysozyme adsorption.
Ma Yi,Zhang Juan,Guo Shanshan,Shi Jie,Du Wenying,Wang Zheng,Ye Ling,Gu Wei
Materials science & engineering. C, Materials for biological applications
Because of its superior biocompatibility, hydroxyapatite (HA) has been widely exploited as a promising vehicle to deliver a broad range of therapeutics in a variety of biological systems. Herein, we report a biomimetic process to prepare nano-sized, colloidal stable HA with needle-like morphology by using carboxymethyl cellulose (CMC) as the template. It was revealed that the needle-like HA was transformed from the spherical amorphous calcium phosphate (ACP) nanoparticles after a 14-day period of aging under ambient conditions. The needle-like HA/CMC exhibited an ultra-high lysozyme adsorption capacity up to 930-940mg/g. Moreover, a sustained and pH-sensitive release of adsorbed lysozyme from HA/CMC was evidenced. Therefore, our biomimetic needle-like HA/CMC nanoparticles hold great potential in serving as an efficient carrier for the delivery and controlled release of lysozyme.
10.1016/j.msec.2016.06.021
Lysozyme loading and release from Se doped hydroxyapatite nanoparticles.
Wang Yanhua,Hao Hang,Zhang Shengmin
Materials science & engineering. C, Materials for biological applications
Element-substituted hydroxyapatite (HA) based nanocomposites have become a promising therapeutic material for improving bone defect repair. Selenium substituted HA nanoparticles can both induce apoptosis of bone tumor cells and enhance osteointegration. However, the effect of selenite ions on the proteins in combination with the HA nanoparticles remains to be elucidated. Here, we investigated the influence of selenium doping concentration on the loading and release of lysozyme (LSM) as a model protein drug. The selenium substituted HA-LSM composites with different doping concentrations were synthesized and characterized. The subsequent delivery of lysozyme was studied in a phosphate buffer solution (PBS). We found that selenium substituted HA-LSM composites with Se:P=10% showed the highest amount of lysozyme loading (41.7%), whereas the amount of lysozyme loaded in undoped HA nanoparticles was the lowest (34.1%). The doped selenium interacts with lysozyme molecules, which leads to the increase of β-sheet and unordered, and the decrease of self-association, α-helix and β-turns in protein structures. Moreover, selenium addition significantly slows the protein release from HA-LSM composites. The composites with Se:P=10% release lysozyme at the slightly slower rate among the samples with different Se doping concentrations. It also shows that the released lysozyme retains most of its enzymatic activity.
10.1016/j.msec.2015.12.060