European Journal of Pharmaceutical Sciences 101 (2017) 167–181

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European Journal of Pharmaceutical Sciences

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Preparation, characterization and in vivo evaluation of a combinationdelivery system based on hyaluronic acid/jeffamine hydrogel loadedwith PHBV/PLGA blend nanoparticles for prolonged deliveryof Teriparatide

Nika Bahari Javan a, Hamed Montazeri b, Leila Rezaie Shirmard c, Nersi Jafary Omid a, Ghullam Reza Barbari a,Mohsen Amini d,e, Mohammad Hossein Ghahremani f, Morteza Rafiee-Tehrani a, Farid Abedin Dorkoosh a,g,⁎a Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iranb School of Pharmacy-International Campus, Iran University of Medical Sciences, Tehran, Iranc Department of Pharmaceutics, School of Pharmacy, Ardabil University of Medical Sciences, Ardabil, Irand Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Irane Drug Design and Development Research Center, Tehran University of Medical Sciences, Tehran, Iranf Department of Toxicology-Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Irang Medical Biomaterial Research Centre (MBRC), Tehran University of Medical Sciences, Tehran, Iran

Abbreviations: PHBV, poly (3-hydroxybutyrate-co-3poly (lactic-co-glycolic acid); NPs, nanoparticles; CDS, cohyaluronic acid; JEF ED-600, Jeffamine ED-600; EE, entrappacity; LE, loading efficiency; HAse, hyaluronidase.⁎ Corresponding author at: Pharmaceutical Products T

1462, Kargar Ave., 1439804448 Tehran, Iran.E-mail address: dorkoosh@tums.ac.ir (F. Abedin Dorko

http://dx.doi.org/10.1016/j.ejps.2017.02.0180928-0987/© 2017 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 31 October 2016Received in revised form 3 February 2017Accepted 9 February 2017Available online 13 February 2017

Chemical compounds studied in this article:Teriparatide (Pubchem CID: 16129682)PLGA (Pubchem CID: 23111554)PHBV (Pubchem CID: 107801)Hyaluronic acid (Pubchem CID: 24728612)Jeffamine ED-600 (Pubchem CID: 16212614)

In the current study, biodegradable PHBV/PLGA blend nanoparticles (NPs) containing Teriparatide were loadedin hyaluronic acid/jeffamine (HA-JEF ED-600) hydrogel to prepare a combination delivery system (CDS) forprolonged delivery of Teriparatide. The principal purpose of the present study was to formulate an effectiveand prolonged Teriparatide delivery system in order to reduce the frequency of injection and thus enhance pa-tient's compliance. Morphological properties, swelling behaviour, crosslinking efficiency and rheological charac-terization ofHA-JEF ED-600 hydrogelwere evaluated. The CDSwas acquired by adding PHBV/PLGANPs toHA-JEFED-600 hydrogel simultaneously with crosslinking reaction. The percentage of NPs incorporation within the hy-drogel as well as the loading capacity andmorphology of Teriparatide loaded CDSwere examined. Intrinsic fluo-rescence and circular dichroism spectroscopy proved that Teriparatide remains stable after processing. Therelease profile represented 63% Teriparatide release from CDSwithin 50 days with lower burst release comparedto NPs and hydrogel. MTT assay was conducted by using NIH3T3 cell line and no sign of reduction in cell viabilitywas observed. Based on Miller and Tainter method, LD50 of Teriparatide loaded CDS was 131.8 mg/kg. In vivostudies demonstrated that Teriparatide loaded CDS could effectively increase serum calcium level after subcuta-neous injection inmice. Favourable results in the current study introduced CDS as a promising candidate for con-trolled delivery of Teriparatide and pave the way for future investigations in the field of designing prolongeddelivery systems for other peptides and proteins.

© 2017 Elsevier B.V. All rights reserved.

Keywords:Combination delivery systemPHBV/PLGA blend nanoparticlesHyaluronic acid/jeffamine hydrogelProlonged deliveryTeriparatide

1. Introduction

Osteoporosis is a bone thinning disorder characterized by progres-sive decline in bone mineral density. This skeletal disease is now

-hydroxyvalerate acid); PLGA,mbination delivery system; HA,ment efficiency; LC, loading ca-

echnology Units Incubator, No.

osh).

identified as themost common cause of bone loss particularly in the el-derly. It is a silent disease because of its symptomless growth until oc-currence of painful and severe fractures in patients. One in threewomen and one in four men aged over 50 will undergo at least one os-teoporotic fracture and other related complications in their life span(Willson et al., 2015). Osteoporosis is also responsible for N8.9 millionbone fragilities that occurs annually worldwide (Hernlund et al.,2013). Due to the universal expansion of osteoporosis, the efficacy andpotency of numerous anti-osteoporotic agents in management of thisskeletal condition have been largely investigated. Bisphosphonatessuch as Alendronate, Etidronate, Risedronate and Ibandronate are

168 N. Bahari Javan et al. / European Journal of Pharmaceutical Sciences 101 (2017) 167–181

known as the first line treatment inmedicinal management of osteopo-rosis (Ott, 2011). Denosumab, Raloxifene, Calcitonin, StrontiumRanelate 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 drugwhich triggers bone formation through stimulation of osteoblasts,whereas the other traditional drugs known as bone resorption inhibi-tors are able to only inhibit osteoclasts. Teriparatide is an anabolicagent mainly administrated for patients suffering from glucocorticoid-induced osteoporosis, postmenopausal osteoporosis and patients whohave failed to response to other anti-osteoporotic agents (Neer et al.,2001). The recommended therapeutic dose of Teriparatide is 20 μginjected subcutaneously once a day for two years (Eriksen et al.,2014). Since most of osteoporotic patients are the elderly, it cannot bedisguised that repeated injection of Teriparatide for a long time is pain-ful and irritating for the patients and it may even cause medicationwithdrawal. Poor patients' acceptance to multiple injections and alsothe exclusive properties of Teriparatide in promoting new bone forma-tion (Eriksen and Robins, 2004) have provoked researchers to work ondevelopment of Teriparatide long acting formulation. Subsequently, thefrequency of injection will be lessened and patient compliance willimprove.

There are only few studies available on Teriparatide sustained re-lease formulation and its characterization (Eswaramoorthy et al.,2012;Wei et al., 2004). Since Teriparatide is a bioactive peptide, generalconcerns such as high sensitivity and poor stability are the big chal-lenges to be considered. Despite these facts, various strategies havebeen employed to achieve long acting formulation of peptides and pro-teins consisting of micro (Gaignaux et al., 2012; Geng et al., 2008) andnanoparticulate delivery systems (Emami et al., 2014; Parajo et al.,2010), hydrogels (Motokawa et al., 2006) and combination delivery sys-tem (CDS) (Peng et al., 2013; Hu et al., 2012). Although particulate de-livery systems are the most widely used carriers in this field of drugdelivery, countless efforts in obviating numerous problems accompa-nied with these systems have led to the break down (Allison, 2008;Yeo and Park, 2004). Poor encapsulation of peptide and protein, whichresults in a suddenburst release and subsequent undesirable adverse ef-fects, is themost troublesome issuewhich should be resolved for furthersuccessful application of particulate carriers in clinics. Owing to thisissue, various strategies such as blending of different biocompatibleand biodegradable polymers with the aim of fabricating core-shell par-ticles 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 inenhancing entrapment efficiency (EE) and reducing initial burst releasein polymeric particulate systems. In spite of various novel designs infabricating more efficient particulate systems, a relatively large amountof drug still remains un-entrapped on the surface of particles and thisleads to unwanted burst release.

Hydrogels also displayed uncontrolled swift release during the initialhours after injection (Wireland et al., 2007). Hence neither particulatesystems nor hydrogels provide an ideal controlled release system fordelivery of peptides and proteins. Thus, the new generation of con-trolled release formulation identified as CDS, which is the hybrid formof previously mentioned carriers, has recently come to the world of re-search. Fabricating a new CDS, keeping desirable properties of both NPsandhydrogels, is a promisingperspective of the current study to achievean ideal controlled release system for delivery of Teriparatide. In otherwords, CDS was prepared as a novel prolonged drug delivery systemfor subcutaneous injection of Teriparatide in patients suffering from os-teoporosis to reduce the number of injections and therefore enhancepatient's acceptance.

Among the polymers used in preparation of NPs, Poly (3-hydroxybutyrate-co-3-hydroxyvalerate acid) (PHBV) is a biocompatibleand biodegradable polymer (Vilos et al., 2012; Lee et al., 2011) which

has attracted pharmaceutical's attention in recent years due to itsfavourable properties in drug delivery and low cost of production(Vilos et al., 2013). The only obstacle in the extensive use of PHBV isits poor thermal stabilitywhichwill be significantly decreased by blend-ing 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 previouslyintroduced as a beneficial strategy in increasing EE of hydrophilicdrugs into hydrophobic polymers to some extent although it could notbe 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 inHyaluronic acid (HA) based hydrogel as a second barrier in order to at-tain a desirable controlled release systemwhichwould not only sustainthe peptide release but also diminish the burst release. HA as an impor-tant ingredient of extracellularmatrix of human body, played amomen-tous role in cartilage lubrication because of its high capacity for waterabsorption whichmakes it an eligible candidate for biomedical applica-tions and in particular for hydrogel preparation (Ouasti et al., 2011; Ohaet al., 2010; Hahn et al., 2006;Motokawa et al., 2006). Subsequently, themorphological properties, swelling ratio, cross-linking efficiency andrheological behaviour of HA hydrogel were investigated to characterizeboth physical and chemical properties of the fabricated hydrogel.

Teriparatide loaded PHBV/PLGA blend NPs were successfully pre-pared and optimized according to our recently published work (BahariJavan et al., 2016) and thereafter Teriparatide NPs were embedded inHA based hydrogel simultaneously with crosslinking reaction usingJeffamine ED-600 (aliphatic diamine derived from a propylene oxidecapped polyethylene glycol) as a cross-linking agent to form CDS. Theconstructed CDS in this study resulted in 63% Teriparatide release over50 days in a more sustained and controlled pattern compared to bothPHBV/PLGA NPs and hydrogels alone. In order to elucidate the in vivoperformance of CDS containing Teriparatide, it was subcutaneouslyinjected in mice and serum calcium level was determined. Increase incalcium level was the proof of effective delivery of Teriparatide aftersubcutaneous injection.

Pursuant to the desirable properties of NPs and hydrogels, the com-bination of these two carriers brings us one step closer to achieving acontrolled release formulation for delivery of peptides and proteins.Thus the current study pays, for the first time, particular attention to de-veloping a novel injectable hyaluronic acid based hydrogel loaded withTeriparatide-PHBV/PLGA NPs to further prolong the biologic effect ofTeriparatide as a result of which the frequency of injection will bereduced.

2. Materials and methods

2.1. Materials

PHBV containing 2–3% polyhydroxyvalerate (PHV) by weight waspurchased from Tianan Biologic Materials Ltd., Hangzhou, China;Teriparatide [rhPTH (1–34)] was obtained from Henan New-SensationChemical Co., Ltd. China. O, O′-Bis (2-aminopropyl) polypropyleneglycol-block-polyethylene glycol-block-polypropylene glycol (JeffamineED-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), morpholinoethanesulfonic acid(MES), ninhydrin and hyaluronidase (HAse) from bovine testes (400–1000 units/mg) were all purchased from Sigma-Aldrich. All other re-agents and solvents used in this work were analytical grade and theywere obtained from Merck.

2.2. Synthesis of chemically crosslinked HA based hydrogel

Initially, 200 mg Jeffamine ED-600(JEF ED-600), as a crosslinkingagent, was accurately weighed, dissolved in 4 ml methylene chloride

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at 25 °C and stirred until a clear solution was obtained. In order to dryJEF ED-600, excess amounts of anhydrous Na2SO4were added to the so-lution and thereafter it was separated by filtration. The organic solventwas removed by rotary evaporator and dried at room temperature for24 h.

After that, 110mgMES and 117mgNaClwere dissolved in 10ml de-ionized water to prepare an appropriate buffer solution. In order to ob-tain 1%w/vHA in buffer solution, 40mgHAwas dissolved in 4ml buffersolution. Then pH was adjusted to 5.5. Afterwards, 174 mg NHS and580 mg EDC were added to the previously prepared solution to activatecarboxyl groups on the backbone of HA. JEF ED-600 was then added toHA solution and continuously stirred at room temperature for approxi-mately 8 h. In the next step, the solution was spread on the Teflon plateand remained at room temperature for 24 h until it completely dried. Fi-nally, HA-JEF ED-600 hydrogel was gently cut into smaller pieces andplaced in deionized water to remove unreacted reagents for furtherassessments.

2.3. Structural characterization of HA-JEF ED-600 hydrogel

The structure of the resulting hydrogel was examined by both Fouri-er Transformed Infrared Spectroscopy (FTIR, Nicolet Magna IR-550,Thermo Electron Corporation, Beverly, Massachusetts, USA) and protonnuclear magnetic resonance (H NMR, 500 MHz, Bruker, Fällanden,Switzerland).FTIR spectra were acquired from samples in solid formusing KBr as a filler, within 400–4000 cm−1 at room temperature. HNMR spectra were conducted at room temperature using D2O as asolvent.

2.4. Degree of swelling ratio

The swelling ratio of both HA-JEF ED-600 hydrogel and CDS (NPsloaded in HA-JEF ED-600 hydrogel) was determined. Hydrogel andCDS were separately weighed and placed in phosphate buffer saline(PBS) at 37 °C for 5 days. At predetermined intervals including 1, 2, 6,12, 24, 48, 72, 96 and 120 h, the wet samples were removed from PBSmedium through filtration and the weight of swollen carriers were de-termined. Finally the percentage of swelling ratio was calculated apply-ing the following formula:

Swelling ratio %ð Þ ¼ Ws−Wd=Wd � 100 ð1Þ

whereWs is theweight of swollen samples (hydrogel or CDS) andWd isthe weight of dry samples prior to immersing in PBS medium.

2.5. Measurement of crosslinking efficiency

The ninhydrin assaywas employed to determine the crosslinking ef-ficiency. Initially, the samples were dispersed in 5 ml of 1 M sodium ac-etate buffer (pH = 5) and then excessive amount of ninhydrin reagentwas appended to the medium. Thereafter, the mixture was gentlystirred at 85 °C for approximately 25min and the test mediumwas cov-ered with a piece of paraffin film to avoid solvent loss. In the next step,75 ml of water/ethanol (1/1, v/v) was added to the mediumwhich wasthen kept in a dark condition and cooled to room temperature in a coldwater bath before measuring the absorbance. The aforementioned pro-cedure was followed for the material remaining on the filter as well asfor the unreacted JEF ED-600 collected from the rinse solution. Thiswas done in order to determine the amount of JEF ED-600 graftedthrough one amino group called “single end anchorage” and to quantifythe amount of crosslinking agent which remained intact and unreactedduring coupling reaction, respectively. Ninhydrin reacted with bothunreacted amino groups and single end anchorage of JEF ED-600 andformed a bluish-red substance whichwas furthermeasured by spectro-photometer (752S UV/VIS Spectrophotometer, Angstrom AdvancedInc., Stoughton, Norfolk County, Massachusetts, USA) at thewavelength

of 570 nm. Eventually, the crosslinking efficiency (%) wasmeasured ac-cording to the following equation:

Crosslinking efficiency %ð Þ ¼ ðJEF ED−600initial−ðJEF ED−600single end anchorage

þJEF ED−600unreactedÞ=JEF ED−600initialÞ � 100

ð2Þ

where JEF ED-600initial indicates the initial amount of jeffamine added tothemedium at the beginning of reaction, JEF ED-600single end anchorage re-fers to the amount of jeffamine chemically grafted only through oneamino group and JEF ED-600unreacted states the amount of jeffamineremained unreacted.

2.6. Rheological characterizations

The rheological behaviour of newly designed HA-JEF ED-600 hydro-gelwas investigated bymeans of a parallel plate rheometer (Anton Paar,Physica MCR 301, Styria, Austria) at 25 °C, angle of 1°. Briefly, the sam-ples were placed on the stationary plate (diameter 15 mm) of rheome-ter and the rheological experiments were carried out after temperatureequilibrium at 0.01–1000 s−1. Themaximum time spent on performingeach test was 20 min. Additionally, small amplitude oscillatory shear(SAOS) method was utilized to evaluate the viscoelastic properties ofthe fabricated hydrogels including shear storage or elastic modulus (G′)and shear loss or viscous modulus (G″). The angular frequency sweepwas conducted from 0.05–500 rad/s at 25 °C.

2.7. Preparation of Teriparatide loaded combination delivery system (CDS)and Teriparatide loaded HA-JEF ED-600 hydrogel

In order to fabricate Teriparatide loaded CDS including Teriparatide-PHBV/PLGA NPs loaded in HA-JEF ED-600 hydrogel, two individualsteps were carried out. Initially, Teriparatide loaded PHBV/PLGA blendNPs were prepared via double emulsion solvent evaporation technique(W1/O/W2) and optimized by using Box-Behnken response surfacemethodology, according to our previous work (Bahari Javan et al.,2016). Thereafter, the optimized Teriparatide-PHBV/PLGA NPs wereembedded in HA-JEF ED-600 hydrogel concurrently to crosslinking re-action to form Teriparatide loaded CDS. In other words, in the utilizedmethod for preparation of CDS, the NPs entrapment within the matrixof hydrogel and the crosslinking occur simultaneously. For this purpose,after activating carboxylic groups on the backbone of HA via EDC andNHS, Teriparatide loaded NPs were added to 4 ml HA solution (1% w/v) under gentle magnetic stirring, accompanied by the addition of200 mg JEF ED-600. Three different weight ratios of NPs: hydrogel(1:1, 2:1 and 4:1) were selected to distinguish the optimum ratio.Thereafter, the percentage of NPs incorporationwithin thematrix of hy-drogel and loading capacity (%LC) of CDS for each ratio were calculatedaccording to the following formulas:

%NPs Incorporation ¼ WCDS−WHydrogel=WInitial NPs � 100 ð3Þ

%LC ¼ WCDS−WHydrogel� ��%LE of NPs� �

=WCDS � 100 ð4Þ

where WCDS, W Hydrogel and W Initial NPs refer to weight of constructedCDS, weight of HA- JEF ED-600 hydrogel alone (without NPs) andweight of initial amount of Teriparatide loadedNPs using in CDS fabrica-tion, respectively. %LE of NPs alluded to the loading efficiency of former-ly optimized PHBV/PLGA NPs loaded with Teriparatide reported in ourprevious work (Bahari Javan et al., 2016).

Teriparatide loaded HA-JEF ED-600 hydrogel was prepared using asimilar method with minor modifications. Briefly, after activating thecarboxylic groups on the backbone of HA with the aid of EDC andNHS,765 μg Teriparatide was added to 4 ml HA solution (1% w/v)under gradual stirring, followed by the addition of 200 mg JEF ED-600

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as a crosslinking agent. The synthesis process was completed accordingto the described method in Section 2.2.

2.8. Morphological characterizations

HA-JEF ED-600 hydrogel and CDS containing Teriparatideweremor-phologically assessed by Field Emission Scanning Electron Microscopy(FESEM, S4160, Hitachi, Japan). The samples were mounted on the alu-minium stubs and were coated by a thin layer of gold with a sputtercoater to prevent them from charging.

2.9. In vitro Teriparatide release studies

The release profile of Teriparatide from CDS was investigated andthe acquired results were compared with both previously optimizedPHBV/PLGA NPs and HA-JEF ED-600 hydrogel containing Teriparatide.In order to measure the initial amount of Teriparatide incorporated inHA-JEF ED-600 hydrogels, 200 μl of HAse solution (500 units/ml) wasutilized to degrade HA-JEF ED-600 hydrogel at 37 °C in 24 h. Thereafter,100 μl of the decomposed solutionwas collected and dilutedwith 300 μlof PBS (pH = 7.4) prior to high performance liquid chromatography(HPLC, Agilent technology, Santa Clara, California, USA) for quantitativemeasurements of Teriparatide (Rane et al., 2012).

In vitro release studies of Teriparatide from both CDS and hydrogelwere conducted using the dialysis method as follows. In order to allowonly the released Teriparatide to permeate into the medium and retainthe polymeric carriers, dialysis bags withmolecular weight cut-off of 12KD were employed. Initially, the samples (50 mg CDS or hydrogel con-taining Teriparatide) were dispersed in 1 ml of PBS (pH = 7.4) andthen added to dialysis bag. Thereafter, the dialysis bag was submergedinto 10ml PBSwhich had been pre-heated to 37 °C. The releasemediumwas shaken under mechanical agitation. At predetermined intervals,100 μl of aliquots were withdrawn and the release medium wasreplenished with freshly prepared PBS to maintain the sink conditionduring the experiment. In order to testify that the dialysis bag had noretaining impact on peptide release, the release pattern of freeTeriparatide was also evaluated.

2.10. Mathematical modelling of release data

The kinetic of Teriparatide release from CDS and the mechanism ofrelease were examined by fitting the data gained from in vitro releasestudies to different mathematical models consisting of zero order,first-order, Higuchi's and Korsmeyer's models. For zero and first ordermodels defined as a constant rate and linear kinetic mechanisms,Eqs. (5) and (6) were used, respectively. The release data was alsoanalysed by the Higuchi's model (Eq. (7)), as well as Korsmeyer'smodel (Eq. (8)).

Mt ¼ K0t ð5Þ

Log Mt ¼ logM0−K1t=2:303 ð6Þ

Mt=M∞ ¼ KHt½ ð7Þ

Mt=M∞ ¼ K0tn ð8Þ

Mt andM0 displays the amount of drug released at time t and the initialamount of drug released, respectively. Mt/M ∞ is the portion of drug re-leased. K0, K1 and KH are the rate constants for zero, first and Higuchimodels, respectively. K′ is the release rate constant identifying geomet-ric properties of the carrier and “n” is the diffusion-power that repre-sents the main mechanism of drug transport.

2.11. Stability of Teriparatide

2.11.1. Intrinsic fluorescence spectroscopyIntrinsic fluorescence spectroscopy was used to confirm that addi-

tion of different excipients and formulation condition processing didnot alter the structure of Teriparatide during CDS preparation. In thismethod, the samples were interspersed in deionized water and subse-quently subjected to spectrofluorometer (LS 55, Perkin Elmer,Waltham,Massachusetts, USA). Intrinsic fluorescence was scanned between 250and 400 nm. The excitation slits, emission slits, excitation wavelengthand emissionwavelengthwere set at 2.5, 5.0, 295.93 and 360.92 nm, re-spectively. Additionally, neat Teriparatide was dissolved in deionizedwater and investigated by spectrofluorometer.

2.11.2. Circular dichroismThe stability of Teriparatide was further proved by circular dichro-

ism (CD) using circular dichroism spectrometer (Model 215, Aviv, Lake-wood, Los Angeles County, California, USA). A CDS containing 0.5mg/mlTeriparatide was utilized for Far-UV CD measurements at 180–250 nmin a 0.02 cm quartz cuvette. The CD signal was the average of five repli-cated scans and the corresponding solvent spectrum was subtractedfrom the sample spectrum. Pure Teriparatide, as a reference, was alsoassessed by circular dichroism spectrometer.

2.12. Cytotoxicity studies

The cytotoxicity of Teriparatide, CDS (drug free) and Teriparatideloaded CDS against NIH3T3 was evaluated using MTT colorimetricassay. In this method, the yellow water soluble tetrazolium salt, MTT(Sigma, Germany), is reduced to purple insoluble formazan crystals.The formazan crystals are then solubilized using an organic solvent,such as DMSO, and the optical density of the resultant solution will beattributed to the number of metabolically active cells.

For this purpose, NIH3T3, the standard fibroblast cell line are obtain-ed from Iranian Biological Resource Center (IBRC, Tehran, Iran). NIH 3T3cells aremouse embryonic cell lines of a non-malignant origin. Althoughit is immortalized andnot diploid, it is considered to be a normal like cellline and a suitable, standard model for cytotoxicity assessment inin vitro experiments (Orsine et al., 2013; Tomankova et al., 2015;Hillegass et al., 2010).

Cells cultured in RPMI 1640 medium containing 10% FBS, 2 mM L-glutamine, 100 units/ml penicillin and 100 μg/ml streptomycin (allfrom Biosera, UK) in humidified air with 5% CO2 at 37 °C. Briefly, thecells were seeded in a 96-well plate at a cell density of 104 cells/well.24 h later, the cells were treated with four different concentrations ofTeriparatide-CDS (0.05, 0.5, 5 and 50 nM) and their correspondingCDS alone. To rule out toxicity of Teriparatide per se, the cells werealso treated with the highest concentration of Teriparatide (50 nM).24 and 48 h after treatment, the medium was removed and 20 μl of5 mg/ml MTT in PBS was added to each well. Later, the plates were in-cubated in darkness for 4 h at 37 °C. Then 60 μl of DMSO was appendedto each well to dissolve the water insoluble formazan crystals and thenthe plates were vigorously shaken to ensure complete solubilisation.The intensity of the colour produced in each well was measured by mi-croplate reader (BioTek ELX800, Winooski, Chittenden County, Ver-mont, USA) at 570 nm wavelength and a reference wavelength of690 nm. The reproducibility of the results was ensured by at leastthree independent experiments with a minimum of eight internal rep-licates. The cell viabilitywas calculated according to the following equa-tion:

Cell viability %ð Þ ¼ Abss=Abscontrolð Þ � 100 ð9Þ

where, Abss is the absorption of incubated samples containing CDS/Teriparatide-CDS and Abscontrol is the absorption of non-treated.

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2.13. In vivo studies

Animal experiments were carried out in compliancewith guidelinesapproved by the Animal Care and Use Committee of Tehran Universityof Medical Sciences. The animals were kept under standard laboratoryconditions (temperature 24 °C and 12 h light-dark cycle) for 7 daysprior to the studies to acclimatize to the experimental environment.Food and tap water was provided ad libitum.

2.13.1. LD50 calculationsLD50 was determined according to Miller and Tainter method

(Randhawa, 2009). The mice fasted for 18 h were injected subcutane-ously by Teriparatide loaded CDS. Five groups of mice (10 animals ineach group) were injected by five different doses including 50, 100,200, 250 and 300 mg/kg. The animals were monitored at 2, 6, 12, 24,48 and 72 h after administration for any toxic symptoms. After 72 h,the number of dead mice in each group and the mortality percentagefrom 0% to 100% were determined. The percentage of mortality for 0and 100 were corrected prior to probits measurements. For correctionof 0% and 100% mortality, 100(0.25/n) and 100(n − 0.25/n) formulaswere used, respectively, where n was considered to be 10 mice(CHANDRA et al., 2014; Randhawa, 2009). Finally, the log-doses wereplotted versus probits. According to Miller and Tainter graphical meth-od, the dose corresponding to probit 5 stating 50%mortalitywasfiguredout as LD50 (Miller and Tainter, 1944).

2.13.2. In vivo Teriparatide bioactivity and efficacyThe mice with free access to water and food were divided into three

groups of 21 mice per group. Group 1, serving as a blank control, wassubcutaneously administrated by blank CDS (PHBV/PLGA NPs loadedin HA-JEF ED-600 hydrogel, without Teriparatide). Group 2 and 3were subcutaneously injected by Teriparatide solution in PBS(0.5 mg/kg) and Teriparatide-CDS (0.5 mg/kg), respectively. Atpredetermined time intervals (0, 1, 7, 15, 30, 40 and 50 days), threemice in each group were anesthetized with an intraperitoneal injectionof ketamine (50 mg/kg) and xylazine (5 mg/kg) and serum calciumlevels were determined by colorimetric assay with o-cresolphthaleincomplexonemethod (Cohen and Sideman, 1979). In addition to calciumdetermination, CDS was recovered from the injection site at each time

Fig. 1. (a) FTIR spectra of (i) HA and (ii) HA-JEF ED-600 hydrogel. (b) H NMR spectra of (i) HA anamide bonds formation were proved by FTIR and H NMR.

point and it was compared in terms of degradation and inflammationto assess the in vivo degradation of CDS (Hahn et al., 2007).

3. Results

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

HA carboxyl groups were activated by appending NHS and EDCusing the method explained above. The activated carboxyl groups be-came ready for covalent crosslinking reaction with amine groups inJEF ED-600. Eventually, the structure of the newly synthesized hydrogelwas examined by FTIR (Fig. 1a). FTIR spectrum of HA displayed a rela-tively intense bond at 1664.86 cm−1 corresponding to acetylatedamide groups in the structure of polysaccharide prior to chemical mod-ifications. The intensity of the aforementioned amide bond in the regionof 1664.86 cm−1 was remarkably enhanced in the FTIR spectrum of HA-JEF ED-600 hydrogel in comparison with neat HA, which was aconfirmative indicator for formation of new amide bonds. Afterwards,the structure of HA-JEF ED-600 hydrogel was affirmed by H NMR asshown in Fig. 1b. According to H NMR spectroscopy, the protons relatedtomethyl groups and sugar rings of HA appeared at 1.9 and 3.3 ppm, re-spectively. After conversion HA to HA-JEF ED-600 hydrogel, the protonsin methyl groups of jeffamine ED-600 became visible at 1 ppm andslightly below 1 ppm in the H NMR spectrum of HA-JEF ED-600 hydro-gel. The conjugation percentage revealed fromHNMR area under curvestatistics was found to be 93.9%.

3.2. Swelling ratio experiments

The swelling ratio of bothhydrogel andCDS as a function of timewasoverviewed. It wasmeasured at predetermined time points including 1,2, 6, 12, 24, 48, 72, 96 and 120 h after immersing samples in PBS. Theswelling ratios of hydrogel were found to be 108%, 112%, 128%, 136%,150.5%, 166%, 177%, 177.6% and 178% at aforementioned intervals, re-spectively, whereas the swelling ratios of CDS were figured out to be105%, 110%, 117%, 120.5%, 124.5%, 127.6%, 133.7%, 134.5% and 135% atpredetermined intervals, respectively.

d (ii) HA-JEF ED-600 hydrogel. Structural characterization of HA-JEF ED-600 hydrogel and

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3.3. Crosslinking efficiency determination

In order to quantify the amount of both unreacted and single-endanchorage JEF ED-600, ninhydrin assay was used. Ninhydrin assaydisplayed that from the total amount of crosslinking agent (JEF ED-600) utilized in the coupling reaction, 5.4% grafted through only oneamino group identified as single-end anchorage and 4.7% did not partic-ipate in the coupling reaction and remained unreacted. Consequently,the crosslinking efficiency determined based on the previously men-tioned formula was equal to 89.9%.

3.4. Rheological characterization of HA-JEF ED-600 hydrogel

In order to assess the rheological properties of HA-JEF ED-600 hydro-gel, stress-strain curve also called flow curve was depicted by plottingshear stress (Ʈ) against velocity gradient of the flow or flow rate (ϒ).The relationship between Ʈ and ϒ is described based on Eq. (10), inwhich “n” value refers to power law index. Fig. 2a exhibits log Ʈ againstlog ϒ for both HA gel and HA-JEF ED-600 hydrogel, individually. Accord-ingly, the “n” values for HA gel and HA-JEF ED-600 hydrogel gained fromthe slopes of the latter plots were found to be 0.86 and 0.91, respectively.

Ʈ ¼ ηϒn ð10Þ

Fig. 2b and c represent the data acquired from SAOS analysis. Shearstorage or elastic (G′) and shear loss or viscous (G″) moduli were plot-ted versus angular frequency for both HA gel prior to covalentcrosslinking reaction and HA-JEF ED-600 hydrogel. Obviously, for HAgel G″ was higher than G′ within almost the whole range of analysis,whereas for HA-JEF ED-600 hydrogel G′ was remarkably higherthan G″, indicating that the rheological behaviour of HA changed asa result of crosslinking reaction. In the following, the phase shift tan-gent described as tan δ = G″ / G′ was plotted versus angular

Fig. 2. Rheological measurements: (a) Stress-strain curve for HA and HA-JEF ED-600 hydrogehydrogel. (d) Phase shift tangent vs. angular frequency for HA and HA-JEF ED-600 hydrogel.

frequency for both HA gel and HA-JEF ED-600 hydrogel (Fig. 2d). Itwas deduced that tanδ for HA-JEF ED-600 hydrogel was significantlylower than HA gel, stating that converting HA gel to HA-JEF ED-600hydrogel led to significant decrease in G″. As a consequence of hy-drogel formation, the rheological properties of the matrix shiftedfrom a viscous (fluid-like) behaviour to more elastic (solid-like)material.

3.5. Preparation of Teriparatide loaded combination delivery system (CDS)

In order to clarify the effect of NPs:hydrogel ratios on the percentageof NPs incorporation within the matrix of hydrogel, three different ra-tios of NPs:hydrogel consisting of 1:1, 2:1 and 4:1 were evaluated dur-ing the CDS preparation. The percentage of NPs incorporation wasfigured out to be 86%, 46% and 26% for the aforementioned ratios, re-spectively. On the other hand, the loading capacity of CDS was foundto be 2.31%, 2.4% and 2.5%, respectively.

3.6. Morphological characterizations

SEM images revealed that both HA-JEF ED-600 hydrogel (Fig. 3a)and CDS containing Teriparatide (Fig. 3b) exhibited porous structure.The only observable distinction between these two carriers lies in thematrix surface. The matrix surface of hydrogel showed a relativelysmooth texture while the matrix surface of CDS is relatively rough dueto the NPs which are adhered to or are accommodated in the matrixof hydrogel.

3.7. In vitro Teriparatide release studies

The release profiles of Teriparatide from PHBV/PLGA blend NPs, HA-JEF ED-600 hydrogel, CDS and free Teriparatide are depicted in Fig. 4.Due to the rapid release of free Teriparatide, which resulted in a 99.5%

l. SAOS tests: G″ and G′ modulus vs. angular frequency for (b) HA and (c) HA-JEF ED-600

Fig. 3. SEM images of (a) HA-JEF ED-600 hydrogel and (b) CDS consisting of PHBV/PLGA NPs loaded in HA-JEF ED-600 hydrogel. Arrows illustrate the loaded NPs.

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release within the first 12 h of peptide release, the retaining effect of di-alysis bag on Teriparatide release was completely eliminated. Therelease pattern of Teriparatide fromHA-JEF ED-600 hydrogel represent-ed a relatively swift release resulted in 25% initial burst release within12 h. 84.8% of total Teriparatide was released from HA-JEF ED-600 hy-drogel within 10 days. The release profile of Teriparatide from formerlyoptimized PHBV/PLGA blend NPs was divided into two main phases. Infirst phase lasting for 5 days, 37.5% of total Teriparatide was released, ofthis amount 22% was rapidly released in 12 h demonstrated the first

burst release and 15.5% was gradually released till the end of day 5. Insecond phase lasting for 25 days, 26.9% of peptide was released, 11.5%in second burst release and 15.4% in second plateau stage. Converselyto PHBV/PLGA blend NPs, in newly constructed CDS the biphasic releasepattern vanished entirely. As it is shown in Fig. 4, 63% of totalTeriparatide was released from the newly designed CDS in a sustainedmanner within 50 days. 57.4% of Teriparatide was released within30 days and only 5.6% was released during the following 20 days in anextremely gradual and sustained pattern. Only one burst release was

Fig. 4. In vitro cumulative release profiles of free Teriparatide, Teriparatide loaded hydrogel, Teriparatide loaded PHBV/PLGA NPs and Teriparatide loaded CDS (n = 3).

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detected in the whole release profile of Teriparatide from CDS resultedin 12% burst release after 12 h, which was remarkably lower than theburst releases observed in both NPs and hydrogels.

3.8. Kinetic analysis of Teriparatide release from CDS

Different mathematical models were employed to elucidate themechanism of Teriparatide release from the fabricated CDS.

The obtained correlation coefficients (R-squares) for the zero order,first order, Higuchi model and Korsmeyer-Peppas model were 0.76,0.34, 0.78 and 0.99, respectively. The “n” value for the constructed CDSwas found to be 0.29.

Fig. 5. Florescence spectroscopy of Teriparatide (….) and Teriparatide loaded CDS (―). The flremained intact indicating structural stability.

3.9. Intrinsic fluorescence spectroscopy

The fluorescence spectrum of Teriparatide loaded CDS in compari-son with the intact Teriparatide was evaluated in order to corroboratethat Teriparatide was not exposed to structural alterations during for-mulation (Fig. 5). Similar shapes of these two spectra revealed that nochanges occurred in the structure of Teriparatide during preparationof CDS.

3.10. Circular dichroism

Circular dichroism spectroscopy in Far-UV region was performed toascertain that formulation processing and adding excipients did not

uorophore groups in Teriparatide loaded CDS compared to pure Teriparatide have been

Fig. 6. Far-UV circular dichroism spectra of Teriparatide (―) and Teriparatide loaded CDS(….). The secondary structure of Teriparatide loaded CDS is extremely similar to the neatTeriparatide stating the structural stability.

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alter the secondary structure of Teriparatide. As it can be seen in Fig. 6,the solvent-subtracted Far-UV CD spectra of Teriparatide loaded CDSand neat Teriparatide were extremely similar to each other. Thus, itcan be deduced that secondary structure of Teriparatide loaded CDSremained intact during formulation.

3.11. Effect of CDS and Teriparatide-CDS on viability of NIH3T3 cells

As it is shown in Fig. 7, MTT assay revealed that no remarkable toxiceffects could be figured out neither in CDS (drug free) nor inTeriparatide loaded CDS in concentrations of 0.05, 0.5 and 5 nM, in com-parisonwith control group, after 24 h.However, the cell viability in bothCDS (drug free) and Teriparatide loaded CDS treatment at concentrationof 50 nM, compared to control group, implies toxicity whereby the de-gree of toxicity is more prominent in cells treated with Teriparatideloaded CDS.

On the other hand, cells incubated for 48 h only showed toxicity at50 nM of Teriparatide loaded CDS and no decrease in cell viability was

Fig. 7. Cytotoxicity analysis of free and Teriparatide loaded CDS. NIH3T3 cells were treatedconcentrations of CDS and Teriparatide (50 nM). The cell proliferation was determined us*P b 0.05, ***P b 0.001 compared to control cells).

observed in other groups. Interestingly, cell density was increased inconcentrations of CDS other than 50 nM, especially at 0.05 nM of bothCDS and Teriparatide loaded CDS.

3.12. LD50 calculations

Common toxicity symptoms such as abnormal gait, dullness, lethar-gy, hair shedding, burying the head in saw dust, salivation, arching androlling, lacrimation, chewing and lickingweremanifested in some treat-ed animals. The mice, which exhibited laboured breathing and gasping,eventually died within 3 days. Initially, at dose of 50 mg/kg no toxicitysymptoms were observed, whereas signs of poisoning were seen insome animals in other doses. The number of dead mice, the percentageof mortality, the corrected values for 0% and 100%mortality and probitsat each dose are tabulated in Table 1. In order to convert percentage ofmortality to probits, standard fixed table previously introduced byMill-er and Tainter for LD50 determinations was used (Table 2). As it is illus-trated in Fig. 8, the log LD50 and LD50 were 2.12 and 131.8 mg/kg,respectively.

3.13. Serum calcium level determination

Mean serum calcium level for each group of animals was plottedagainst time-points. In the control group, mean serum calcium levelremained relatively constant and no significant alteration was detectedin all time-points. In group 2 injected by Teriparatide solution in PBS,serum calcium level reached 10.9 mg/dl in day one, which was signifi-cantly higher than control group at the corresponding time (P-value ≤ 0.05). Afterwards, calcium level fell in all time-points until it fi-nally reached 7.57 mg/dl. Serum calcium level exhibited a completelydifferent trend in group 3 injected by Teriparatide loaded CDS. In thisgroup, calcium level reached 10.23 mg/dl in day one, which was re-markably higher than control group (P-value ≤ 0.05) but it representedno significant difference in comparison with group 2 (P-value ≥ 0.05).Contrary to group 2, serum calcium level was increased in group 3 andit reached 12.10 mg/dl at the end of day 50, which was significantlyhigher than both groups 1 and 2 at the similar time-point (P-value ≤ 0.05) (Fig. 9). After 50 days, most of the CDS was degradedand only partial residue of the fabricated CDS could be recovered fromthe injection site in comparison with the first day of injection.

with different concentrations of Teriparatide loaded CDS (0.05–50 nM), correspondinging MTT assay at 24 h and 48 h after treatment (One way ANOVA, Dunnett's post test

Table 1Results of the lethal doses of Teriparatide loaded CDS for the determination of LD50 after subcutaneous injection inmice. Five groups of mice (10 animals in each group) were injected byfive different doses of Teriparatide loaded CDS (50, 100, 200, 250 and 300 mg/kg) and toxic symptoms were monitored for up to 72 h after administration.

Group Dose (mg/kg) Log dose Dead %Dead %Corrected Probits

1 50 1.7 0 0 2.5 3.042 100 2.0 3 30 30 4.483 200 2.3 7 70 70 5.524 250 2.4 9 90 90 6.285 300 2.5 10 100 97.5 6.96

Table 2Standard fixed table for transformation of percentage mortalities to probits introduced byMiller and Tainter.

Transformation(%)

0 1 2 3 4 5 6 7 8 9

– 2.67 2.95 3.12 3.25 3.36 3.45 3.52 3.59 3.6610 3.72 3.77 3.82 3.87 3.92 3.96 4.01 4.05 4.08 4.1220 4.16 4.19 4.23 4.26 4.29 4.33 4.36 4.39 4.42 4.4530 4.48 4.50 4.53 4.56 4.59 4.61 4.64 4.67 4.69 4.7240 4.75 4.77 4.80 4.82 4.85 4.87 4.90 4.92 4.95 4.9750 5.00 5.03 5.05 5.08 5.10 5.13 5.15 5.18 5.20 5.2360 5.25 5.28 5.31 5.33 5.36 5.39 5.41 5.44 5.47 5.5070 5.52 5.55 5.58 5.61 5.64 5.67 5.71 5.74 5.77 5.8180 5.84 5.88 5.92 5.95 5.99 6.04 6.08 6.13 6.18 6.2390 6.28 6.34 6.41 6.48 6.55 6.64 6.75 6.88 7.05 7.33

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4. Discussion

In recent decades, various strategies have been employed to designprolonged delivery systems for different peptides and proteins (Sinhaand Trehan, 2003; Fogueri and Singh, 2009;Malik et al., 2007; Jayamantiet al., 2014). Nanoparticulate delivery systems and hydrogels have beenextensively studied for this purpose but they still have some problemat-ic issues which restrict their widespread application. Undesirable burstrelease, which could lead to adverse effects, is the principal obstacle formost of these carriers loading with peptides and proteins (Zhang et al.,2005). Nevertheless, NPs and hydrogels still represent favourable prop-erties in designing prolonged delivery systems which cannot be dis-guised (Segura et al., 2005; Ouasti et al., 2011; Leach and Schmidt,2005; Gan and Wang, 2007; Estrada et al., 2014; Emami et al., 2014).Thus, combining NPs and hydrogels to make a novel prolonged deliverysystem has highly attracted pharmaceutical's attention in the recentyears. CDS has hopefully demonstrated favourable results in achievingideal controlled release systems for different therapeutic agents suchas dexamethasone, rifampicin and insulin (Bhardwaj et al., 2010; Huet al., 2012; Peng et al., 2012). In view of this, previously optimizedPHBV/PLGA blend NPs (Bahari Javan et al., 2016) were successfully em-bedded in newly constructed HA-JEF ED-600 hydrogel.

Initially, HA-JEF ED-600 hydrogel was synthesized by reacting thecarboxylic groups on the backbone of HA formerly activated by EDCand NHS, with amine groups on Jeffamine ED-600. FTIR and H NMRfindings ascertained the formation of amide bonds in chemicallycrosslinked hydrogel. In order to assess the hydrogel behaviour, swell-ing ratio, crosslinking efficiency and rheological characteristics of thefabricated hydrogel were examined in advance to CDS preparation.

In general, swelling behaviour of hydrogels is known to be a criticalproperty which regulates the release of therapeutic agents (Hamidiet al., 2008). In order to achieve equilibrium swelling ratio, the weightof swollen hydrogel was examined at specific intervals. As soon as near-ly constantweightwas obtained for swollen hydrogel, it was consideredas ameasure of equilibrium swelling ratio in PBS. Consequently, equilib-rium swelling ratiowas found to be 177% after 3 days and it did not altersignificantly after that and onlyminor swellingwas observed in thema-trix of hydrogel. The data gained from in vitro release studies ofTeriparatide from HA-JEF ED-600 hydrogel further testified this result.As HA-JEF ED-600 hydrogel swelled in PBS medium, the pore sizes ofhydrogel became slightly larger and subsequently the peptide releasewas facilitated. In the first 3 days of Teriparatide release from hydrogel,53.4% of peptide was released, which was coincident with the signifi-cant swelling of hydrogel, while in the 7 days left, 31.4% of the remain-ing Teriparatide was released, which was notably less than the amountreleased within the first 3 days. As the swelling of hydrogel started todecrease after 3 days, Teriparatide release from hydrogel was alsodiminished.

The swelling ratio of CDS was also determined in order to comparethe swelling ratio of CDS with hydrogel as well as investigate the effectof NPs incorporation within the matrix of hydrogel on swelling behav-iour. The swelling ratio of the fabricated CDSwas remarkably decreasedcompared to hydrogel and it was found to be 135% after 5 days. Interest-ingly, we found that introduction of PHBV/PLGA nanoparticles into thematrix of hydrogel affected the swelling ratio, probably due to their in-terferencewith the formation of crosslinking.Moreover, the structure of

CDS is more compact and dense compared to that of hydrogel alone,which could restrict movement and relaxation, resulted in less swelling(Hezaveh and Muhamad, 2012). The results are also in agreement witha previous study inwhich it was illustrated that further incorporation ofAu nanospheres within thematrix of carrageenan hydrogels resulted inadditional strengthening of polymer network which restricted theswelling of the nanocomposite (Salgueiro et al., 2013).

The extent of crosslinking efficiency of HA-JEF ED-600 hydrogel wasspecified by subtracting single-end anchorage and unreacted aminogroups of JEF ED-600 from the total amount of JEF ED-600 initiallyadded as a bi-functional crosslinking agent to the medium of reaction.For this purpose, ninhydrin assay extensively used in quantitative deter-mination of α-amino acids was utilized. Briefly, ninhydrin reacted withfreeα-amino groups and produced a purple blue dyewhichwas furtherdetected by UV spectrophotometer at 570 nm (Jeon et al., 2007).Pursuant to ninhydrin assay, the percentage of unreacted JEF ED-600,single-end HA-JEF ED-600 and the crosslinking efficiencywere estimat-ed to be 4.7%, 5.4% and 89.9%, respectively. By summing single-end HA-JEF ED-600 and the crosslinking efficiency values, the extent of conjuga-tion percentage was found out to be 95.3%, which was further con-firmed by H NMR area under curve. H NMR area under curve revealedthat HA-JEF ED-600 conjugation percentage, including both single-endHA-JEF ED-600 and crosslinked HA-JEF ED-600, was 93.9%.The differ-ence between these two values (1.4%) corresponded to the inevitableerrors of the two methods.

Both the stress-strain curve and the SAOS analysis were exerted toevaluate the mechanical and viscoelastic properties of HA-JEF ED-600hydrogel in comparison with HA gel. Stress-strain curve is identifiedas a consequential graphical determination of material's mechanical be-haviour (Kaur et al., 2012). The power law index, “n”, gained fromstress-strain curve is beneficial to interpret a material flow under asmall range of shear rate. If “n” value is smaller than one (n b 1), mate-rial behaves as a shear thinning or pseudo plastic matrix in which theviscosity decreases as a function of increasing in shear rate, but If “n”value is greater than one (n N 1), material behaves as a shear thickeningor dilatancy matrix in which the viscosity increases as a function of in-creasing in shear rate (Vorvolakos et al., 2011). In this work, the “n”values for both HA gel and HA-JEF ED-600 hydrogel are smaller thanone (n b 1), thus it can be inferred that both HA gel and HA-JEF ED-600 hydrogel show pseudo plastic behaviour even after crosslinking re-action, although the pseudo plastic property of hydrogel was

Fig. 8. Plot of log-doses vs. probits from Table 1 for calculation of LD50 of Teriparatide loaded CDS administrated subcutaneously. LD50 of Teriparatide loaded CDSwas determined based onMiller and Tainter method.

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significantly decreased in comparisonwith HA gel. The “n” value gainedfor hydrogel was significantly higher than the “n” value of HA gel. Thehigher the “n” value, the less pseudo plastic behaviour will be obtainedwhich signifies that the hydrogel exhibits more resistance to flow andshape alterations in comparisonwithHAgel and also decrease in viscos-ity due to the employed stress occurs with more delay. SAOS analysisdemonstrates that HA gel treated as a viscous or fluid-like materialbased on the greater values achieved for G″ in comparison with G′ in al-most all range of experiments (G″ N G′) but as the HA gel converted toHA-JEF ED-600 hydrogel, the viscoelasticity altered towards a moreelastic or solid-like material and exceedingly greater values were ob-tained for G′ compared to G″ (G′ N G″) (Mayol et al., 2008; Kim et al.,2007). Afterwards, phase shift (δ) corresponded to tanδ = G″/G′ wasplotted against angular frequency. Phase shift is an indicator to displaythe behaviour of material between purely viscous (δ=90°) and purelyelastic (δ= 0°) condition (Kreger and Voytik-Harbin, 2009). Since tanδin the constructed hydrogel is manifestly lower than the gel, the formerresults about shifting the viscoelasticity from viscous or fluid-like mate-rial in HA gel to elastic or solid-like material in hydrogel were re-substantiated. In general, hydrogels with greater values of G′, common-ly exhibitmore resistance against deformation, possess longer retention

Fig. 9. Serum calcium level vs. time inmicewithin 50 days after subcutaneous injection of blank30, 40 and 50 days after injection, three mice in each group were anesthetized and serum camethod. The animals injected by Teriparatide loaded CDS had significantly higher (*P b 0.05) s

time after application and they are firmer and harder than hydrogelswith lower G′. The results are in accordance with a previous study inwhich Tommaso Iannitti et al. claimed that Varriofil as a crosslinkedhyaluronan hydrogel possessed elastic modulus higher than viscousmodulus resulting in more resistance to shape alterations, bettervolumizing effect in the soft tissues and long-lasting retention(Iannitti et al., 2013). In a recent study, tyramine modified hyaluronicacid (HA–Tyr) hydrogels with different degrees of substitution wereprepared and thereafter rheological measurements revealed that elasticmoduluswas significantly higher than viscousmodulus in all specimens(Loebel et al., 2015). Additionally, in amide derivatives of HAwith linearalkyl-amines previously synthesized byMauro Pavan et al. themechan-ical properties of conjugates were shifted towards more elastic mate-rials after chain polymerization (Pavan et al., 2013).

In order to prepare CDS, NPs incorporation within the matrix of hy-drogel fell out concurrently to the crosslinking procedure. CDS structureimaged by SEM displayed a porous structure in which NPs either ad-hered to or incorporated within the matrix of hydrogel, whereas therewas no sign of NPs in the structure of HA-JEF ED-600 hydrogel. Thus,the discrepancy between the structure of CDS and hydrogel was con-firmed by SEM topographies.

CDS, free Teriparatide (0.5 mg/kg) and Teriparatide loaded CDS (0.5mg/kg). At 0, 1, 7, 15,lcium levels were determined by colorimetric assay with o-cresolphthalein complexoneerum calcium level than the other groups after 50 days.

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According to the results achieved in this study, an increase in theNPs:hydrogel ratio did not show significant enhancement in %LC ofCDS. Additionally, increment in the ratio of NPs:hydrogel led to anunfavourable high decrease in the percentage of NPs incorporation.These results are in favour of this hypothesize that hydrogel structureis likely to have limited capacity for accommodating NPs within its ma-trix. Thus, the amount of NPs whichwould be able to incorporate in thehydrogel is restricted by limited capacity of hydrogel and afterwards bythe employed method for fabricating CDS. Since embedding the NPswithin the matrix of hydrogel and crosslinking reaction occur simulta-neously, it might be a deterrent factor for more incorporation of NPs.As a result, some NPs remained un-entrapped during the preparationof CDS and even increasing in the ratio of NPs:hydrogel did not illustratefavourable results. Finally, it was derived that increasing inNPs:hydrogel ratio is a vain attempt which does not produce any desir-able results in improving the percentage of NPs incorporation and %LCof CDS. Hence, ratio 1:1 of NPs:hydrogel was chosen to be the bestratio for CDS preparation.

In the current study, the release profile of Teriparatide fromCDSwascompared with both HA-JEF ED-600 hydrogel and PHBV/PLGA NPs. Therelease profile of Teriparatide fromHA-JEF ED-600 hydrogel exhibited aswift initial release, which might be due to the amount of Teriparatideadsorbed on the surface of hydrogel, thus led to faster release. Addition-ally, the concentration gradient of peptide created inside and outside ofthe hydrogel might be responsible for the latter phenomenon causedTeriparatide located in the superficial part of the hydrogel to be spreadout of the hydrogel swiftly. Totally 84.8% of Teriparatide was releasedfrom hydrogel within 10 days in an uncontrolled manner implyingthat the hydrogel alone was not able to thoroughly retard the peptiderelease over a long period of time. The results are well in consistencywith those found for Erythropoietin loaded HA hydrogels, in which25% burst release was distinguished in the release profile continued by90% Erythropoietin release within 10 days (Hahn et al., 2006). In anoth-er study conducted by Fan Lee et al., 25.8% of α-amylase from HA-tyramine hydrogel was released within only 2 h (Lee et al., 2009). Inspite of favourable properties of hydrogels for delivery of peptides andproteins such as low tendency for protein aggregations and denatur-ation (Hamidi et al., 2008), desirable controlled release profile is notfully achieved with these carriers. The same obstacle also exists in therelease profile obtained from nanoparticulate systems. In PHBV/PLGAblend NPs, the first and second burst releases were ascribed to the ad-hered drug on the surface shell of NPs and the rapid degradation ofPLGA in comparison with PHBV, respectively (Bahari Javan et al.,2016). Although fabricating core-shell like NPs reduced the suddenburst release due to the presence of shell around the NPs, relativelylarge amount of therapeutic agent which still remained on the surfaceof NPs led to uncontrolled burst release (Zhu et al., 2009). In contrastto HA-JEF ED-600 hydrogel and PHBV/PLGANPs, the initial burst releasesubstantially decreased from 25% in hydrogel and 22% in NPs to 12% inthe CDS, indicating the pivotal role of CDS in diminishing the undesir-able burst release. Besides this, the second burst release observed inPHBV/PLGA NPs apparently disappeared in CDS. Pursuant to the obtain-ed results in this work, it seems that dispersing NPswithin thematrix ofhydrogel represents promising results in both lessening unfavourableburst release generally seen in controlled delivery systems andprolonging peptide release. Evidence based results confirmed thisclaim. 64.4% of Teriparatide was released from NPs within 30 dayswhereas 63% (relatively the same amount) of peptide was releasedfromCDSwithin 50 days. Thus, themore retarding effect of CDS in com-parison with NPs was testified. It is hypothesized that after swelling ofhydrogel, PHBV/PLGA NPs started to be released from the matrix ofCDS. As the swelling of hydrogel increased over time, the more NPsgot released from the CDS. Afterwards, the surrounding aqueous medi-um penetrated into the matrix of NPs and Teriparatide was releasedbased on diffusion mechanism. The slower Teriparatide release fromday 30 to 50 might be due to the formation of intermolecular linkages

in PHBV/PLGA NPs which could postpone Teriparatide release. More-over, the acquired findings in the present study are in agreement withthe previous studies, in which HA hydrogels containing vascular endo-thelial growth factor (VEGF) loaded PLGA microspheres were recog-nized as a potential carrier for controlled delivery of biofactors (Wanget al., 2011).

Two mechanisms might be involved in partial and incomplete re-lease of Teriparatide from NPs.

Firstly, Teriparatide molecules might unfavourably be entrappedwithin the long polymeric chains of PHBV and never become exposedto be released. Since PHBV has a higher molecular weight and longerpolymeric chains than PLGA, Teriparatide entrapment within the poly-meric chains of PHBV is more probable. This phenomenon could alsobe attributed to the interactions between Teriparatide and copolymersduring NPs preparation. Undesirable interactions between someamounts of Teriparatide and copolymers might decrease the amountsof drug accessible for release. These interactions (mainly van derWaals and partially chemical interactions)might be additional inhibito-ry factors in releasing some amounts of Teriparatide. Chemical and vander Waals interactions have been previously introduced as the reasonsof partial release of various therapeutic agents (Ukmar et al., 2011b;Ukmar et al., 2011a; Peng et al., 2013).

Secondly, stronger hydrophobicity and slower degradability of PHBVin comparison to PLGA might have been a delaying factor for facile dif-fusion of aqueous medium to the matrix of copolymer causing rigidityin the skeleton of NPs. As a result, bulk degradation and subsequentpores formation occurred slowly, which might have been the reasonsfor the intensification of Teriparatide entrapment within the intermo-lecular linkages of copolymer during diffusion. Insufficient pores in thestructure of NPs might be an inhibitory factor in drug release.

Our findings disclosed that the swelling ratio of CDS (TeriparatideNPs loaded in hydrogel) was decreased by incorporating NPs withinthe matrix of hydrogel compared to blank hydrogel. As a result, CDSretained some amounts of NPs. It is also hypothesized that NH2 groupsin HA- JEF ED 600 hydrogel might be adsorbed by the surface of thePHBV/PLGA NPs and cause interaction between NPs and hydrogel,thereby resulting in retained effect. Hence, the fabricated CDS couldnot release all the amounts (100%) of the NPs initially incorporatedwithin the matrix of hydrogel probably due to two reasons, firstly lessswelling ratio of CDS compared to blank hydrogel and secondly interac-tions between NPs and hydrogel. The retaining effect of CDS on NPs re-lease might lead to reach the relatively same percentage of release inlonger time (63% release from CDS in 50 days vs. 64.4% release fromNPs within 30 days).

After NPs release from the matrix of hydrogel, the limiting factorspreviously discussed such as Teriparatide entrapmentwithin both poly-meric chains and intermolecular linkages and drug interactionswith co-polymers, resulted in Teriparatide partial release from NPs. Theretaining effect of CDS on NPs could not be disguised in manifestationof incomplete release, since it has been extensively studied in previousresearches. Peng and colleagues demonstrated that only 19.11% of insu-lin was released from PHBHHx (poly (3-hydroxybutyrate-co-3-hydroxyhexanoate)) nanoparticles loaded chitosan hydrogel within31 days. Peng et al. proposed that drug release was dually impeded byboth NPs and hydrogel and eventually led to incomplete release. Theyintroduced strong hydrophobicity and slow degradability of PHBV asthe rate limiting factors in insulin release (Peng et al., 2013). Stanwickand colleagues fabricated a composite drug delivery system consistingof PLGA NPs dispersed in an injectable methyl cellulose hyaluronan hy-drogel for controlled delivery of Neurotrophin-3 (NT-3). They assumedthat the interaction between PLGANPs and hyaluronan hydrogel result-ed in plateau phase in release profile of NT-3 aswell as in an incompleterelease. They also indicated that methyl cellulose in hyaluronan hydro-gel adsorbed to the surface of PLGA NPs which led to about 65% releasewithin 30 days (Stanwick et al., 2012). In another study conducted by Liet al., honokiol release from composite drug delivery system consisting

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of pluronic F127 nanoparticles loaded in chitosan hydrogel resulted in35% release in 15 days. They assumed that honokiol release from com-posite was concentration dependent and higher drug loading resultedin less release (Li et al., 2009). E. Tous et al. showed that there was nodifference in the rate of drug release between the gel only and the com-posite drug delivery system (PLGA microspheres loaded in hyaluronicacid based hydrogel) (Tous et al., 2012).

The data acquired from in vitro release studies were fitted to differ-ent mathematical models to anticipate the mechanism of Teriparatiderelease fromCDS. In this study, the bestfittingmodel for Teriparatide re-lease was Korsmeyer Peppas model since the highest R-square value(r2 = 0.99) was achieved with this model stating the favourable linear-ity of themodel. In General, the diffusional exponent, “n”, in KorsmeyerPappas model is utilized to clarify the conquering mechanism involvingin drug release from a carrier. Universally, the drug transport is catego-rized as Fickian diffusion (n = 0.5), Quasi-Fickian diffusion (n b 0.5),Case II transport (n = 1), Non-Fickian or anomalous transport(0.5 b n b 1) and Super Case II transport (n N 1), according to the “n”power. In this experiment, it was figured out that the “n” value was0.29 (n b 0.5). As a result, Quasi-Fickian diffusion could be the funda-mental mechanism of Teriparatide release from CDS. The results are inaccordance with former reports in which “n” value lower than 0.5 wasconsidered as an indicator for Quasi-Fickian diffusion mechanism(Halder et al., 2012; Lao et al., 2011; El-Leithy et al., 2010).

Intrinsic fluorescence and circular dichroism spectroscopy wereemployed to affirm that Teriparatide structure remained intactduring formulation and elevated temperature, organic solventsand that the addition of various excipients had no destructive ef-fects on peptide (Johnson, 1990; Capelle et al., 2007; Shoyeleet al., 2011). The fluorescence spectroscopy provides evidence toprove that the location of the fluorophore groups consisting oftryptophan and phenylalanine in the structure of Teriparatide re-mains preserved during formulation. In this technique, any degreeof either red-shift (left shift) or blue-shift (right shift) in the spec-tra of peptides and proteins could be a proof of undesirable struc-tural alterations in these compounds (Vivian and Callis, 2001). Inthe current study, no changes in florescence intensity werediscerned and neither right shift nor left shift was distinguishedin the two spectra, hence the location of fluorophore amino acidsin the structure of Teriparatide were maintained during formula-tion. These results were further confirmed by CD spectroscopy.CD spectroscopy is known as a valuable technique which providesuseful information about the secondary structure of different pep-tides (Zull et al., 1990). The Far-UV CD spectra of both neatTeriparatide and Teriparatide loaded CDS were comparable toeach other stating that secondary structure of Teriparatide wasnot altered during the formulation process.

According to MTT assay results, the fabricated CDS not only in drugfree form but also in drug loaded form, displayed no cytotoxic effectson NIH3T3 cell line in concentrations of 0.05, 0.5 and 5 nm after 24 h.The obtained results are well in consistency with a previous study inwhich PTH (1–34) loaded PLGAmicrospheres exhibited no cytotoxic ef-fects on MC3T3-E1 cell line after 24 h (Eswaramoorthy et al., 2012). Inanother study, the cytotoxicity of PTH (1–34) loaded chitosan NPswere evaluated using Saos-2, UMR 106, L 929, and NIH3T3 cell linesand it was concluded that relative cell viability was unaffected by thefabricated nanoformulation after 24 h (Narayanan et al., 2012). In thecurrent study, the significant toxicity observed for both CDS (drugfree) and Teriparatide loaded CDS in concentration of 50 nM in compar-ison with control group is not controversial and it does not cause anyconcern for application in clinics since this concentration is approxi-mately 107 times higher than physiologic concentration of Teriparatide.After 48 h, decline in the number of viable cells was only detected inTeriparatide loaded CDS at concentration of 50 nM compared to thecontrol group. The cell viability of both CDS (drug free) and Teriparatideloaded CDS in all concentrations, except for 50 nM CDS, was

significantly increased compared to the control group. SinceTeriparatide alone did not disclose significance increase in the numberof viable cells after 48 h, it seems that the rise in cell viability seen inother groups is related to the constructed CDS. It can be hypothesizedthat the fabricated CDS forms a scaffold like structure which resembles3D cell cultures platforms and mimics the native extracellular matrix(Tibbitt and Anseth, 2009).

The LD50 test was initially developed in 1927 and it is now part of allgovernmental guidelineswhich adjust andmanage toxicological assess-ments of various compounds (Zbinden and Flury-Roversi, 1981).Knowledge of LD50 is also known as a valuable tool to measure theacute toxicity of substances (Arome and Chinedu, 2013). In the presentstudy, LD50 value of Teriparatide loaded CDS was achieved based onMiller and Tainter method (Miller and Tainter, 1944; Randhawa,2009). In order to measure the LD50 of Teriparatide after loading inCDS and evaluate whether the constructed CDS could affect the LD50

of Teriparatide, LD50 determinationwas conducted. LD50 value achievedwas 131.8 mg/kg for Teriparatide loaded CDS and it was confirmed thatloading Teriparatide in the newly fabricated CDS did not affect the LD50

of neat Teriparatide.Besides achieving favourable release profile, the principal challenge

of peptide delivery is to preserve the bioactivity of the peptide after ad-ministration (Brown, 2005). Fabricating a controlled release formula-tion without evaluating its bioactivity and effectiveness in in vivo is avain attempt. Teriparatide is the primary regulator of calcium homeo-stasis in bone and kidney. Itmainly acts through renal tubular reabsorp-tion of calcium and intestinal calcium absorption (Brown and Juppner,2006). Hence, serum calcium level measurement could give useful in-formation about the effective delivery of Teriparatide. Results gainedin the present study reveal that serum calcium level was immediatelyincreased after subcutaneous injection of Teriparatide alone in compar-isonwith the control group. But it started to fall down after day 1.This isprobably due to the short half-life and rapid metabolic degradation ofTeriparatide (Wei et al., 2004). These two reasonsmight makemultipleinjections of Teriparatide unavoidable tomaintain its effectiveness. Sim-ilar to Teriparatide alone, we inferred that serum calcium level was in-creased after administration of Teriparatide loaded CDS indicating thatthe biological activity of Teriparatide had been maintained. Further-more, serum calcium level was significantly elevated in all time-pointsduring delivery of the peptide stating that the constructed Teriparatideloaded CDS could act effectively. Eventually, we deduced thatTeriparatide alone should be administrated frequently to maintain theappropriate serum calcium level since calcium concentration returnedto baseline level promptly after injection of Teriparatide. But serum cal-cium level followed an ascending trend after administration ofTeriparatide loaded CDS and it reached an approximately constantlevel after 50 days. Therefore, there was no need for daily injection.Most importantly, designing such an effective prolonged release formu-lation of Teriparatide will enhance patient's compliance by reducing theproblems and discomfort of frequent injections, which is the valuableachievement of this study.

The remnants of CDS after 50 days was ascribed to a combination ofthe impact of local tissue response on degradation and the suspectedhydrophobic interactions as previously discussed by E. Tous et al. Simi-lar to our work, they represented that nanocomposite containing PLGAmicrospheres embedded in hyaluronic acid hydrogel had remnants ofhydrogel composite present between infiltrated tissues after 4 weeks(Tous et al., 2012). Additionally, the site of injection was monitoredfor symptoms of undesirable adverse effects after subcutaneous admin-istration in mice and no sign of redness or swelling at the injection sitewas identified during 50 days.

5. Conclusion

A novel CDS consisting of PHBV/PLGA NPs loaded in HA-JEF ED-600 hydrogel with the aim of preparing injectable formulation for

180 N. Bahari Javan et al. / European Journal of Pharmaceutical Sciences 101 (2017) 167–181

prolonged delivery of Teriparatide was successfully designed andcharacterized. Swelling behaviour, crosslinking efficiency and rhe-ological properties were all confirmative indicators for convertingHA gel to HA-JEF ED-600 hydrogel. Morphological assessments ofCDS revealed that the incorporation of NPs within the matrix ofhydrogel took place. The more prolonging effect of CDS onTeriparatide release in comparison with NPs and hydrogel wasproven by the obtained results. Intrinsic florescence and circulardichroism spectroscopy were all in favour of keeping the structuralstability of Teriparatide after formulation. The toxicity of CDS wasruled out based on MTT assay results. The elevation trend ofserum calcium level acquired after administration of Teriparatideloaded CDS in mice demonstrated that this drug delivery systemcould be effective in in vivo and the bioactivity of Teriparatidewas preserved. In addition, the application of biocompatible, bio-degradable, non-toxic and non-irritant polymers will hopefullymake this novel delivery system safe and one step closer to clinicaluse.

Declaration of interest

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

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