Injectable PLGA/Hydroxyapatite/Chitosan Microcapsules Produced by Supercritical Emulsion Extraction Technology: An In Vitro Study on Teriparatide/Gentamicin Controlled Release

Abstract

Supercritical emulsion extraction (SEE) is proposed as a green and effective strategy for the fabrication of chitosan-covered poly-lactic-co-glycolic acid (chi-PLGA) injectable microcapsules for the controlled release of teriparatide (THA) and teriparatide/gentamicin sulfate (THA/Gen). These formulations can be used for locally bone pathologies treatment or in complex fracture healing of aged patients. Several oil-water (o-w) and water-oil-water (w-o-w) emulsions were processed by SEE to produce multifunctional microcapsules containing hydroxyapatite (HA) within a poly-lactic-co-glycolic acid (PLGA) matrix (up to 24 mg/g) and with both THA (0.45 mg/g) and Gen (up to 9 mg/g). Chitosan coating was also successfully added, as external layer (0.4 μm). SEE-fabricated microcapsules showed good encapsulation efficiency (up to 90%) for all the drugs tested and a mean size ranging between 1.4 (±0.4) μm and 2.2 (±0.5) μm. Different drug amounts loaded and microcapsules compositions assured a controlled drug release over a wide range of times and concentrations, as in vitro monitored in PBS medium at 37°C for 15/20 days. HA embedded into the biopolymer structure delayed the THA release profile; chitosan coating strongly reduced the initial drug “burst” release. In addition, the coencapsulation of both THA and Gen, which have very different water solubility, accelerated the release profile of the less water-soluble drug. No drugs degradation was also monitored after the SEE manufacturing. Apparent drug diffusivities (D) were calculated by fitting of the release profiles. In the case of Gen, D ranged between 2.9 × 10⁻⁸ and 1.6 × 10⁻⁹ cm²s⁻¹ if the drug was entrapped in simple PLGA or in the chitosan-coated microcapsules, respectively. In the case of THA, the calculated values ranged between 8.1 × 10⁻⁹ and 7.4 × 10⁻¹⁰ cm²s⁻¹ when the drug was entrapped in PLGA/HA microcapsules or in the chitosan-coated ones, respectively. These mass transfer values are consistent with the different release behaviors observed and confirmed the possibility of multicomponent microcapsules fabrication by SEE.

Introduction

Osteoporosis affects more than 75 million people in USA, Europe, and Japan,¹ with a consequent increase in bone fragility and susceptibility to fracture at any skeletal site,2, 3 and an enormous economic burden on health care systems in Europe and worldwide. Teriparatide (THA) is currently Food and Drug Administration–approved for the treatment of osteoporosis and osteoarthritis⁴ and may strongly improve fracture healing or repair bone defects.⁵ THA has been reported to accelerate and improve healing at the standard available dose of 20-40 mg/day, especially for aged patients with a fracture.6, 7, 8 THA is the recombinant 1-34 amino acid segment of the parathyroid hormone (PHT) with a molecular weight of 4117.72 g/mol. THA binds to the receptor for PHT present in bone and stimulates the G-protein-related production of cyclic adenosine monophosphate.⁹ During treatment, an increase in trabecular connectivity and cortical thickness is observed, with consequent improvement of the microarchitectural strength of bone¹⁰; THA may also stimulate mesenchymal stem cell proliferation and stem cell maturation in the musculoskeletal lineage.¹¹

Despite the demonstrated clinical efficacy of THA, patient acceptance and compliance is limited by the need for daily subcutaneous injections. The development of an equally efficient formulation not requiring daily injections would significantly expand the present market.12, 13 Moreover, among all the possible drug administration, a local therapy, if indicated, has many potential advantages such as circumventing possible adverse side effects resulting from systemic administration, decreasing dose or number of dosages, and maintaining local agent levels within a desirable range.¹⁴ Several devices have been already explored for THA (and PHT)-controlled delivery such as, multipulse subcutaneous implantable devices15, 16 or microchip,¹⁷ coated microneedle patch system,¹⁸ or implantable biopolymer scaffold.¹⁹ Eli Lilly recently has launched a new THA formulation in the injectable form and Daiichi and West Pharmaceuticals are working on nasal formulations.

Poly-lactic-co-glycolic acid (PLGA) excellent biocompatibility and biodegradability makes this biopolymer an appropriate carrier for drug delivery; in addition, PLGA has been approved by the Food and Drug Administration as a safe biomaterial for clinical applications. The intraarticular and local injection of microcarriers, fabricated with PLGA and able of controlled and local THA delivery may be an interesting option.²⁰ Recently, it was reported a successful suppression of osteoarthritis progression in rats replacing the single-dose injection, required every 3 days, by using PLGA (65:35) microspheres able to release the drug for 19 days with a concentration range of 5-100 nM (covering 10 nM) at 37°C. The in vivo study showed after intraarticular treatment with either 3 days/injection or PLGA microspheres (15 days/injection) for 5 weeks a similar effect on suppressing papain-induced osteoarthritis changes, by decreasing production of GAG and Col II and increasing Col III in rat knee cartilage.²¹

An engineered size distribution, as well as, excellent drug encapsulation efficiency are very important fabrication parameters for microcapsules industrial production to ensure a controlled and reproducible drug sustained release formulation. In this sense, supercritical fluid technologies may offer an improved control over the morphology and composition of microdevices and nanodevices coupled with good fabrication process robustness.22, 23, 24, 25 The basic idea of use supercritical carbon dioxide (SC-CO₂) as the extracting agent of the oily phase of an emulsion, leading solvent-free microspheres was proposed in 2006²⁶; the original process was performed using a batch operative layout, then was later improved by other authors and named supercritical emulsion extraction (SEE).27, 28, 29 After these pioneering articles, new SEE continuous process layout was proposed using a countercurrent-packed tower to reduce the emulsion processing time (few minutes of residence time), improve the encapsulation efficiency (especially if double emulsions are treated), and assured higher process reproducibility.³⁰

In this work, we will explore the SEE technology (continuous layout) to fabricate an engineered, versatile, and multiloaded PLGA microdevice for THA sustained release. Particularly, hydroxyapatite (HA) will be embedded into the microcarrier structure because it is widely used in bone tissue engineering and is reported to be good matrix for bone cell differentiation and mineralization.³¹ Indeed, in vitro and in vivo studies of biopolymer/HA composites found that osteoblast cells adhered to HA preferentially, leading to subsequent cell proliferation, and the composite implants displayed good bone integration and osteoconductivity in rabbit models.32, 33 Artificial defects induced in the osteoporotic bone of the rat mandible were also successfully reconstructed following implantation of HA nanoparticles coated with cholecalciferol-loaded PLGA devices,³⁴ whereas hollow HA microspheres loaded with clinically safe doses of bone morphogenetic protein–2 could provide promising implants for healing nonloaded bone defects.³⁵ Chitosan (Chi) will be also chosen because it was already used to repair critical bone defects36, 37, 38; some authors also indicated that Chi/PLGA microsphere combined with appropriate drug delivery can achieve accelerated bone healing both in vitro and in vivo and were able to guide bone formation in a rabbit ulnar critical-sized-defect model.³⁹ In our hypothesis, the basic inorganic component of HA could interfere with the acidic degradation products of PLGA polymer, affecting the drug release, whereas the chitosan coating would additionally delay the drug release. Gentamicin (Gen) is largely used in the treatment of osteomyelitis,⁴⁰ and it will be also coencapsulated in the microcarriers to understand SEE versatility in producing microcapsules with a simultaneous release of different molecules.

Different oil-in-water (o-w) and water-in-oil-in-water (w-o-w) emulsions will be processed by SEE and the fabricated microcapsule (1) morphology will be observed using scanning electron microscopy, (2) particle size distribution (PSD) and zeta potential measured by a laser scattering. HA loading and its dispersion in the biopolymer matter will be measured by thermogravimetric analysis (TGA) and energy-dispersive X-ray analysis (EDX), respectively, whereas drugs loading and encapsulation efficiency will be monitored by chemical analysis. The effect of HA embedding and chitosan coating on drugs release will be both explored by monitor the in vitro drug release profiles obtained in PBS medium at 37°C. THA and Gen apparent diffusivities will be calculated by fitting the release profiles obtained from the different microcapsules.