Growth of hydroxyapatite coatings on biodegradable polymer microspheres

Abstract

Mineral-coated microspheres were prepared via a bioinspired, heterogeneous nucleation process at physiological temperature. Poly(d,l-lactide-co-glycolide) (PLG) microspheres were fabricated via a water-in-oil-in-water emulsion method and were mineral-coated via incubation in a modified simulated body fluid (mSBF). X-ray diffraction, Fourier transform infrared spectroscopy, and scanning electron microscopy with associated energy-dispersive X-ray spectroscopy confirmed the presence of a continuous mineral coating on the microspheres. The mineral grown on the PLG microsphere surface has characteristics analogous to those of bone mineral (termed "bonelike" mineral), with a carbonate-containing hydroxyapatite phase and a porous structure of platelike crystals at the nanometer scale. The assembly of mineral-coated microspheres into aggregates was observed when microsphere concentrations above 0.50 mg/mL were incubated in mSBF for 7 days, and the size of the aggregates was dependent on the microsphere concentration in solution. In vitro mineral dissolution studies performed in Tris-buffered saline confirmed that the mineral formed was resorbable. A surfactant additive (Tween 20) was incorporated into mSBF to gain insight into the mineral growth process, and Tween 20 not only prevented aggregation but also significantly inhibited mineral formation and influenced the characteristics of the mineral formed on the surface of PLG microspheres. Taken together, these findings indicate that mineral-coated PLG microspheres or mineral-coated microsphere aggregates can be synthesized in a controllable manner using a bioinspired process. These materials may be useful in a range of applications, including controlled drug delivery and biomolecule purification.

Keywords: HAP chromatography; PLG microsphere; bioinspired; drug delivery; mineralization.

Figure 1

PLG microsphere (A) and mineral-coated microsphere after incubation in mSBF for 7 days (B).

Figure 2

Zeta potential of (A) PLG microspheres in buffers PBS, mSBF, and mSBF + 0.1%v/v Tween20™, and (B) non-hydrolysed and hydrolysed PLG films in comparison with PLG microspheres.

Figure 3

SEM images of mineral-coated microspheres after a 7 day incubation in mSBF solution, (A) 0.25% w/v, (B) 0.50% w/v, (C) 0.75% w/v, and (D) 1.00% w/v. (E) Relationship between the microsphere concentration in solution during mineral growth, and the size of mineral-coated microsphere aggregates.

Figure 4

(A) X-ray diffraction analysis of mineral-coated microspheres and hydroxyapatite powder (included for comparison), (B) Fourier transform infrared analysis of mineral-coated microspheres. Peaks associated with carbonate are denoted by *, and peaks associated with phosphate are denoted by †. (C) EDS spectrum of mineral-coated microspheres after a 7 day incubation in mSBF.

Figure 5

The process of mineral nucleation and growth on PLG microspheres. SEM images of microspheres after: (A) the first day of immersion in mSBF, (B) day 3 of incubation, (C) day 5 of incubation, (D) day 7 of incubation.

Figure 6

(A) Cumulative dissolution of Ca²⁺ and PO₄³⁻ during a 25 day incubation in tris-buffered saline (TBS), and (B) SEM images of mineral-coated microspheres after the 25 day TBS incubation. (C) Cumulative dissolution of PO₄³⁻ during a 25 day incubation in DMEM, and (D) SEM images of mineral-coated microspheres after the 25 day DMEM incubation.

Figure 7

Effect of surfactant (Tween 20™) on the mineral formed on the PLG microsphere surfaces (A) after 3 days, (B) after 7 days, (C) after 14 days, and (D) after 21 days. (E) FTIR spectrum of PLG microspheres coated with mineral via a 21 days mSBF incubation in the presence of 0.1%v/v Tween20™. A spectrum of commercial HA powder is included for comparison.

Figure 8

Nanometer-scale mineral morphology on the surface of microspheres formed (A) in the presence of 0.1% v/v Tween20™, and (B) in the absence of Tween20™.