Injectable nanoparticle-loaded hydrogel system for local delivery of sodium alendronate

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Abstract

Systemic administration of bisphosphonates, e.g. sodium alendronate (Aln) is characterized by extremely low bioavailability and high toxicity. To omit aforementioned drawbacks an injectable system for the intra-bone delivery of Aln based on Aln-loaded nanoparticles (NPs-Aln) suspended in a hydrogel matrix (gellan gum, GG) was developed. Aln was encapsulated in poly(lactide-co-glycolide) (PLGA 85:15) by solid–oil–water emulsification. Drug release tests showed that within 25 days all the encapsulated drug was released from NPs-Aln and the release rate was highest at the beginning and decreased with time. In contrast, by suspending NPs-Aln in a GG matrix, the release rate was significantly lower and more constant in time. The GG–NPs-Aln system was engineered to be easily injectable and was able to reassemble its structure after extrusion as shown by rheological measurements. In vitro studies showed that the GG–NPs-Aln was cytocompatible with MG-63 osteoblast-like cells and it inhibited RANKL-mediated osteoclastic differentiation of RAW 264.7 cells. The injectability, the sustained local delivery of small doses of Aln and the biological activity render the GG–NPs-Aln system promising for the local treatment of osteoporosis and other bone tissue disorders.

Introduction

Bisphosphonates, e.g. sodium alendronate (Aln), are one of the most important classes of drugs for the treatment of bone tissue pathologies. It is known that the Aln prevents hydroxyapatite loss and inhibits osteoclast-mediated bone resorption (Hayden et al., 2014). This drug is mainly used for the treatment of osteoporosis, bone metastasis and Paget’s disease (Ezzati Nazhad Dolatabadi et al., 2014). More recently it has been proposed as adjuvant analgesic in combination with opioids in the management of cancer pain symptoms (Mitra and Jones, 2012) and as a bone antitumour agent (Cornelis et al., 2014). However, its systemic application beside low bioavailability (<1% when administered orally) evokes numerous side effects such as fever, esophageal erosions, ulcers and problems with gastrointestinal tract (Marshall et al., 2000).

The side effects and limitations of oral administration may be overcome by local delivery of Aln. So far, there have been few reports in the literature describing systems for local bisphosphonates administration; they were based on hydroxyapatite microspheres (Lee et al., 2013), calcium phosphate microspheres (Kim et al., 2010), poly(β-hydroxybutyrate-co- β-hydroxyvalerate)–hydroxyapatite composites (Huang et al., 2009) or carbopol gels (Reddy et al., 2005). Attempts to coat titanium implants with bisphosphonates to improve osteointegration have also been described (Harmankaya et al., 2013). Nevertheless the approach of local delivery of Aln is still poorly explored.

There is a need to develop a local delivery system for Aln which will be easy to handle by the surgeon, and will remain at the targeted area of the affected tissue. We hypothesize that such a system will assure local controlled delivery of small doses of the drug, so systemic side effects typical for oral Aln administration will be reduced. Moreover, the system will be administered via injection so it will be less-invasive and traumatic than typical surgical intervention performed to fill in the defect or treat affected area with bone graft. To this end, we developed an injectable material consisting of Aln-loaded nanoparticles suspended in a hydrogel matrix.

A resorbable polymer, poly(lactide-co-glycolide) (PLGA), was chosen to encapsulate Aln because this FDA-approved copolymer has a tunable degradation rate (i.e., by molar ratio of lactide to glycolide, chain structure and molecular weight) and can be easily processed into nanocarriers using emulsification (Shive and Anderson, 1997). As a hydrogel matrix gellan gum (GG) was used. GG is an anionic polysaccharide produced by bacteria (Sphingomonas elodea) during aerobic fermentation (Doner and Douds, 1995). GG is widely applied in the food and pharmaceutical industries (Babu et al., 2010) and more recently it has been proposed for cartilage (Oliveira et al., 2010) and bone (Douglas et al., 2014) regeneration. Further advantages of GG are low cost, biodegradability and low toxicity of its degradation products, as well as the fact that it is not animal-derived, avoiding regulatory concerns (Gantar et al., 2014).

The aim of this study was first to manufacture and characterize PLGA nanoparticles loaded with Aln. In the second step, drug delivery system based on Aln-loaded nanoparticles and GG hydrogel was produced and characterized in terms of its mechanical properties (rheology), surgical handiness (injectability, swelling) and Aln release kinetics. Finally, to confirm the feasibility of the developed system in the treatment of bone tissue defects and diseases, in vitro tests in contact with osteoblast-like cells and osteoclast-like cells were conducted.

Section snippets

Materials and chemicals

Sodium alendronate (Aln) was a gift from Polpharma S.A. Poland (batch no.: 504041227). Polyvinyl alcohol (PVA, Mowiol® 4-88, Mn = 31,000 Da) and gellan gum (Gelzan™ CM, classified as low-acyl gellan by the manufacturer) were bought from Sigma–Aldrich, Poland. Dichloromethane, methanol, 2-mercaptoethanol, isopropanol, orthophthaldialdehyde (OPA), phosphate buffered saline (PBS) buffer (concentrate), calcium chloride, potassium hydroxide and borate acid were all of analytical grade and were

Properties of nanoparticles

Nanoparticles were formulated with an average diameter of ca. 270 nm and ca. 230 nm for empty and Aln-loaded batches, respectively (Table 1). The polydispersity was low (0.2 and 0.3 for Aln-loaded and empty nanoparticles, respectively), which corresponded to a narrow size distribution (Fig. 1A and B). Laser electrophoresis demonstrated a negative zeta potential in the range of −28 and −30 mV (Table 1). The solubilization efficiency and loading efficiencies of Aln in the nanoparticles were ca. 70%

Discussion

We aimed at manufacturing a new type of Aln delivery system for the local treatment of osteoporosis and other bone diseases. The system, intended to be administered inside affected bone tissue as a minimally invasive and handy injection, was based on hydrogel containing Aln encapsulated within PLGA nanoparticles. Our hypothesis was that the system would assure local and sustained release of drug, which is known to inhibit osteoclasts maturation. In the study NPs-Aln were obtained and their

Conclusion

Our system based on Aln-loaded PLGA nanoparticles suspended in gellan gum hydrogel matrix: (i) was found to be injectable and restored its elastic structure after extrusion, (ii) assured local and uniform drug delivery, and (iii) inhibited osteoclasts differentiation without affecting osteoblasts functions. The results show the potential of our approach for the local treatment of osteoporosis and other bone tissue pathologies.

Conflict of interest

The authors have no conflict of interest to declare.

Acknowledgments

The authors would like to thank to Professor Piotr Dobrzynski (Center of Polymer and Carbon Materials, Polish Academy of Sciences) for providing us with degradable copolymer, Polpharma S.A. for providing sodium alendronate and Dr. Lukasz Zych (Department of Ceramics and Refractories, Faculty of Materials Science and Ceramics) for the access to the Zetasizer. Polish National Science Centre (Grant no: 2012/05/B/ST8/00129) and Ministry of Health of the Czech Republic (Grant No. NT13297-4/2012)

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