Development of paclitaxel-loaded poly(lactic acid)/hydroxyapatite core-shell nanoparticles as a stimuli-responsive drug delivery system.

Biodegradable nanoparticles have been well studied as biocompatible delivery systems. Nanoparticles of less than 200 nm in size can facilitate the passive targeting of drugs to tumour tissues and their accumulation therein via the enhanced permeability and retention (EPR) effect. Recent studies have focused on stimuli-responsive drug delivery systems (DDS) for improving the effectiveness of chemotherapy; for example, pH-sensitive DDS depend on the weakly acidic and neutral extracellular pH of tumour and normal tissues, respectively. In our previous work, core-shell nanoparticles composed of the biodegradable polymer poly(lactic acid) (PLA) and the widely used inorganic biomaterial hydroxyapatite (HAp, which exhibits pH sensitivity) were prepared using a surfactant-free method. These PLA/HAp core-shell nanoparticles could load 750 wt% of a hydrophobic model drug. In this work, the properties of the PLA/HAp core-shell nanoparticles loaded with the anti-cancer drug paclitaxel (PTX) were thoroughly investigated in vitro. Because the PTX-containing nanoparticles were approximately 80 nm in size, they can be expected to facilitate efficient drug delivery via the EPR effect. The core-shell nanoparticles were cytotoxic towards cancer cells (4T1). This was due to the pH sensitivity of the HAp shell, which is stable in neutral conditions and dissolves in acidic conditions. The cytotoxic activity of the PTX-loaded nanoparticles was sustained for up to 48 h, which was suitable for tumour growth inhibition. These results suggest that the core-shell nanoparticles can be suitable drug carriers for various water-insoluble drugs.

Evaluation of Drug-Loading Ability of Poly(Lactic Acid)/Hydroxyapatite Core-Shell Particles.

Poly(lactic acid)/hydroxyapatite (PLA/HAp) core-shell particles are prepared using the emulsification method. These particles are safe for living organisms because they are composed of biodegradable polymers and biocompatible ceramics. These particles are approximately 50-100 nm in size, and their hydrophobic substance loading can be controlled. Hence, PLA/HAp core-shell particles are expected to be used as drug delivery carriers for hydrophobic drugs. In this work, PLA/HAp core-shell particles with a loading of vitamin K1 were prepared, and their drug-loading ability was evaluated. The particles were 40-80 nm in diameter with a PLA core and a HAp shell. The particle size increased with an increase in the vitamin K1 loading. The drug-loading capacity (LC) value of the particles, an indicator of their drug-loading ability, was approximately 250%, which is higher than the previously reported values. The amount of vitamin K1 released from the particles increased as the pH of the soaking solution decreased because the HAp shell easily dissolved under the acidic conditions. The PLA/HAp particles prepared in this work were found to be promising candidates for drug delivery carriers because of their excellent drug-loading ability and pH sensitivity.

Structures and Dissolution Behaviors of Quaternary CaO-SrO-P2O5-TiO2 Glasses.

Calcium phosphate glasses have a high potential for use as biomaterials because their composition is similar to that of the mineral phase of bone. Phosphate glasses can dissolve completely in aqueous solution and can contain various elements owing to their acidity. Thus, the glass can be a candidate for therapeutic ion carriers. Recently, we focused on the effect of strontium ions for bone formation, which exhibited dual effects of stimulating bone formation and inhibiting bone resorption. However, large amounts of strontium ions may induce a cytotoxic effect, and there is a need to control their releasing amount. This work reports fundamental data for designing quaternary CaO-SrO-P2O5-TiO2 glasses with pyro- and meta-phosphate compositions to control strontium ion-releasing behavior. The glasses were prepared by substituting CaO by SrO using the melt-quenching method. The SrO/CaO mixed composition exhibited a mixed cation effect on the glassification degree and ion-releasing behavior, which showed non-linear properties with mixed cation compositions of the glasses. Sr2+ ions have smaller field strength than Ca2+ ions, and the glass network structure may be weakened by the substitution of CaO by SrO. However, glassification degree and chemical durability of pyro- and meta-phosphate glasses increased with substituted all CaO by SrO. This is because titanium groups in the glasses are closely related to their glass network structure by SrO substitution. The P-O-Ti bonds in pyrophosphate glass series and TiO4 tetrahedra in metaphosphate glass series increased with substitution by SrO. The titanium groups in the glasses were crosslink and/or coordinate phosphate groups to improve glassification degree and chemical durability. Sr2+ ion releasing amount of pyrophosphate glasses with >83% SrO substitution was larger than 0.1 mM at day seven, an amount that reported enhanced bone formation by stimulation of osteogenic markers.

Development of orthophosphosilicate glass/poly(lactic acid) composite anisotropic scaffolds for simultaneous reconstruction of bone quality and quantity.

Reconstruction of organ-specific architecture is necessary to recover the original organ function. The anisotropic structure of bone tissue is strongly related to the collagen fibril alignment and bone apatite crystal direction. Bone regeneration indicates following two main process; first, restoration of bone mineral density (BMD; bone quantity), and second, restoring bone apatite c-axis orientation (bone quality). In addition to BMD, bone quality is the most important factor among bone mechanical properties. Recovery of the original bone function requires development of novel scaffolds with simultaneous reconstruction of bone quality and quantity. Herein, novel orthophosphosilicate glass (PSG)/poly(lactic acid) composite anisotropic scaffolds were developed to control cell alignment and enhance bone formation, which are important for the simultaneous reconstruction of bone quality and quantity. The strategy to control cell alignment and bone formation involved designing anisotropic scaffolds in combination with the release of therapeutic ions by PSGs. The morphology of fibrous scaffolds containing PSGs was quantitatively designed using electrospinning. This successfully modulated cell alignment and subsequent bone apatite c-axis orientation along the fiber-oriented direction. The released silicate and Mg2+ ions from PSGs in scaffolds improved cell adhesion, proliferation, and calcification. To best of our knowledge, this is the first report demonstrating that the anisotropic scaffolds containing bioactive glasses regenerate bone tissues with simultaneous reconstruction of bone quality and quantity via stimulating osteoblasts by inorganic ions and designing morphology of scaffolds.

Preparation of Protein-Peptide-Calcium Phosphate Composites for Controlled Protein Release.

Protein-peptide-calcium phosphate composites were developed for achieving sustainable and controlled protein release. Bovine serum albumin (BSA) as a model acidic protein was efficiently encapsulated with basic polypeptides such as polylysine and polyarginine during the precipitation of calcium phosphate (CaP). The prepared composites were fully characterized in terms of their morphologies, crystallinities, and the porosity of their structures, and from these analyses, it was observed that there are no significant differences between the composites. Scanning transmission electron microscopy and energy dispersive X-ray spectroscopy analysis indicated a homogeneous distribution of nitrogen and sulfur, confirming the uniform distribution of BSA and polypeptide in the CaP composite. In vitro release studies demonstrated that the composite prepared with the peptides α-polylysine and polyarginine were suitable for the gradual release of the protein BSA, while those containing ε-polylysine and no peptide were unsuitable for protein release. Additionally, these composites showed high hemocompatibility for mouse red blood cells, and the osteoblast-like cell proliferation and spread in media with the composites prepared using BSA and α-polylysine showed similar tendencies to medium with no composite. From these results, protein-peptide-CaP composites are expected to be useful as highly biocompatible protein delivery agents.

Bone apatite anisotropic structure control via designing fibrous scaffolds.

Bone tissue has an anisotropic structure, associated with the collagen fibrils' orientation and the c-axis direction of the bone apatite crystal. The bone regeneration process comprises two main phases: bone mineral density restoration (bone quantity), and subsequent recovery of bone apatite c-axis orientation (bone quality). Bone quality is the determinant factor for mechanical properties of bone. Control of osteoblast alignment is one of the strategies for reconstructing bone quality since the collagen/apatite matrix orientation in calcified tissues is dependent on the osteoblast orientation. In this work, fibrous scaffolds designed for reconstruction of bone quality via cell alignment control was investigated. The fibrous scaffolds were fabricated using the electrospinning method with poly(lactic acid) at various fiber collecting speeds. The degree of fiber alignment in the prepared fibrous scaffolds increased with increasing fiber collecting speed, indicating that the fibers were oriented in a single direction. The alignment of osteoblasts on the fibrous scaffolds as well as the subsequent apatite c-axis orientation increased with increasing fiber collecting speed. We successfully controlled cell alignment and apatite c-axis orientation using the designed morphology of fibrous scaffolds. To the best of our knowledge, this is the first report demonstrating that adjusting the degree of fiber orientation for fibrous scaffolds can manipulate the regeneration of bone quality.

Hydroxyapatite Formation on Self-Assembling Peptides with Differing Secondary Structures and Their Selective Adsorption for Proteins.

Self-assembling peptides have been employed as biotemplates for biomineralization, as the morphologies and sizes of the inorganic materials can be easily controlled. We synthesized two types of highly ordered self-assembling peptides with different secondary structures and investigated the effects of secondary structures on hydroxyapatite (HAp) biomineralization of peptide templates. All as-synthesized HAp-peptides have a selective protein adsorption capacity for basic protein (e.g., cytochrome c and lysozyme). Moreover, the selectivity was improved as peptide amounts increased. In particular, peptide-HAp templated on ß-sheet peptides adsorbed more cytochrome c than peptide-HAp with α-helix structures, due to the greater than 2-times carboxyl group density at their surfaces. It can be expected that self-assembled peptide-templated HAp may be used as carriers for protein immobilization in biosensing and bioseparation applications and as enzyme-stabilizing agents.

Enzyme immobilisation on poly-l-lysine-containing calcium phosphate particles for highly sensitive glucose detection.

High catalytic activities of enzymes are necessary for enzyme immobilising technology for the development of glucose sensors. The aim of this study is to synthesise two types of poly(l-lysine)-containing calcium phosphate particles (pLys-HAp) and to achieve the immobilisation of glucose oxidase (GOX) on them. The oxidation activity of GOX immobilised on these particles was more than 80% compared to that of native GOX (considered to be 100%). Additionally, the relative activity of GOX immobilised on poly-ε-lysine-containing HAp (ε-pLys-HAp) remained approximately 70% after ten cycles. Moreover, glucose detection was able to be performed in the linear range of 4-400 µM using GOX immobilised on pLys-HAp composites. In the direct electrochemistry measurement using the cyclic voltammetry (CV) method, a glassy carbon electrode (GCE) modified by ε-pLys-HAp was a good enzyme electrode and can be used for glucose detection with high sensitivity. From these results, poly(l-lysine)-containing HAp composites can be expected to be enzyme immobilisation agents with high stability and biosensors with high sensitivity.

Adsorption and desorption characteristics of DNA onto the surface of amino functional mesoporous silica with various particle morphologies.

Recently, deoxyribonucleic acid (DNA) adsorption on solid materials has been reported for applications such as genetic diagnosis of diseases, gene delivery, and biosensors. Mesoporous silica (MPS) is an excellent carrier because of its high surface area and large pore volume. Functionalization of the MPS surface can be controlled by silane coupling reagents, and the MPS particle morphology can be easily changed by the synthetic conditions. In this study, to evaluate the ability of DNA adsorption on MPS, the MPS surface was functionalized using four reagents, 3-aminopropyltriethoxysilane (-NH2), N-(2-aminoethyl)-3-aminopropyltriethoxysilane (-2ENH2), N-(6-aminohexyl)aminopropyltrimethoxysilane (-2HNH2), and (3-trimethoxysilylpropyl)diethylenetriamine (-3NH2), each having a different number of amino groups and alkyl chain lengths. Moreover, we prepared three types of MPSs with different particle morphologies: sheet-type structure (MPS sheet), spherical MPS (MCM-41s), and nonporous spherical silica. A high adsorption capacity was observed in MPS sheets with -2HNH2 (sheet-2HNH2) and -3NH2 (sheet-3NH2), as well as MCM-41s with -3NH2 (41s-3NH2). The adsorption and desorption rates of DNA on these three MPSs were then examined and the best results were obtained with 41s-3NH2. These results demonstrate that the amino functionalized MPS materials are useful DNA adsorbents.

Morphology control of calcium phosphate by mineralization on the beta-sheet peptide template.

To investigate the influence of the spatial placement of the organic functional groups in mineralization, an amphiphilic peptide assembled monolayer with strictly arrayed carboxyl groups was applied to a mineralization system of calcium phosphate.