Research paper
Different modalities of NaCl osmogen in biodegradable microspheres for bone deposition of risedronate sodium by alveolar targeting

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Abstract

Risedronate sodium was formulated into polylactide-co-glycolic acid microspheres for pulmonary delivery using the w/o/w double emulsion technique. Sodium chloride was used as osmogen in either the internal or external aqueous phase to surface-engineer the particles to achieve favorable properties. The prepared microspheres were characterized for the surface morphology, entrapment efficiency, in vitro release behavior, particle size, surface area, aerodynamic as well as powder flow properties. Furthermore, the safety of the drug and the selected formula were assessed by MTT viability test performed on Calu-3 cell line as well as histopathological lung tissue examination. A novel in vivo approach based on the radiolabeling of risedronate sodium with I125 was developed in order to assess its deposition in the bones of male albino rats. The majority of the prepared microspheres exhibited high entrapment efficiency, sustained release profile up to 15 days, suitable geometric and aerodynamic particle sizes as well as good flow properties. The safety of the drug and the selected formula were proven by the high cell viability percentage of Calu-3 cells as well as the normal lung histology after intra-tracheal administration. The in vivo study showed high bone deposition for risedronate sodium following the pulmonary route, suggesting that it could be utilized as an alternative route of administration for delivery of bisphosphonates.

Graphical abstract

Effect of varying NaCl concentration in either internal/external phase of the w/o/w emulsion on the morphology of risedronate sodium loaded PLGA microspheres.

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Introduction

Porous or surface modified polylactide-co-glycolide (PLGA) microspheres are commonly employed as a suitable carrier for pulmonary administered drugs owing to their biocompatibility, controlled release behavior, and their well-known safety profile [1].

Various approaches have been attempted in the literature to incorporate water soluble drugs into biodegradable microspheres at high loading. However, the double (w/o/w) emulsion method remains the most commonly used method for achieving such purpose [2]. Upon using osmogens as NaCl in the preparation of these microparticles, the organic phase of a w/o/w emulsion acts as semipermeable membrane allowing the passage of water across the organic phase [3], [4], which in turn leads to the production of diverse modalities of microspheres, differing in their morphology, entrapment efficiency properties, release capabilities, and aerodynamic deposition in the lung.

Bisphosphonates (BPs), the gold-standard pharmacological treatment of osteoporosis [5], suffer extremely low bioavailability (less than 1%) upon their oral intake owing to their high polarity and hydrophilicity. Furthermore, they exhibit several gastric and esophageal side effects such as erosive esophagitis, gastritis, and ulcers. These adverse effects are hypothesized to be caused by the reflex of the acidic gastric contents along with the undissolved drug crystals back to the esophagus, exposing it to the free acid form of the drug, or due to the exacerbation of an existing esophageal disorder [6], [7]. They might also be caused by the local reaction of the mucosa upon contact with the concentrated form of the drug [8], [9], [10]. The local tissue damage and irritation at the sites of injection precluded the use of intra-muscular and subcutaneous routes of administration for delivery of BPs. Slow release of BPs is also critical, since it was reported that rapid injection of BPs can lead to renal failure due to the formation of complexes with calcium in the blood, which are held back in the kidney [10]. A model BP, risedronate sodium (RS), was used in our study, as it possessed a high anti-resorptive activity while causing less incidence of gastric damage [11].

Therefore, the aim of our work was to test the possibility for delivery of RS through an alternative route (the pulmonary route), taking advantage of the neutral pH of the lung environment with the possibility of using small dose to minimize possible local mucosal irritation. Different modalities for NaCl as osmogen through changing its concentration in the internal and external aqueous phases were attempted, in which the influence of the relative difference in osmotic pressure on the produced microspheres was studied through several experimental parameters. Sodium chloride was used as the osmogen of choice based on preliminary study conducted in our laboratory [12]. Cellular toxicity using MTT assay on Calu-3 cells as well as histological examination of lung tissue was performed in order to assess the safety of the optimized formula. In addition, instead of using the traditional chromatographic methods to test the bioavailability of our optimized formula, a novel radiolabeling technique was utilized to calculate the percentage of RS directly deposited in the bones after pulmonary administration.

Section snippets

Materials

Risedronate sodium was gifted by SPIC Pharma. Co., India. PLGA 50:50 and 75:25 of molecular weights 150,000 and 95,000 g/mole, respectively, were gifted by PURAC Biomaterials Co., the Netherlands. Polyvinyl alcohol (Mowiol®4-88) Molecular weight 31,000, MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide, HEPES buffer, fetal calf serum, gentamycin, phosphate buffered saline tablets (PBS), dimethyl sulfoxide (DMSO), sodium dodecyl sulfate (SLS), sodium pentobarbital, and

Preparation of RS loaded PLGA microspheres using NaCl as osmogen

Sodium chloride is commonly used as porogen owing to its osmotic effects. It is well known that water molecules may pass from one aqueous phase to the other, based on the difference in osmotic pressure [4]. Materials such as electrolytes and drugs in either aqueous phases are also known to exert a similar osmotic effect [3]. Therefore, in our study, the osmotic gradient was varied through changing the amount of NaCl in the internal and external aqueous phases, in order to study its effect on

Conclusions

The utilization of NaCl as an osmogen in the w/o/w double emulsion technique leads to morphologically diverse microspheres. The increased concentration of NaCl in the external phase was proven to be advantageous in producing dimpled microspheres of high EE%, suitable particle size both geometrically and aerodynamically, good flow properties, and a sustained release profile. While more work is needed to extrapolate these findings to better anti-osteoporotic efficacy by pulmonary route, regarding

Acknowledgment

The authors would like to thank SPIC Pharma Co., India, and PURAC company, the Netherlands, for their kind supply of risedronate and PLGA polymers, respectively.

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