Hollow hydroxyapatite microspheres as a device for controlled delivery of proteins

Hollow hydroxyapatite (HA) microspheres were prepared by reacting solid microspheres of Li₂O–CaO–B₂O₃ glass (106–150 μm) in K₂HPO₄ solution, and evaluated as a controlled delivery device for a model protein, bovine serum albumin (BSA). Reaction of the glass microspheres for 2 days in 0.02 M K₂HPO₄ solution (pH = 9) at 37°C resulted in the formation of biocompatible HA microspheres with a hollow core diameter equal to 0.6 the external diameter, high surface area (~100 m²/g), and a mesoporous shell wall (pore size ≈13 nm). After loading with a solution of BSA in phosphate-buffered saline (PBS) (5 mg BSA/ml), the release kinetics of BSA from the HA microspheres into a PBS medium were measured using a micro bicinchoninic acid (BCA) protein assay. Release of BSA initially increased linearly with time, but almost ceased after 24–48 h. Modification of the BSA release kinetics was achieved by modifying the microstructure of the as-prepared HA microspheres using a controlled heat treatment (1–24 h at 600–900°C). Sustained release of BSA was achieved over 7–14 days from HA microspheres heated for 5 h at 600°C. The amount of BSA released at a given time was dependent on the concentration of BSA initially loaded into the HA microspheres. These hollow HA microspheres could provide a novel inorganic device for controlled local delivery of proteins and drugs.     

Tricalcium phosphate and tricalcium phosphate/polycaprolactone particulate composite for controlled release of protein

β-Tricalcium phosphate (β-TCP) with three different particle size ranges was used to study the effects of particle size and surface area on protein adsorption and release. Polycaprolactone (PCL) coating was applied on the particle systems to investigate its effect on particulate system properties from both structural and application aspects. The maximum loading of 27 mg/g was achieved for 100 nm particles. Bovine serum albumin (BSA) loading amount was controlled by varying the BSA loading solution concentration, as well as the sample powder's surface area. Increasing the surface area of the delivery powder significantly increased loading and release yield. Unlike the samples with low surface area, the lowest particle size samples showed sigmoidal release profile. This indicated that release was governed by different mechanisms for particles with different sizes. While the majority of samples showed no more than 50% release, the 550 nm particles demonstrated 100% release. PCL coating showed no significant ability to attenuate burst release in PBS. However, it led to a steadier release profile as compared to the bare TCP particles. FTIR analysis also proved that the secondary structure of BSA did not change significantly during the adsorption; however, minor denaturation was found during the release. The same results were found when PCL coating was applied on the TCP particles. We envision potential use of TCP and TCP + PCL systems in bone growth factor or orthopedic drug delivery applications in future bone tissue engineering application.     

Recognition of four structurally resembled benzoic acid derivatives by albumin-crosslinked poly(acrylamide) hydrogel

Poly(acrylamide) hydrogels crosslinked by bovine serum albumin (BSA) were prepared by the introduction of vinyl groups into BSA and subsequent co-polymerization with acrylamide (AAm). The hydrogels were loaded with four structurally resembled benzoic acid derivatives such as salicylic acid, o-anisic acid, salicylamide and sodium benzoate, and their release from the hydrogel was investigated. The affinity of these four compounds for BSA gradually decreased in a following order; salicylic acid > o-anisic acid > salicylamide > sodium benzoate. The amounts of compounds loaded on a hydrogel of high BSA content and the duration of their release were found to be dependent on the affinity for BSA clearly reflecting the subtle difference in BSA affinity of each compound. Therefore, this hydrogel would be used not only as a sustained drug release carrier, but also a facile tool to evaluate the binding of various compounds to serum albumin.     

Preparation of open porous polycaprolactone microspheres and their applications as effective cell carriers in hydrogel system

Common hydrogel, composed of synthetic polymers or natural polysaccharides could not support the adhesion of anchorage-dependent cells due to the lack of cell affinitive interface and high cell constraint. The use of porous polyester microspheres as cell-carriers and introduction of cell-loaded microspheres into the hydrogel system might overcome the problem. However, the preparation of the open porous microsphere especially using polycaprolactone (PCL) has been rarely reported. Here, the open porous PCL microspheres were fabricated via the combined emulsion/solvent evaporation and particle leaching method. The microspheres exhibited porous surface and inter-connective pore structure. Additionally, the pore structure could be easily controlled by adjusting the processing parameters. The surface pore size could be altered from 20 μm to 80 μm and the internal porosities were varied from 30% to 70%. The obtained microspheres were evaluated to delivery mesenchymal stem cells (MSCs) and showed the improved cell adhesion and growth when compared with the non-porous microspheres. Then, the MSCs loaded microspheres were introduced into agarose hydrogel. MSCs remained alive and sustained proliferation in microsphere/agarose composite in 5-day incubation while a decrement of MSCs viabilities was found in agarose hydrogel without microspheres. The results indicated that the microsphere/hydrogel composite had a great potential in cell therapy and injectable system for tissue regeneration.     

Osteogenic effect of hyaluronic acid sodium salt in the pores of a hydroxyapatite scaffold

According to previous reports, a large volume of bone marrow cells (1 × 10⁷ cells/ml) is required for bone regeneration in the pores of a scaffold in vivo. We theorized that immersion of a porous hydroxyapatite (HA) scaffold in hyaluronic acid solution would facilitate bone formation in the scaffold at 1 × 10⁶ cells/ml density of bone marrow cells. The cells were respectively seeded into pores of the cylindrical HA scaffolds with a hollow center after immersion in hyaluronic acid solution or in culture medium. The scaffolds were implanted in the dorsal subcutis of rats for 4 weeks. Thereafter, serially sectioned paraffin specimens were made and observed histologically. Bone formation was observed in many pores of HA scaffold by immersion in hyaluronic acid solution. However, there were no or less pores with new bone formation in the scaffold by immersion in culture medium. The cells were cultured with and without hyaluronic acid in vitro. There was no significant difference in bone formation in vitro with and without hyaluronic acid. The results of this study suggest that hyaluronic acid binds to the cells on the wall of three-dimensional structure and effectively promotes new bone formation.     

The production of volvox spheres and their potential application in multi-drugs encapsulation and release

Volvox sphere is a bio-mimicking concept of an innovative biomaterial structure of a sphere that contains smaller microspheres which then encapsulate chemicals, drugs and/or cells. The volvox spheres were produced via a high-voltage electrostatic field system, using alginate as the primary material. Encapsulated materials tested in this study include staining dyes, nuclear fast red and trypan blue, and model drugs, bovine serum albumin (BSA) and cytochrome c (CytC). The external morphology of the volvox spheres was observed via electron microscopy whereas the internal structure of the volvox spheres was observed via an optical microscope with the aid of the staining dyes, since alginate is colorless and transparent. The diameter of the microspheres was about 200 to 300 μm, whereas the diameter of the volvox spheres was about 1500 μm. Volvox spheres were durable, retaining about 95% of their mass after 4 weeks. Factors affecting entrapment efficiency, such as temperature and concentration of the bivalent cross-linker, were compared followed by a 7-day in vitro release study. The encapsulation efficiency of CytC within the microspheres was higher at cold (~ 4 °C) and warm (~ 50 °C) temperatures whereas temperature has no obvious effect on the BSA encapsulation. High crosslinking concentration (25% w/v) of calcium chloride has resulted higher entrapment efficiency for BSA but not for CytC. Furthermore, volvox spheres showed a different release pattern of BSA and CytC when compared to microspheres encapsulating BSA and CytC. Despite the fact that the mechanisms behind remain unclear and further investigation is required, this study demonstrates the potential of the volvox spheres for drug delivery.     

Fabrication of calcined hierarchical porous hollow silicate micro-size spheres via double emulsion process

Silicate (7 to 12 μm) microspheres with porous shell were prepared via modified double emulsion (water₁/oil/water₂) method mediated with N₂ pressure filtration and calcination to completely remove the organic components. With the addition of sodium polymethacrylate (Na-PA) into the aqueous solution of water₁/oil/water₂ emulsion system then calcined, led to the formation of stable hierarchical macroporous (surface area: 42.94 m²/g) from mesoporous (surface area: 259.2 m²/g) shell wall of silicate hollow microspheres.     

Synthesis of aligned porous poly(ε-caprolactone) (PCL)/hydroxyapatite (HA) composite microspheres

We synthesized poly(ε-caprolactone) (PCL)/hydroxyapatite (HA) composite microspheres with an aligned porous structure and evaluated their potential applications in bone tissue engineering. A range of HA particles (0, 5, 10 and 20 wt.% in relation to the PCL polymer) were added to a PCL solution in order to improve the biocompatibility of the porous PCL/HA composite microspheres. All the synthesized microspheres showed that the HA particles were distributed well in the PCL matrix, while preserving their aligned porous structure. The average size of the PCL/HA composite microspheres increased from 62 ± 7 to 179 ± 95 μm with increasing HA content from 0 to 20 wt.%. The incorporation of the HA particles to the PCL polymer led to a considerable improvement in in vitro bioactivity, which was assessed by immersing the PCL/HA composite microspheres in simulated body fluid (SBF). A number of apatite crystals could be precipitated on the surface of the aligned porous PCL/HA composite microspheres after soaking in the SBF for 7 days.     

Calcium carbonate microspheres as carriers for the anticancer drug camptothecin

Biogenic calcium carbonate has come to the attention of many researchers as a promising drug delivery system due to its safety, pH sensitivity and the large volume of information already in existence on its medical use. In this study, we employed bovine serum albumin (BSA) as an additive to synthesize a series of porous calcium carbonate microspheres (CCMS). These spheres, identified as vaterite, are stable both in aqueous solutions and organic solvents. Camptothecin, an effective anticancer agent, was loaded into the CCMS by simple diffusion and adsorption. The camptothecin loaded CCMS showed sustained cell growth inhibitory activity and a pH dependent release of camptothecin. With a few hours, the release is negligible under physiological conditions (pH = 7.4) but almost complete at pH 4 to 6 (i.e. pHs found in lysosomes and solid tumor tissue respectively). These findings suggest that porous, biogenic calcium carbonate microspheres could be promising carriers for the safe and efficient delivery of anticancer drugs of low aqueous solubility.     

Preparation of grafted microspheres CPVA-g-PSSS and studies on their drug-carrying and colon-specific drug delivery properties

Sodium 4-styrene sulfonate (SSS) was graft-polymerized on the surfaces of crosslinked polyvinyl alcohol (CPVA) microspheres in a manner of surface-initiated graft-polymerization by using cerium salt-hydroxyl group redox initiation system, obtaining the grafted microspheres CPVA-g-PSSS. The chemical structure and physicochemical characters of CPVA-g-PSSS microspheres were fully characterized with infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and zeta potential determination. The aim of this work is to constitute a novel colon-specific drug delivery system via molecular design by using CPVA-g-PSSS microspheres as the drug-carrying material and by taking metronidazole (MTZ) as the model drug. The drug-carrying ability and mechanism of the grafted microspheres CPVA-g-PSSS for MTZ were investigated. Finally, in-vitro release tests for the drug-carrying microspheres were conducted. The experimental results show that in an acidic medium, the grafted microspheres CPVA-g-PSSS exhibit strong adsorption ability for MTZ by driving of electrostatic interaction, and have an adsorption capacity of 112 mg/g, displaying the high efficiency of drug-carrying. The in-vitro release behavior of the drug-carried microspheres is highly pH-sensitive. In the medium of pH = 1, the drug-carrying microspheres do not release the drug, whereas in the medium of pH = 7.4, a sudden delivery phenomenon of the drug will occur, displaying an excellent colon-specific drug delivery behavior.     

Factors influencing the deposition of hydroxyapatite coating onto hollow glass microspheres

Hydroxyapatite (HA) and HA coated microcarriers for cell culture and delivery have attracted more attention recently, owing to the rapid progress in the field of tissue engineering. In this research, a dense and uniform HA coating with the thickness of about 2 μm was successfully deposited on hollow glass microspheres (HGM) by biomimetic process. The influences of SBF concentration, immersion time, solid/liquid ratio and activation of HGM on the deposition rate and coating characteristics were discussed. X-ray diffraction (XRD) and Fourier transform infrared spectrum (FTIR) analyses revealed that the deposited HA is poorly crystalline. The thickness of HA coating showed almost no increase after immersion in 1.5SBF for more than 15 days with the solid/liquid ratio of 1:150. At the same time, SBF concentration, solid/liquid ratio and activation treatment played vital roles in the formation of HA coating on HGM. This poorly crystallized HA coated HGM could have potential use as microcarrier for cell culture.     

Fabrication and evaluation of a sustained-release chitosan-based scaffold embedded with PLGA microspheres

Nutrient depletion within three-dimensional (3D) scaffolds is one of the major hurdles in the use of this technology to grow cells for applications in tissue engineering. In order to help in addressing it, we herein propose to use the controlled release of encapsulated nutrients within polymer microspheres into chitosan-based 3D scaffolds, wherein the microspheres are embedded. This method has allowed maintaining a stable concentration of nutrients within the scaffolds over the long term. The polymer microspheres were prepared using multiple emulsions (w/o/w), in which bovine serum albumin (BSA) and poly (lactic-co-glycolic) acid (PLGA) were regarded as the protein pattern and the exoperidium material, respectively. These were then mixed with a chitosan solution in order to form the scaffolds by cryo-desiccation. The release of BSA, entrapped within the embedded microspheres, was monitored with time using a BCA kit. The morphology and structure of the PLGA microspheres containing BSA before and after embedding within the scaffold were observed under a scanning electron microscope (SEM). These had a round shape with diameters in the range of 27–55 μm, whereas the chitosan-based scaffolds had a uniform porous structure with the microspheres uniformly dispersed within their 3D structure and without any morphological change. In addition, the porosity, water absorption and degradation rate at 37 °C in an aqueous environment of 1% chitosan-based scaffolds were (92.99 ± 2.51) %, (89.66 ± 0.66) % and (73.77 ± 3.21) %, respectively. The studies of BSA release from the embedded microspheres have shown a sustained and cumulative tendency with little initial burst, with (20.24 ± 0.83) % of the initial amount released after 168 h (an average rate of 0.12%/h). The protein concentration within the chitosan-based scaffolds after 168 h was found to be (11.44 ± 1.81) × 10^? 2 mg/mL. This novel chitosan-based scaffold embedded with PLGA microspheres has proven to be a promising technique for the development of new and improved tissue engineering scaffolds.     

Formulation and characterization of naked DNA and complexed DNA loaded polymer films

Sustained release of DNA from polymeric films is of considerable interest for enhanced and prolonged gene therapy. This report describes the detailed studies of the formulation of naked plasmid DNA (pDNA) and complexed DNA-incorporated polymer films and in vitro release of the DNA. The effect of hydrophilic polymers (PEG, HA) also studied to modulate the release of pDNA and complexed DNA (lipoplex) from slow biodegradable polymer (PCL) films. The polymer system consists of a biodegradable semi-crystalline polymer (PCL) blended with a hydrophilic polymer such as poly (ethylene glycol) (PEG) or hyaluronic acid (HA). For the release of pDNA (naked DNA), a burst effect was always seen, and the addition of HA and PEG did not suppress the burst release of pDNA from PCL films. For complexed pDNA (lipoplex), the release was slow, but it could be accelerated using additives such as PEG or HA. The transfection efficiency of the complexed DNA and the naked pDNA was determined in vitro using COS 7 cells to evaluate the bioactivity of the released DNA. Transfection was observed from released lipoplexes samples from PCL/HA film. Overall, this work suggested that these polymeric DNA delivery systems are promising for the local sustained release of DNA from implanted films.     

Quasi-static uni-axial compression behaviour of hollow glass microspheres/epoxy based syntactic foams

Hollow glass microspheres/epoxy foams of different densities were prepared by stir casting process in order to investigate their mechanical properties. The effect of hollow spheres content and wall thickness of the microspheres on the mechanical response of these foams is studied extensively through a series of quasi-static uni-axial compression tests performed at a constant strain rate of 0.001 s^?1. It is found that strength of these foams decreases linearly from 105 MPa (for the pure resin) to 25 MPa (for foam reinforced with 60 vol.% hollow microspheres) with increase in hollow spheres content. However, foams prepared using hollow spheres with a higher density possess higher strength than those prepared with a lower one. The energy absorption capacity increases till a critical volume fraction (40 vol.% of the hollow microspheres content) and then decreases. Failure and fracture of these materials occur through shear yielding of the matrix followed by axial splitting beyond a critical volume fraction.     

Hollow microspheres fabricated from instant adhesive

Non-invasive surgery techniques and drug delivery system with acoustic characteristics of ultrasound contrast agent have been studied intensively in recent years. Many ultrasound contrast agents are commercially available, and they are usually composed of a microbubble coated by a surfactant or lipid bilayer, i.e., a hollow microsphere. We show that the hollow microsphere with polymer shell can be fabricated just blowing vapor of commonly-used instant adhesive into water as microbubbles. The hollow microspheres are composed of cyanoacrylate, a biocompatible material used for adhesion to human tissue such as skin, blood vessels, and organs. Moreover these are less than 10 μm in diameter, which is smaller than a blood capillary (approximately 10 μm). Therefore, with their stable polymer shells, the hollow microspheres are possible ultrasound contrast agents, and are expected to be generated at low cost on the medical frontline, and not in a pharmaceutical factory.     

Controlled release of brefeldin A from electrospun PEG–PLLA nanofibers and their in vitro antitumor activity against HepG2 cells

In this work, PEG–PLLA electrospun fibers were developed as a new controlled release system for macrolide antibiotic drug brefeldin A (BFA). SEM and XRD analyses of the BFA-loaded PEG–PLLA fibers revealed that the average diameter of fibers was below 950 nm with smooth surfaces, and the drug was well incorporated into the fibers in amorphous form. The release profiles of BFA in PBS were measured by HPLC, demonstrating that the controlled release of BFA could be gained for long time. The in vitro antitumor activity against human liver carcinoma HepG2 cells of the fibers containing 3%, 6%, 9%, 12% and 15% BFA were examined by MTT method, and the results showed that cell growth inhibition rates at 72 h were 64%, 77%, 80%, 81% and 85%, respectively. These results strongly suggested that the BFA/PEG–PLLA fibers had an effect of controlled release of BFA and were suitable for postoperative chemotherapy of cancers.     

Preparation and characterization of hollow Fe3O4/SiO2@PEG–PLA nanoparticles for drug delivery

In this study we present a novel targeted anticancer drug delivery, which was size controlled Fe₃O₄/SiO₂ hollow microspheres (HMS) as magnetic core and poly (ethylene glycol)-poly–(d,l-lactide) (PEG–PLA) surface coating (HMS@PEG–PLA). And investigations were to test a new convenient method, which is one-step precipitation polymerization on HMS, forming magnetic hollow polymer microspheres. The HMS@PEG–PLA which have hollow structure and uniform size were characterized by Transmission Electron Microscopy (TEM). Vibrating Sample Magnetometer (VSM) showed a characteristic of super paramagnetic with saturation magnetization value of about 19.78 emu/g. In vitro cytotoxicity of Fe₃O₄/SiO₂@PEG–PLA (HMS@PEG–PLA) hollow microspheres were of low toxicity, so it can be used as a drug carrier, and cisplatin (CDDP) as the model drug release behavior was researched. The results have exhibited preferable release properties.     

In vitro study of hydroxyapatite/polycaprolactone (HA/PCL) nanocomposite synthesized by an in situ sol–gel process

Hydroxyapatite (HA) is the most substantial mineral constituent of a bone which has been extensively used in medicine as implantable materials, owing to its good biocompatibility, bioactivity high osteoconductive, and/or osteoinductive properties. Nevertheless, its mechanical property is not utmost appropriate for a bone substitution. Therefore, a composite consist of HA and a biodegradable polymer is usually prepared to generate an apt bone scaffold. In the present work polycaprolactone (PCL), a newly remarkable biocompatible and biodegradable polymer, was employed as a matrix and hydroxyapatite nanoparticles were used as a reinforcement element of the composite. HA/PCL nanocomposites were synthesized by a new in situ sol–gel process using calcium hydroxide and phosphoric acid precursors in the presence of Tetrahydrofuran (THF) as a solvent. Chemical and physical characteristics of the nanocomposite were studied by X-ray diffraction (XRD), field emission scanning electron microscopy (FE-SEM) and Fourier transform infrared (FTIR) analyses. The results indicated that pure HA nanoparticles were well-incorporated and homogenously dispersed in the PCL matrix. It was found that the mechanical property of PCL was improved by addition of 20 wt.% HA nanoparticles. Furthermore, the biological property of nanocomposites was investigated under in vitro condition. For this purpose, HA/PCL scaffolds were prepared through a salt leaching process and immersed in a saturated simulated body fluid (SBF) after 3 and 7 days. It was found that a uniform layer of biomimetic HA could be deposited on the surface of HA/PCL scaffolds. Therefore, the prepared HA/PCL scaffolds showed good potential for bone tissue engineering and could be used for many clinical applications in orthopedic and maxillofacial surgery.     

中空羟基磷灰石微球作为rhBMP-2缓释载体的研究

利用锂钙硼玻璃在磷酸盐溶液中的原位转化反应制备表面多孔且中空的羟基磷灰石(HA)微球, 将重组人骨形态发生蛋白2(rhBMP-2)装载到微球中, 研究了微球中rhBMP-2的体外缓释行为, 并采用体外细胞培养技术, 将载有rhBMP-2的微球和大鼠骨髓间充质干细胞(MSCs)一起培养, 测定细胞的碱性磷酸酶(ALP)活性, 并与单纯rhBMP-2的作用进行比较. 结果显示, 微球中所装载的rhBMP-2具有明显的缓释效应, 体外释放周期达到1000h以上, 该微球缓释系统具有一定的生物活性, 其作用效果优于单纯使用rhBMP-2.     

Mineral phase deposition on pectin microspheres

The nucleation of apatite and calcium phosphates onto natural polysaccharides containing carboxyl groups can be obtained by immersion in biomimetic solutions. The deposition of hydroxyapatite has been recently described also on pectin, and pectin–apatite hybrid gels were proposed as scaffolds for bone tissue regeneration. In this work, injectable calcium phosphate/pectin microspheres were prepared to promote both the natural process of biomineralization and the controlled release of encapsulated cells, genes, enzymes, proteins or drugs in the pathological situ. Pectin microspheres were prepared with different formulations from aqueous solutions using an extrusion system, and incubated in H₂O or SBF for 14 days. The presence of an excess of CaCl₂, deriving from the preparation process, was determinant in promoting the deposition of calcium phosphates from SBF, whereas no mineral deposition was detected on pectin microspheres extensively purified after preparation. The nucleation of calcium phosphates occurred on pectin microspheres incubated in SBF for 14 days and was evaluated through ESEM–EDS analysis and FT-IR spectroscopy.