Preparation, characterization and in vivo evaluation of a combination delivery system based on hyaluronic acid/jeffamine hydrogel loaded with PHBV/PLGA blend nanoparticles for prolonged delivery of Teriparatide

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Abstract

In the current study, biodegradable PHBV/PLGA blend nanoparticles (NPs) containing Teriparatide were loaded in hyaluronic acid/jeffamine (HA-JEF ED-600) hydrogel to prepare a combination delivery system (CDS) for prolonged delivery of Teriparatide. The principal purpose of the present study was to formulate an effective and prolonged Teriparatide delivery system in order to reduce the frequency of injection and thus enhance patient's compliance. Morphological properties, swelling behaviour, crosslinking efficiency and rheological characterization of HA-JEF ED-600 hydrogel were evaluated. The CDS was acquired by adding PHBV/PLGA NPs to HA-JEF ED-600 hydrogel simultaneously with crosslinking reaction. The percentage of NPs incorporation within the hydrogel as well as the loading capacity and morphology of Teriparatide loaded CDS were examined. Intrinsic fluorescence and circular dichroism spectroscopy proved that Teriparatide remains stable after processing. The release profile represented 63% Teriparatide release from CDS within 50 days with lower burst release compared to NPs and hydrogel. MTT assay was conducted by using NIH3T3 cell line and no sign of reduction in cell viability was observed. Based on Miller and Tainter method, LD50 of Teriparatide loaded CDS was 131.8 mg/kg. In vivo studies demonstrated that Teriparatide loaded CDS could effectively increase serum calcium level after subcutaneous injection in mice. Favourable results in the current study introduced CDS as a promising candidate for controlled delivery of Teriparatide and pave the way for future investigations in the field of designing prolonged delivery systems for other peptides and proteins.

Introduction

Osteoporosis is a bone thinning disorder characterized by progressive decline in bone mineral density. This skeletal disease is now identified as the most common cause of bone loss particularly in the elderly. It is a silent disease because of its symptomless growth until occurrence of painful and severe fractures in patients. One in three women and one in four men aged over 50 will undergo at least one osteoporotic fracture and other related complications in their life span (Willson et al., 2015). Osteoporosis is also responsible for > 8.9 million bone fragilities that occurs annually worldwide (Hernlund et al., 2013). Due to the universal expansion of osteoporosis, the efficacy and potency of numerous anti-osteoporotic agents in management of this skeletal condition have been largely investigated. Bisphosphonates such as Alendronate, Etidronate, Risedronate and Ibandronate are known as the first line treatment in medicinal management of osteoporosis (Ott, 2011). Denosumab, Raloxifene, Calcitonin, Strontium Ranelate and Teriparatide (recombinant human parathyroid hormone) [rhPTH (1–34)] are the other recommended therapies (Iwamoto et al., 2006, Riggs and Parfitt, 2005). Among these treatment options, Teriparatide has unique effects. It is the only FDA approved drug which triggers bone formation through stimulation of osteoblasts, whereas the other traditional drugs known as bone resorption inhibitors are able to only inhibit osteoclasts. Teriparatide is an anabolic agent mainly administrated for patients suffering from glucocorticoid-induced osteoporosis, postmenopausal osteoporosis and patients who have failed to response to other anti-osteoporotic agents (Neer et al., 2001). The recommended therapeutic dose of Teriparatide is 20 μg injected subcutaneously once a day for two years (Eriksen et al., 2014). Since most of osteoporotic patients are the elderly, it cannot be disguised that repeated injection of Teriparatide for a long time is painful and irritating for the patients and it may even cause medication withdrawal. Poor patients' acceptance to multiple injections and also the exclusive properties of Teriparatide in promoting new bone formation (Eriksen and Robins, 2004) have provoked researchers to work on development of Teriparatide long acting formulation. Subsequently, the frequency of injection will be lessened and patient compliance will improve.

There are only few studies available on Teriparatide sustained release formulation and its characterization (Eswaramoorthy et al., 2012, Wei et al., 2004). Since Teriparatide is a bioactive peptide, general concerns such as high sensitivity and poor stability are the big challenges to be considered. Despite these facts, various strategies have been employed to achieve long acting formulation of peptides and proteins consisting of micro (Gaignaux et al., 2012, Geng et al., 2008) and nanoparticulate delivery systems (Emami et al., 2014, Parajo et al., 2010), hydrogels (Motokawa et al., 2006) and combination delivery system (CDS) (Peng et al., 2013, Hu et al., 2012). Although particulate delivery systems are the most widely used carriers in this field of drug delivery, countless efforts in obviating numerous problems accompanied with these systems have led to the break down (Allison, 2008, Yeo and Park, 2004). Poor encapsulation of peptide and protein, which results in a sudden burst release and subsequent undesirable adverse effects, is the most troublesome issue which should be resolved for further successful application of particulate carriers in clinics. Owing to this issue, various strategies such as blending of different biocompatible and biodegradable polymers with the aim of fabricating core-shell particles were employed (Chatterjee et al., 2014, Vukomanovic et al., 2011, Vukomanovi'ca et al., 2011, Zhu et al., 2009). Unfortunately, even core-shell nanoparticles (NPs) did not completely show promising results in enhancing entrapment efficiency (EE) and reducing initial burst release in polymeric particulate systems. In spite of various novel designs in fabricating more efficient particulate systems, a relatively large amount of drug still remains un-entrapped on the surface of particles and this leads to unwanted burst release.

Hydrogels also displayed uncontrolled swift release during the initial hours after injection (Wireland et al., 2007). Hence neither particulate systems nor hydrogels provide an ideal controlled release system for delivery of peptides and proteins. Thus, the new generation of controlled release formulation identified as CDS, which is the hybrid form of previously mentioned carriers, has recently come to the world of research. Fabricating a new CDS, keeping desirable properties of both NPs and hydrogels, is a promising perspective of the current study to achieve an ideal controlled release system for delivery of Teriparatide. In other words, CDS was prepared as a novel prolonged drug delivery system for subcutaneous injection of Teriparatide in patients suffering from osteoporosis to reduce the number of injections and therefore enhance patient's acceptance.

Among the polymers used in preparation of NPs, Poly (3-hydroxybutyrate-co-3-hydroxyvalerate acid) (PHBV) is a biocompatible and biodegradable polymer (Vilos et al., 2012, Lee et al., 2011) which has attracted pharmaceutical's attention in recent years due to its favourable properties in drug delivery and low cost of production (Vilos et al., 2013). The only obstacle in the extensive use of PHBV is its poor thermal stability which will be significantly decreased by blending this polymer with other polymers like Poly (lactic-co-glycolic acid) (PLGA) (Bazzo et al., 2012, Huang et al., 2009, Emiliomendes et al., 2012, Zhu et al., 2009). Besides this, polymer blending was previously introduced as a beneficial strategy in increasing EE of hydrophilic drugs into hydrophobic polymers to some extent although it could not be fully an effective solution (Zhu et al., 2009, Santander-Ortega et al., 2007, Csaba et al., 2006).

In the present study, PHBV/PLGA blend NPs were loaded in Hyaluronic acid (HA) based hydrogel as a second barrier in order to attain a desirable controlled release system which would not only sustain the peptide release but also diminish the burst release. HA as an important ingredient of extracellular matrix of human body, played a momentous role in cartilage lubrication because of its high capacity for water absorption which makes it an eligible candidate for biomedical applications and in particular for hydrogel preparation (Ouasti et al., 2011, Oha et al., 2010, Hahn et al., 2006, Motokawa et al., 2006). Subsequently, the morphological properties, swelling ratio, cross-linking efficiency and rheological behaviour of HA hydrogel were investigated to characterize both physical and chemical properties of the fabricated hydrogel.

Teriparatide loaded PHBV/PLGA blend NPs were successfully prepared and optimized according to our recently published work (Bahari Javan et al., 2016) and thereafter Teriparatide NPs were embedded in HA based hydrogel simultaneously with crosslinking reaction using Jeffamine ED-600 (aliphatic diamine derived from a propylene oxide capped polyethylene glycol) as a cross-linking agent to form CDS. The constructed CDS in this study resulted in 63% Teriparatide release over 50 days in a more sustained and controlled pattern compared to both PHBV/PLGA NPs and hydrogels alone. In order to elucidate the in vivo performance of CDS containing Teriparatide, it was subcutaneously injected in mice and serum calcium level was determined. Increase in calcium level was the proof of effective delivery of Teriparatide after subcutaneous injection.

Pursuant to the desirable properties of NPs and hydrogels, the combination of these two carriers brings us one step closer to achieving a controlled release formulation for delivery of peptides and proteins. Thus the current study pays, for the first time, particular attention to developing a novel injectable hyaluronic acid based hydrogel loaded with Teriparatide-PHBV/PLGA NPs to further prolong the biologic effect of Teriparatide as a result of which the frequency of injection will be reduced.

Section snippets

Materials

PHBV containing 2–3% polyhydroxyvalerate (PHV) by weight was purchased from Tianan Biologic Materials Ltd., Hangzhou, China; Teriparatide [rhPTH (1–34)] was obtained from Henan New-Sensation Chemical Co., Ltd. China. O, O′-Bis (2-aminopropyl) polypropylene glycol-block-polyethylene glycol-block-polypropylene glycol (Jeffamine ED-600), PLGA(50:50), polyvinyl alcohol (PVA) (average mol wt. 30,000–70,000), 1-Ethyl-3-[3-(dimethylamino) propyl] carbodiimide (EDC), N-hydroxysuccinimide (NHS),

Synthesis of HA-JEF ED-600 hydrogel and chemical characterizations

HA carboxyl groups were activated by appending NHS and EDC using the method explained above. The activated carboxyl groups became ready for covalent crosslinking reaction with amine groups in JEF ED-600. Eventually, the structure of the newly synthesized hydrogel was examined by FTIR (Fig. 1a). FTIR spectrum of HA displayed a relatively intense bond at 1664.86 cm 1 corresponding to acetylated amide groups in the structure of polysaccharide prior to chemical modifications. The intensity of the

Discussion

In recent decades, various strategies have been employed to design prolonged delivery systems for different peptides and proteins (Sinha and Trehan, 2003, Fogueri and Singh, 2009, Malik et al., 2007, Jayamanti et al., 2014). Nanoparticulate delivery systems and hydrogels have been extensively studied for this purpose but they still have some problematic issues which restrict their widespread application. Undesirable burst release, which could lead to adverse effects, is the principal obstacle

Conclusion

A novel CDS consisting of PHBV/PLGA NPs loaded in HA-JEF ED-600 hydrogel with the aim of preparing injectable formulation for prolonged delivery of Teriparatide was successfully designed and characterized. Swelling behaviour, crosslinking efficiency and rheological properties were all confirmative indicators for converting HA gel to HA-JEF ED-600 hydrogel. Morphological assessments of CDS revealed that the incorporation of NPs within the matrix of hydrogel took place. The more prolonging effect

Declaration of interest

This work was supported by Tehran University of Medical Sciences [grant number 93-03-33-27074, 2016]. The authors report no conflict of interest.

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