Bisphosphonate–polyaspartamide conjugates as bone targeted drug delivery systems

Journal ofMaterials Chemistry B

PAPER

Bisphosphonate–

aDepartment of Health Sciences, #IRC FSH

Safety &, Health, University of Catanzaro

“S. Venuta”, Viale S. Venuta, Germaneto, I-8bDepartment of Biological, Chemical and Ph

(STEBICEF), Sezione di Chimica e Tecnolog

di Palermo, Via Archira 32, 90123 Pale

unipa.itcDepartment of Pharmacy, University of Ch

Vestini 31, Chieti 66013, ItalydDepartment of Nanomedicine, The Hous

Bertner Avenue, Houston, TX, 77030, USA

† Electronic supplementary informa10.1039/c4tb00955j

Cite this: J. Mater. Chem. B, 2015, 3,250

Received 13th June 2014Accepted 23rd October 2014

DOI: 10.1039/c4tb00955j

www.rsc.org/MaterialsB

250 | J. Mater. Chem. B, 2015, 3, 250–

polyaspartamide conjugates asbone targeted drug delivery systems†

D. Paolino,a M. Licciardi,b C. Celia,cd G. Giammona,b M. Frestaa and G. Cavallaro*b

Poly-hydroxy-aspartamide was used as a backbone to synthesize bisphosphonate derivatives thus

achieving macromolecular carriers to be potentially used as targeting agents for bone drug delivery.

Molecules bearing bisphosphonate groups, such as aminobisphosphonate (ABP) and neridronate (NRD),

have been conjugated to polyaspartamide (a,b-poly(N-2-hydroxyethyl)-DL-aspartamide, PHEA), with or

without a spacer (succinic acid or 6-aminocaproic acid) thus obtaining PHEA-succinate-ABP and PHEA-

caproylcarbamate-ABP and PHEA-ABP and PHEA-NRD, respectively. Bisphosphonate–polymer

conjugates were physico-chemically characterized using size exclusion chromatography and 1H-NMR;

and their in vitro and ex vivo affinity for bone tissue has been further tested using the hydroxylapatite and

rabbit bone binding assays, respectively. In vivo studies were carried out using rats to evaluate the

biodistribution features of bisphosphonate–polymer conjugates in comparison with the starting PHEA. In

vivo findings evidenced a suitable selectivity of bisphosphonate–polymer conjugates toward the bone

tissues also as a function of time.

Introduction

Bisphosphonates are the rst line treatment for differentdiseases such as bone pathologies, osteoporosis, Paget'sdisease, and metastatic bone cancers. Bisphosphonates, due totheir chemical structure, accumulate in bones and specicallyinhibit osteoclast growth, with the mechanism still not wellknown.

Bisphosphonates are metabolically stable and analogous topyrophosphate (P–O–P), an endogenous mediator of bonemineralization, capable to be adsorbed onto hydroxyapatitecrystals, thus providing bone stabilization and inhibiting itsdissolution. The change of the central oxygen group in thebisphosphonate structure with a carbon unit (P–C–P) improvesthe bisphosphonate resistance against acid hydrolysiscompared to other pyrophosphates.1 Furthermore, its chemicalstability allows accumulation of bisphosphonates in the bonecells, in particular osteoclasts. Bisphosphonate adhesion to

-Interregional Research Center for Food

“Magna Graecia”, Campus Universitario

8100 Catanzaro, Italy

armaceutical Sciences and Technologies,

ie Farmaceutiche, Universita degli Studi

rmo, Italy. E-mail: gennara.cavallaro@

ieti – Pescara “G. d'Annunzio”, Via dei

ton Methodist Research Institute, 6670

tion (ESI) available. See DOI:

259

hydroxyapatite may be able to: (i) inhibit osteoclast recruitmentand adhesion to the bone matrix; (ii) reduce osteoclast half-lifeand (iii) inhibit its activity directly.1

To date, bisphosphonates have been the front line treatmentto prevent osteolysis and pain occurring in bone metastases.Despite their therapeutic effect, bisphosphonates are currentlyused in clinics as suitable targeting agents in radiotherapy.Bisphosphonate-radio-labeled-drug conjugates are used asspecic markers in imaging diagnosis to detect bone patholo-gies.2 Bisphosphonate-radio-labeled-drug complexes, such as99mTc-hydroxyethylidene bisphosphonate, 99mTc-methylenebisphosphonate and 99mTc-hydroxymethylene bisphospho-nate,2 are currently used in bone scanning to detect and eval-uate metastases, tumors, metabolic diseases, Paget's disease,infections, traumatisms, arthritis, etc. Samarium (153Sm) lexi-dronam, another bisphosphonate-radio-labeled-drug complex,has been approved by U.S. Food and Drug Administration (FDA)for treating bone metastasis pain.3 All these drugs showed thathigh affinity and selectivity for bone tissue depend on thebisphosphonate unit conjugated to co-polymers. In 2009,United States patent4 demonstrated the potentiality ofbisphosphonates as suitable targeting agents to synthesizeuorescent and non-radioactive molecular probes for molecularimaging and detection.

Concerning the conjugation of bisphosphonate to polymersit was reported that a single molecule of alendronate wassuccessfully conjugated either to polyethylene glycol (PEG) orhydroxypropylmethacrylamide (HPMA)5 to obtain two alendro-nate–polymer conjugates showing high affinity for thehydroxyapatite matrix compared to native polymers and good in

This journal is © The Royal Society of Chemistry 2015

http://crossmark.crossref.org/dialog/?doi=10.1039/c4tb00955j&domain=pdf&date_stamp=2014-12-06

Paper Journal of Materials Chemistry B

vivo bone affinity.5 The synthesis and the characterization offunctional poly(benzyl-L-glutamate) (PBLG) derivatives bearinglinked alendronate molecules to develop nanoparticle formu-lations for bone targeting were also reported.6 Moreover, Milleret al. reported that the N-(2-hydroxypropyl)methacrylamidecopolymer–paclitaxel–alendronate conjugate is an effectivebone-delivery system.7,8 However, to the best of our knowledge,alendronate molecules mainly in single or in multiple mole-cules have been linked to linear or branched polymers to obtainwater-soluble or nanoparticle devices for bone targeting.5–9

For many years our research group explored the possibility ofusing a,b-poly(N-2-hydroxyethyl)-DL-aspartamide (PHEA)10–12 toobtainmacromolecular targeting systems that are able to addressdrug molecules to specic tissues.13–15 The advantages of PHEAcompared to other copolymers are its safety, biocompatibility,ease of production, high yields, reproducibility and low costs.

Polymeric micelles that are potentially able to carry a riba-virin (RBV) prodrug to hepatocytes, exploiting the presence ofthe asialoglycoprotein receptor, were prepared starting from agalactosylated polylactide–polyaminoacid conjugate based onPHEA.13 The obtained copolymer bearing ethylenediaminegroups (EDA), polylactic chains (PLA) and galactose residues(GAL) named PHEA-EDA-PLA-GAL gave nanometric liver-tar-geted RBV tripalmitate-loaded micelles in aqueous media at lowcopolymer concentration. By in vitro experiments, the specicityof RBV tripalmitate-loaded PHEA-EDA-PLA-GALmicelles towardHepG2 was demonstrated by using a competitive inhibitionassay in the presence of free GAL.13

For antitumor therapy, liver-targeted sorafenib-loadedmicelles obtained by the PHEA-EDA-PLA-GAL copolymershowed, aer oral administration, by in vivo biodistributionstudies the preferential sorafenib accumulation in the liver;14

conversely, a different study demonstrated that a folate- andmagnetic-targeted nanocarrier based on 10 nm iron oxidenanodomains coated with a folic acid (FA)-functionalizedamphiphilic copolymer PHEA allowed to synthesize dual-tar-geted drug delivery systems for antitumor therapy.15

Considering the high versatility of PHEA to obtain targetedcarriers to different specic tissues, due to the high chemicalmultifunctionality of this copolymer and the unexploredpossibility to evaluate the targeting ability of differentbisphosphonate molecules linked to PHEA, the aim of this workwas to prepare four different – bisphosphonate PHEA conju-gates in the presence, or not, of spacers, between the polymerand bisphosphonates, such as aminobisphosphonate (ABP) andneridronate (NRD), and to test the ability of these conjugates tobe targeted to the bone by some in vitro and in vivo studies.

The main application of these systems consists in their useas targeted macromolecular carriers for macromolecular pro-drugs of antitumor drugs to be directed to the bone.

Results and discussionSynthesis of PHEA–bisphosphonate conjugates

PHEA–bisphosphonate conjugates obtained by direct reactionof ABP and NRD were synthesized starting from PHEA (Fig. 1and 2) by using a single step reaction. The hydroxyl nucleophilic


groups of PHEA (a single group for each repeating unit), whichare soluble both in water and organic solvents, can react,through their side chains, with different molecules at consec-utive and multiple reaction steps. The hydroxyl groups presenton the PHEA side chain were initially activated by reacting withBNPC, in anhydrous DMF solution at 40 �C. The molar ratiobetween PHEA repeating units (RU) and the moles of activatingagent (RU/BNPC), and the reaction time determined the acti-vation degree of the hydroxyl groups of the polymer. Namely, anactivation degree of the hydroxyl groups of PHEA equal to 15%was obtained by using a RU/BNPC molar ratio of 0.5 and anactivation time of 1 h. Subsequently, activated PHEA wasmodied by conjugating a certain number of bisphosphonategroups on the polymer side chain by reaction with an excess ofABP (Fig. 1A) or NRD (Fig. 2A) and obtaining the PHEA-ABP(Fig. 1) or PHEA-NRD (Fig. 2) polymer conjugate, respectively.

The conjugation degree of ABP to PHEA was determined bycolorimetric determination of phosphate groups (Amesassay)16 and was found to be 4.4 mol% with respect to thehydroxyethyl-aspartamide repeating units of PHEA. Differ-ently, the 1H-NMR allowed easy quantication of PHEA func-tionalized with NRD by comparing the integral of the peakattributable to the methylene groups of NRD at d 1.3–1.8 to thatbelonging to NH–CH–CH2–CO of the PHEA backbone at d 2.82(Fig. 2C). The molar percent of the NRD groups covalentlybound to PHEA was 5 mol% referred to the PHEA repeatingunits as well.

Our ndings demonstrate that the number of bisphospho-nate moieties covalently bound to PHEA-ABP and PHEA-NRDranged from 9 (ABP) to 14 (NRD) for the different polymericchains, respectively. Following the functional reaction, thePHEA–bisphosphonate conjugates still maintain a largenumber of hydroxyl groups (about 95 mol%), available forfurther activation and conjugation with other macromolecules,co-polymers or bioactive compounds.

Synthesis of PHEA–bisphosphonate conjugates via spacers

In the design of macromolecular conjugates the role of thespacer has been oen considered in coupling between drugmolecules and polymeric carriers and in many cases severaldifferences in drug performance have been recorded and someadvantages considered depending on the presence of spacergroups.17

In the case of targeting groups, several advantages can occurin terms of improved accessibility to the target by conjugatingthe targeting portion to the carrier polymer through specicspacers. In particular, spacers can properly space out the tar-geting portion from the polymeric backbone and promote thebiological interaction between these compounds and cellmembrane receptors.18

In this paper two different spacers were used to link the ABPtargeting group to PHEA macromolecules, succinic and 6-ami-nocaproic acid groups. The main differences between these twocompounds are the length of the spacer groups and the natureof the linkage between the spacer portion and the polymericchain.

J. Mater. Chem. B, 2015, 3, 250–259 | 251

Fig. 1 Schematic synthesis of the PHEA-ABP (a,b-poly(N-2-hydroxyethyl)-co-[N-2-(aminobisphosphonate-N-carbamate)ethylene]-DL-aspar-tamide) conjugate (A) starting from the PHEA (a,b-poly(N-2-hydroxyethyl)-DL-aspartamide) polymer; size exclusion chromatography (SEC) peakof PHEA-ABP (B) and 1H-NMR spectrum (C).

Journal of Materials Chemistry B Paper

In the case of PHEA-CS-ABP (Fig. 3), the succinic spacer wasinitially linked to the PHEA polymer by the reaction with suc-cinic anhydride via the ester linkage and then the conjugationreaction of ABP to PHEA-CS was carried out in bi-distilled waterin the presence of the carboxyl groups activating agents HOBTand EDC-HCl (Fig. 3A). The pendant carboxyl groups of PHEA-CS could be partially derivatized with ABP moieties by suitablymodulating the molar ratios between the moles of pendantcarboxyl groups of PHEA-CS and the moles of activating agentsand ABP. In particular, an ABP derivatization degree of 15% ofcarboxyl groups of PHEA-CS (5 mol% with respect to PHEA RU)was obtained by using the carboxyl groups of PHEA-CS/HOBTand EDC-HCl molar ratio of 2 and the carboxyl groups of PHEA-CS/ABP molar ratio of 1.7.

Conversely, 6-aminocaproic acid was the spacer used toconjugate ABP with PHEA-caproylcarbamate (Fig. 4). This spacerwas conjugated to a PHEA backbone using BNPC as thecondensing agent. The conjugation reaction of ABP to PHEA-caproylcarbamate was carried out in double-distilled water inthe presence of EDC-HCl as the condensing agent (Fig. 4A). Inthis case, the derivatization degree of ABP, with respect to thecarboxyl groups of PHEA-caproylcarbamate was 30% (5 mol%with respect to PHEA RU), by using the carboxyl groups of PHEA-caproylcarbamate/EDC-HCl molar ratio of 2 and the carboxylgroups of PHEA-caproylcarbamate/ABP molar ratio of 3.

252 | J. Mater. Chem. B, 2015, 3, 250–259

Evaluation of the affinity of PHEA–bisphosphonate derivativeswith hydroxyapatite and bone tissue

The hydroxyapatite-binding assay provided information on theinteraction between polymers–bisphosphonate conjugates andthe mineral components of the bone tissue, i.e. calcium phos-phate or hydroxylapatite.19 As shown in Fig. 5A, all PHEA–bisphosphonate co-polymers have an ability to bind to hydroxylapatite component ve times greater than the native PHEApolymer.

The binding assay of PHEA–bisphosphonate conjugates wasalso carried out using ex vivo lyophilized bone tissues collectedfrom healthy rabbits (Fig. 5B). These experiments conrmed thendings already observed for the hydroxylapatite-binding assay;namely, PHEA–bisphosphonate conjugates demonstrated anability to bind the bone tissue 5–6 times greater than the nativePHEA polymer.

In vivo biodistribution of PHEA–bisphosphonate derivatives

Considering that in vitro and ex vivo assays relating to bindingaffinity to bone components provided no signicant differencebetween the various PHEA–bisphosphonate conjugates,including that containing spacer groups, we decided to inves-tigate the in vivo biodistribution only on PHEA conjugateshaving two different bisphosphonates as targeting portions, but


Fig. 2 Schematic synthesis of the PHEA-NRD (a,b-poly(N-2-hydroxyethyl)-co-[N-2-(6-amino-caproyl-bisphosphonate-N-carbamate)ethylene]-DL-aspartamide) conjugate (A) starting from the PHEA (a,b-poly(N-2-hydroxyethyl)-DL-aspartamide) polymer; size exclusionchromatography (SEC) peak of PHEA-NRD (B) and 1H-NMR spectrum (C).


without a spacer, i.e. PHEA-NRD and PHEA-ABP in comparisonwith the native PHEA polymer and as a function of time. Theexperiments were performed using PHEA derivatives containinga suitable amount of 1,2-3H-ethanolamine (see the Experi-mental section).

As shown in Fig. 6, the in vivo experiments are in agreementwith the in vitro and ex vivo ndings (Fig. 5); in fact, PHEA–bisphosphonate conjugates showed a greater bone tissueaffinity/selectivity than the native PHEA polymer at all theinvestigated biodistribution times. In particular, PHEA-NRDprovided just aer 1 h from the i.v. injection a greater accu-mulation in the bone tissue (sternum and femur) than PHEA-ABP and native PHEA. Conversely, PHEA-ABP showed a signif-icantly greater accumulation in bone tissues (sternum andfemur) from 4 h aer its administration in male Wistar rats, upto 24 h, than the native PHEA polymer (Fig. 6). The slow rate ofaccumulation of the PHEA–ABP conjugate in bone tissues wasevidenced by the fact that it reached the maximum concentra-tion in the bones (summing both the amount accumulated insternum and femur) only aer 24 h from administration.

Interesting results in terms of bone accumulation arereported in the literature for N-(2-hydroxypropyl)-meth-acrylamide (HPMA) copolymer–alendronate conjugates in


comparison with untargeted derivatives.20 In particular, theHPMA copolymer–alendronate conjugates were found in theoverall skeleton, represented by tibia and femur, within 15 minaer administration and their accumulation increased up to48 h of incubation.20 Moreover in this case the effect of the boneaccumulation was found to be dependent on the molecularweight of polymeric derivatives. With our polymeric conjugateswe detected a difference in the bone accumulation betweenNRD and ABP PHEA conjugates.

Our results demonstrate that the NRD moiety improved thebone affinity of the PHEA-NRD when compared to ABP, thusincreasing both short and long period accumulation of PHEAconjugates in the bone tissue (Fig. 6). These differences couldbe explained considering the structural differences betweenPHEA-NRD and PHEA-ABP copolymers with reference to thenumber of bisphosphonate moieties covalently bound to PHEA,and to the possible different conformational arrangementsdepending on different side chain moieties, ABP and NRD.Some experimental evidence has also been recorded for PHEAcopolymers, supporting the hypothesis that slight physico-chemical differences may reect strong biological differences.21

Following intravenous injection PHEA-ABP and PHEA-NRDconjugates were rapidly accumulated in the liver (1 h aer

J. Mater. Chem. B, 2015, 3, 250–259 | 253

Fig. 3 Schematic synthesis of the PHEA-CS-ABP (a,b-poly(N-2-hydroxyethyl)-co-[N-2-(20-carboxysuccinate)ethylene]-co-[N-2-(20-amino-bisphosphonate-succinylamide)ethylene]-DL-aspartamide) conjugate (A) starting from the PHEA (a,b-poly(N-2-hydroxyethyl)-DL-aspartamide)polymer; size exclusion chromatography (SEC) peak of PHEA-CS-ABP (B) and 1H-NMR spectrum (C).


administration) and then their accumulation in perfusedorgans, particularly liver, decreased with time (4–24 h); whilethe accumulation in the bone increased or remained elevatedup to 24 h (Fig. 6). This effect could depend on the polymericcarrier (PHEA) conjugated to bisphosphonates which, similar tocolloidal drug delivery devices, could saturate hepatic macro-phages, thus justifying the rapid liver accumulation.22,23 Actu-ally, the accumulation and retention of macromoleculartherapeutic agents in the liver is a problem connected with thepolymer carrier approach. Nevertheless, although the use of lowmolecular weight bisphosphonate conjugates, for example withtherapeutic radionuclides,24,25 provided higher bone/liver accu-mulation ratios, the use of a multifunctional polymer like PHEAas a carrier, provides other advantages, such as a reducedclearance from plasma and a high molar ratio between thebisphosphonate content and polymer amount, because eachPHEA macromolecule can simultaneously conjugate multiplemolecules of bisphosphonate in the same polymer chain.

The level of PHEA-NRD and PHEA-ABP conjugate accumu-lation in the bone tissue aer 24 h in comparison with thenative PHEA polymer further proved that bisphosphonates playan essential role in the bone uptake since forming stable

254 | J. Mater. Chem. B, 2015, 3, 250–259

complexes with bone and the bone mineral matrix, even iffollowing different kinetics as a function of the bisphosphonatemoiety (Fig. 6). These ndings are in good agreement withrecently reported26 in vivo tissue distribution of zoledronic acid-loaded PEGylated liposomes and suggest to continue thedevelopment of these conjugates as carriers to specicallytransport other kinds of drugs to bone tissues.

ExperimentalMaterials and chemicals

Bis(4-nitrophenyl)carbonate (BNPC), succinic anhydride (SA), 6-aminocaproic acid, and hydroxylapatite were purchased fromAldrich (Milan, Italy). Hydroxybenzotriazole (HOBT) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride(EDC-HCl) were purchased from Fluka (Switzerland). Aminobi-sphosphonate (ABP) and neridronate (NRD) were obtained fromAbiogen Pharma S.p.A. Cholesterol hexadecyl ether [cholesteryl-1,2-3H(N)] (3H-CHE) (TLC purity $ 97%) was obtained fromPerkin Elmer-Italia (Monza (MI), Italy). Beckman Tissue Sol-ubilizer (BTS-450) (0.5 N quaternary ammonium hydroxide intoluene) and Ready Organic™, a liquid scintillation cocktail fororganic sample and tissue, were purchased from Beckman


Fig. 4 Schematic synthesis of the PHEA-caproylcarbamate-ABP (a,b-poly(N-2-hydroxyethyl)-co-[N-2-(N0-6-carboxycaproyl-carbamate)ethylene]-co-[N-2-(N0-6-bisphosphonatecarboxyamidecaproyl-carbamate)ethylene]-DL aspartamide) conjugate (A) starting from the PHEA(a,b-poly(N-2-hydroxyethyl)-DL-aspartamide) polymer; size exclusion chromatography (SEC) peak of PHEA-caproylcarbamate-ABP (B) and 1

H-NMR spectrum (C).


(Beckman Instruments, Inc., Fullerton, Netherlands). Sterilesaline solution was a Fresenius Kabi product (Potenza S.r.l.,Verona, Italy). Samples were analyzed using a Wallac WinSpectral™ 1414 liquid scintillation counter (PerkinElmer LifeAnd Analytical Sciences, Inc. Waltham, Massachusetts, USA). Allthe other reagents were of analytical grade.

Preparation and characterization of PHEA

PHEA was prepared and puried as previously reported.10–12

Briey, polysuccinimide was rstly synthesized by poly-condensation reaction of DL-aspartic acid (Fluka) in the pres-ence of phosphoric acid (Aldrich) at 220 �C. Polysuccinimidewas isolated by precipitation in water, collected and dried.PHEA was obtained by reaction of polysuccinimide with anexcess of ethanolamine in DMF solution, isolated by precipita-tion in butanol, collected and washed in acetone. Then, anaqueous solution of PHEA was puried through exhaustivedialysis against water using a Spectrapor dialysis tube with amolecular weight cut-off of 12–14 kDa. The PHEA polymer wascharacterized using 1H-NMR. Results agreed with the datapublished in the literature.10–12 The average molecular weight


(Mw) of PHEA was 49 kDa (Mw/Mn ¼ 1.68), the analysis wascarried out using polyethylene oxide (PEO)/PEG as standards.The molecular weight of PHEA was determined by size exclu-sion chromatography (SEC). The 1H-NMR spectra were recordedin D2O (Aldrich) using a Bruker Avance II 300 spectrometeroperating at 300 MHz. UV Spectroscopy was performed using aShimadzu UV160U spectrophotometer. Centrifugations wereperformed using a Centra MP4R IEC centrifuge. SEC was carriedout using two Ultrahydrogel columns fromWater (500 and 250 A)(Milford, MA, USA) connected to a Water 2410 refractive indexdetector. Phosphate buffer pH 9 solution was used as an eluentat 37 �C with a ux of 0.8 mL min�1, and PEO standards (145.0–1.0 kDa) were used to set up the calibration curve.

Synthesis of a,b-poly(N-2-hydroxyethyl)-co-[N-2-(aminobisphosphonate-N-carbamate)ethylene]-DL-aspartamide (PHEA-ABP)

PHEA (175 mg, 1.10 mmol) dissolved in anhydrous DMF(1.5 mL) was added to BNPC (168 mg, 0.55 mmol) dissolved in 1mL of anhydrous DMF. The mixture was maintained undercontinuous stirring for 1 h at 40 �C. The solution of activated

J. Mater. Chem. B, 2015, 3, 250–259 | 255

Fig. 5 Affinity of the various PHEA–bisphosphonate conjugates (A)with hydroxyapatite (mg of conjugate/50mg of hydroxyapatite) and (B)with ex vivo bone tissue (mg of conjugate/50 mg of bone tissue).Statistical analysis: *, data are significant (p < 0.001) in comparison withthe PHEA treatment; **, data are significant (p < 0.05) in comparisonwith PHEA-ABP, PHEA-CS-ABP and PHEA-caproylcarbamate-ABP.

Fig. 6 Biodistribution of [3H]-radiolabeled PHEA, PHEA-NRD andPHEA-ABP following i.v. injection into Wistar rats as a function of time.Results are expressed as the mean � standard deviation from a groupof six rats.


PHEA was added dropwise to an ABP solution (210 mg; 1.10mmol, in 6 mL of water at pH 8–9) and the mixture was main-tained at 25 �C for 24 h. The obtained product was puriedthrough exhaustive dialysis against water and then freeze-dried.PHEA-ABP showed a yield of 95% compared to the startingPHEA (Fig. 1A). The degree of functionalization of ABP to PHEAwas quantied using phosphates assay (Ames assay) and a valueof 4.4 mol% was obtained. The Mw of the PHEA-ABP conjugate(30 kDa) was determined by SEC in pH 9 aqueous phosphatebuffer (Fig. 1B). A calibration curve using PEG standards(molecular weight ranging from 1 to 145 kDa) was used for theanalysis. The characteristic 1H-NMR signals of PHEA-ABPconjugates in D2O were the following: d 2.79 (m, 2H, –CO–CH–

CH2–CO–NH–), 3.11 (m, 1H, –CH–(PO3H)2), 3.36 (m, 2H, –NH–

CH2–CH2–OH), 3.63 (m, 2H, –NH–CH2–CH2–OH), 4.09–4.22

256 | J. Mater. Chem. B, 2015, 3, 250–259

(m, 4H, –NH–CH2–CH2–O(CO)NH–), 4.71 (m, 1H, –NH–CH(CO)CH2–) (Fig. 1C).

Synthesis of a,b-poly(N-2-hydroxyethyl)-co-[N-2-(6-amino-caproyl-bisphosphonate-N-carbamate)ethylene]-DL-aspartamide (PHEA-NRD)

The PHEA-NRD co-polymer was synthesized starting from PHEA(Mw ¼ 50 kDa). PHEA (250 mg, 1.58 mmol) dissolved in 3 mL of



anhydrous DMF was added to BNPC (239 mg, 0.78 mmol) dis-solved in 1 mL of anhydrous DMF. The obtained mixture wasmaintained under continuous stirring for 1 h at 40 �C. Themixture solution was then added dropwise to NRD solution (150mg, 0.54 mM) dissolved in 7 mL of water (pH 7). The reactionwas carried out for 24 h at room temperature. PHEA-NRD wasthen puried by exhaustive dialysis against water and freeze-dried. PHEA-NRD was obtained with a yield of 98% compared tostarting PHEA (Fig. 2A). The conjugation degree of NRD inPHEA-NRD conjugates was determined by 1H-NMR (Fig. 2C)showing a value of 5 mol%. The PHEA-NRD conjugate has a Mw

of 45 kDa as determined by SEC in pH 9 phosphate buffer. Alsoin this case a calibration curve using PEG standards was used(Fig. 2B). The characteristic 1H-NMR signals of PHEA-NRDconjugates in D2O were the following: d 1.3–1.8 (m, 8H, –CH2–CH2–CH2–CH2–C(OH)–), 2.82 (m, 2H, –CO–CH–CH2–CO–NH–),3.36 (m, 2H, –NH–CH2–CH2–OH), 3.66 (m, 2H, –NH–CH2–CH2–

OH), 4.09–4.22 (m, 4H, –NH–CH2–CH2–O(CO)NH–), 4.54 (m, 1H,–NH–CH(CO)CH2–) (Fig. 2C).

Synthesis of a,b-poly(N-2-hydroxyethyl)-co-[N-2-(20-carboxysuccinate)ethylene]-co-[N-2-(20-aminobisphosphonate-succinylamide)ethylene]-DL-aspartamide (PHEA-CS-ABP)

The a,b-poly(N-2-hydroxyethyl)-co-[N-2-(20-carboxysuccinate)eth-ylene]-DL-aspartamide (PHEA-CS) co-polymer was synthesized byreacting PHEA (Mw ¼ 50 kDa) with succinic anhydride in thepresence of triethylamine and anhydrous DMF solution at 25�C. A RU/anhydride molar ratio of 0.4 and a reaction time of 24h (Fig. 3A) provided a derivatization degree of hydroxyl groupsof PHEA of 35% as determined by 1H-NMR (Fig. 3C). The PHEA-CS co-polymer was precipitated in ethyl ether, puried throughexhaustive dialysis and then lyophilized. PHEA-CS was obtainedwith a yield of 85% with respect to PHEA and it was thencharacterized by using 1H-NMR analysis. The PHEA-CS conju-gate has a Mw of 45 kDa as determined by SEC in 0.05 M CaCl2aqueous solution (calibration curve using PEG standards). Thecharacteristic 1H-NMR signals of PHEA-CS conjugates in D2Owere the following: 1H-NMR (D2O): d 2.55–2.66 (m, 4H, OOC–(CH2)2–COOH), 2.79 (m, 2H, –CO–CH–CH2–CO–NH–), 3.36 (m,2H, –NH–CH2–CH2–OH), 3.63 (m, 2H, –NH–CH2–CH2–OH), 4.16(m, 2H, –NH–CH2–CH2–O(CO)–), 4.70 (m, 1H, –NH–CH(CO)CH2–).

The PHEA-CS-ABP conjugate was then synthesized by react-ing the PHEA-CS copolymer with ABP. PHEA-CS (100 mg) wasdissolved in 4 mL of water (pH 6.8) and added with 40 mg (0.296mmol) of HOBT, ABP (50 mg; 0.263 mmol) dissolved in 1 mL ofwater (pH 6) and 58 mg (0.30 mmol) of EDC-HCl. The mixturewas maintained under stirring for 18 h at 25 �C, and pH wasadjusted to 6.8 using HCl (0.1 N) during the rst 2 h of incu-bation (Fig. 3A). The nal product was then puried byexhaustive dialysis against distilled water. The yield of PHEA-CS-ABP was 85% with respect to the starting PHEA-CS and thenal product was characterized using 1H-NMR analysis(Fig. 3C). The ABP conjugation degree in PHEA-CS-ABP was5 mol% as determined by the phosphate assay.15 The Mw of thePHEA-CS-ABP conjugate was 55 kDa as determined by SEC in


pH 9 aqueous phosphate buffer (calibration curve using PEGstandards) (Fig. 3B). The characteristic 1H-NMR signals ofPHEA-CS-ABP conjugates in D2O were the following: d 2.45–2.60(m, 4H, OOC–(CH2)2–CO), 2.79 (m, 2H, –CO–CH–CH2–CO–NH–),3.11 (m, 1H, –CH–(PO3H)2), 3.36 (m, 2H, –NH–CH2–CH2–OH),3.45 (m, 2H, –NH–CH2–CH2–O(CO)–), 3.63 (m, 2H, –NH–CH2–

CH2–OH), 4.16 (m, 2H, –NH–CH2–CH2–O(CO)–), 4.68 (m, 1H,–NH–CH(CO)CH2–) (Fig. 3C).

Synthesis of a,b-poly(N-2-hydroxyethyl)-co-[N-2-(N0-6-carboxycaproyl-carbamate)ethylene]-co-[N-2-(N0-6-bisphosphonatecarboxyamidecaproyl-carbamate) ethylene]-DL-aspartamide (PHEA-caproylcarbamate-ABP)

The a,b-poly(N-2-hydroxyethyl)-co-[N-2-(N0-6-carboxycaproyl-car-bamate)ethylene]-DL-aspartamide (PHEA-caproylcarbamate) co-polymer was synthesized starting from PHEA (Mw ¼ 50 kDa).The pendant hydroxyl groups of PHEA were activated by react-ing copolymers with BNPC, in anhydrous DMF solution at 40 �Cfor 1 h. The solution of activated PHEA was added dropwise to a0.56 mM solution of 6-aminocaproic acid in double-distilledwater and this mixture was maintained at 25 �C for 24 h(Fig. 4A). The copolymer was then precipitated in acetone,puried through dialysis in double-distilled water and nallylyophilized. The yield of PHEA-caproylcarbamate was 75% withrespect to the starting PHEA. PHEA-caproylcarbamate wascharacterized using 1H-NMR. The conjugation degree of 6-aminocaproic acid in PHEA-caproylcarbamate was 5 mol% asdetermined by 1H-NMR. TheMw of the PHEA-caproylcarbamateconjugate was 50 kDa as determined by SEC in pH 9 phosphatebuffer (calibration curve using PEG standards). The character-istic 1H-NMR signals of the PHEA-caproylcarbamate copolymerin D2O were the following: d 1.34 (m, 2H, –CH2–CH2–CH2–),1.45–1.62 (m, 4H, –CH2–CH2–CH2–), 2.25 (m, 2H, –CH2–COOH),2.82 (m, 2H, –CO–CH–CH2–CO–NH–), 3.35 (m, 2H, –NH–CH2–

CH2–OH), 3.65 (m, 2H, –NH–CH2–CH2–OH), 4.11 (m, 2H, –CH2–

O–CO–NH–CH2–), 4.63 (m, 1H, –NH–CH(CO)CH2–).The PHEA-caproylcarbamate-ABP copolymer was then

synthesized from PHEA-caproylcarbamate. PHEA-caproylcarba-mate (115 mg) was dissolved in 3 mL of double-distilled water(pH 7.2) and mixed with 2 mL of a double-distilled water(pH 7.2) solution of ABP (50 mg; 0.263 mmol) and EDC-HCl(45 mg; 0.234 mmol). This mixture was maintained undercontinuous stirring for 18 h at 25 �C by adjusting pH to 7.2 withHCl 0.1 N during the rst 3 h of the reaction (Fig. 4A). PHEA-caproylcarbamate-ABP was puried using dialysis in double-distilled water and then lyophilized. The yield of PHEA-cap-roylcarbamate-ABP was 90% with respect to the starting PHEA-caproylcarbamate. The PHEA-caproylcarbamate-ABP was char-acterized using 1H-NMR (Fig. 4C). The 30% of carboxyl groupsof PHEA-caproylcarbamate were conjugated with ABP, asdetermined by the Ames assay, thus resulting in an ABPconjugation degree of 5 mol% with respect to PHEA RU. TheMw

of PHEA-caproylcarbamate-ABP was 54 kDa, as determined bySEC in pH 9 phosphate buffer (calibration curve using PEGstandards) (Fig. 4B). The characteristic 1H-NMR signals of thePHEA-caproylcarbamate-ABP copolymer in D2O were the

J. Mater. Chem. B, 2015, 3, 250–259 | 257


following: 1H-NMR (D2O): d 1.31 (m, 2H, –CH2–CH2–CH2–),1.45–1.62 (m, 4H, –CH2–CH2–CH2–), 2.25 (m, 2H, –CH2–COOH),2.82 (m, 2H, –CO–CH–CH2–CO–NH–), 3.09 (m, 1H, –CH–

(PO3H)2), 3.35 (m, 2H, –NH–CH2–CH2–OH), 3.62 (m, 2H, –NH–

CH2–CH2–OH), 4.10 (m, 2H, –CH2–O–CO–NH–CH2–), 4.63 (m,1H, –NH–CH(CO)CH2–) (Fig. 4C).

The in vitro hydroxyapatite binding assay

Different conjugates (PHEA-ABP, PHEA-NRD, PHEA-CS-ABP andPHEA-caproylcarbamate-ABP) (50 mg) showing bisphosphonatelinkage ranging from 3 to 7 weight% or the PHEA polymer (usedas a control) were dissolved in 2 mL of pH 7.4 phosphate buffersolution and then 50 mg of pure hydroxylapatite were added.The obtained suspensions were gently stirred at 37 �C for 4 hand then centrifuged (5 min at 1000 rpm) using a Centra MP4RIEC centrifuge. Supernatants were puried by exhaustive dial-ysis (2 days) in double-distilled water, using a Spectrapor dial-ysis tube with a molecular weight cut-off of 12–14 kDa. Theobtained polymer dispersions were freeze-dried and theobtained residues were weighed. Hydroxylapatite residues weredissolved in 1 mL of phosphoric acid solution (8.5% w/v) andthe obtained dispersions were puried by dialysis as reportedabove. The puried solutions were then freeze-dried and thesolid residues were weighed in order to evaluate the amount ofcopolymer that was bound to hydroxyapatite.

Animals

Male Wistar rats (250–300 g) were purchased from Harlan, Italy(Correzzana, Milan, Italy). Healthy male rabbits (300–350 g)were obtained from the Istituto Zooprolattico della Sicilia,Palermo, Italy. Animal experiments followed the main proce-dures outlined by the local Ethics Committee, Italian law andthe accepted international standards for biomedical research.Care and handling of the animals followed the EuropeanEconomic Community Council Directive 86/209, recognizedand adopted by Italian Government (D.M. no. 95/2003-D).Animals were housed in metabolic cages and maintained atdark/light cycle of 12 h as previously reported.22,27

Binding assay with rabbit bone

Similar to the hydroxylapatite binding assay, the solutions ofthe different conjugates were incubated with 100 mg of rabbitbone (femur) separated from organic tissues, lyophilised andnally crushed thus achieving a ne powder. This suspensionwas gently shaken at 37 �C for 4 h and then processed asdescribed above. Bone residues were rinsed using doubledistilled water and freeze-dried. The amount of polymer (mg)bound to bone was calculated from the difference between theamount of conjugate initially added and that recovered in thesupernatant and rinsing water upon removing the solid powder.

Biodistribution studies in rats

Biodistribution of different conjugates was carried out in maleWistar rats. PHEA-NRD and PHEA-ABP conjugates were intra-venously injected through the tail vein and bone tissue

258 | J. Mater. Chem. B, 2015, 3, 250–259

(sternum and femur) and perfused organs (heart, lung, spleenand kidney) were collected at different incubation times. Thebone and systemic biodistribution of PHEA-NRD and PHEA-ABP was carried out using radiolabeled derivatives synthesizedstarting from a PHEA polymer containing 1,2-3H-ethanolaminehydrochloride. PHEA-NRD and PHEA-ABP conjugates were thendiluted using isotonic sterile solution to obtain a stockconcentration of 8 mg mL�1 (200 mCi per formulations).

Co-polymer suspension (1 mL), showing an activity of 40 mCiper formulations, was i.v. injected into rats (six per group)through the tail vein. Animals were sacriced at different timeintervals by inhaling CO2 and organs, i.e. heart, lungs, liver,spleen, kidneys and bone tissues (sternum and femur) werecollected and stored at�80 �C before analysis (Table S1†). Blood(2 mL) was harvested through retro-orbital plexus before CO2

inhalation. Different samples were weighed and the averageweight was recorded, as reported in Table S1 (see ESI).†

Organs were cut in regular stripes (0.4 g) and each single stripwas transferred into polypropylene vials for radioactive analysisand then digested using 2mL of BeckamTissue Solubilizer (BTS-450; 0.5 N ammonium hydroxide in toluene) under continuousstirring (100 rpm for 2 h, 60 �C). The samples were then addedwith 0.5 mL of sulphuric acid solution (1 N) and further incu-bated for 1 h thus obtaining the complete blood and tissuedissolution. Dissolved tissues and blood were decoloured bytreatment with 1mL of 24% v/vH2O2 (5min at 25 �C) and dilutedwith scintillography liquid for organic samples and tissues(Ready Organic™, Beckam) to obtain a nal volume of 7mL. Thesamples were analysed using a scintillation counter Wallac WinSpectral™ 1414 Perkin Elmer as previously reported.28

The radioactivity of various tissues (Xtissue) coming from theuptake of 3H-copolymers was normalized according to theradioactivity derived from radio-labeled copolymers accumu-lated into the vascular space and nally calculated using thefollowing equation:

Xtissue ¼ Xorgan � (V0 � C(t)) (1)

where Xorgan is the radioactivity measured in the differentsamples; V0 is the total volume of the vascular space andinterstitial uid determined 10 min aer the i.v. injection of the3H-radiolabeled PHEA, PHEA-NRD and PHEA-ABP and C(t) is theblood concentration at the different incubation times.21 Aquenching correction factor was also applied to quantify theradioactivity of blank tissues from non-injected mice spikedwith amounts of 3H-CHE (0.0030 mCi/mmol of co-polymers).22

Data were quantied using the following equation:

mCi ¼ DPM/(2.22 � 106) (2)

where DPM represents the disintegration per minute that wasobtained using the following equation:

DPM ¼ (CPM/40%) (3)

where 40% is an indicative parameter for radionuclide andinstrumental radioactive sensitivity as previously reported.20 Datawere reported as a percentage of [3H]-co-polymer per tissue. Bone



tissue and blood distribution values of 3H-radiolabelled PHEA,PHEA-NRD and PHEA-ABP were normalized using the bloodvolume distribution (13mL) and the skeletal weight (90 g) of rats.

Statistical analysis

A one way analysis of variance (ANOVA) was applied to comparedifferent groups. Data were considered statistically signicantwith a value of p < 0.05 and differences between different groupswere compared using a Bonferroni t-test. All values are theaverage of three experiments � standard deviation.

Conclusions

PHEA–bisphosphonate conjugates have been synthesized andproposed as macromolecular carriers for bone targeting. Thesynthetic procedure was optimized to obtain pure derivatives,stable and soluble in aqueous, as well as organic solvents. Theobtained bisphosphonate–polymer conjugates were physico-chemically characterized using size exclusion chromatographyand 1H-NMR, and their in vitro and ex vivo affinity for bonetissue was primarily conrmed using the hydroxylapatite andthe rabbit bone binding assays. In vivo biodistribution studiesdemonstrated that the rate of bone accumulation of variousPHEA–bisphosphonate conjugates mainly depended on thebisphosphonate moiety linked to PHEA. In particular, thePHEA-NRD conjugate showed greater affinity for the hydroxyl-apatite crystals forming the mineral component of bones,which surround the bone surface, thus leading to a greater bonetissue uptake. Results obtained with both PHEA–bisphospho-nate conjugates support the hypothesis that they could beuseful as drug bone targeted carriers.

Acknowledgements

This work was supported by the PON a3_00359, IRC FSH –

Interregional Research Center for Food Safety & Health andPRIN 2010–2011 – prot. 2010H834LS_006. The authors alsothank the MIUR and the University of Palermo for funding. Theauthors are grateful to Dr Renato Carmine Barbacane, M.D., forhis kind language revision of the manuscript.

References

1 H. Fleisch, A. Reszka, G. A. Rodan and M. Rogers, Principlesof Bone Biology, ed. J. P. Bilezikian, L. G. Raisz and G. A.Rodan, Academic Press, San Diego, 2nd edn, 2002, ch. 10,pp. 1361–1385.

2 M. D. Fancis and I. Fogelman, Bone Scanning in ClinicalPactice, ed. I. Fogelman, Springer-Verlag, New York, 1987,ch. 4, pp. 7–17.

3 H. M. Lamb and D. Faulds, Drugs Aging, 1997, 11, 413–418.4 J. Frangioni, US Pat. Application 20090123383, 2009.5 J. D. Wang, S. C. Miller, M. Sima, P. Kopeckova andJ. Kopecek, Bioconjugate Chem., 2003, 14, 853–859.

6 I. Ozcan, K. Bouchemal, F. Segura-Sanchez, O. Ozer, T. Guneriand G. Ponchel, J. Pharm. Sci., 2011, 100, 4877–4887.


7 K. Miller, A. Eldar-Boock, D. Polyak, E. Segal, L. Benayoun,Y. Shaked and R. Satchi-Fainaro, Mol. Pharmaceutics, 2011,8, 1052–1056.

8 K. Miller, R. Erez, E. Segal, D. Shabat and R. Satchi-Fainaro,Angew. Chem., Int. Ed., 2009, 48, 2949–2954.

9 H. Chen, G. Li, H. Chi, D. Wang, C. Tu, L. Pan, L. Zhu, F. Qiu,F. Guo and X. Zhu, Bioconjugate Chem., 2012, 23, 1915–1924.

10 G. Giammona, G. Carlisi and S. Palazzo, J. Polym. Sci., Part A:Polym. Chem., 1987, 25, 2813–2818.

11 R. Mendichi, A. Giacometti Schieroni, G. Cavallaro,M. Licciardi and G. Giammona, Polymer, 2003, 44, 4871–4879.

12 M. Licciardi, M. Campisi, G. Cavallaro, M. Cervello,A. Azzolina and G. Giammona, Biomaterials, 2006, 27,2066–2075.

13 E. F. Craparo, D. Triolo, G. Pitarresi, G. Giammona andG. Cavallaro, Biomacromolecules, 2013, 14, 1838–1849.

14 E. F. Craparo, C. Sardo, R. Serio, M. G. Zizzo, M. L. Bondı,G. Giammona and G. Cavallaro, Int. J. Pharm., 2014, 466,172–180.

15 M. Licciardi, C. Scialabba, G. Cavallaro, C. Sangregorio,E. Fantechi and G. Giammona, J. Biomed. Nanotechnol.,2013, 9, 949–964.

16 B. N. Ames, inMethods in Enzymology, S. P. Colowick, and N.O. Kaplan, Academic Press, NewYork, 1966, vol. 8, pp. 115–118.

17 J. Khandare and T. Minko, Prog. Polym. Sci., 2006, 31, 359–397.

18 Rohini, A. Neeraj, J. Anupam and M. Alok, J. AntiviralsAntiretrovirals, 2013, S15, 4172–4184.

19 R. Fujisawa, Y. Wada, Y. Nodasaka and Y. Kuboki, Biochim.Biophys. Acta, 1996, 1292, 53–60.

20 H. Pan, M. Sima, P. Kopeckova, K. Wu, S. Gao, J. Liu,D. Wang, S. C. Miller and J. Kopecek, Mol. Pharmaceutics,2008, 5, 548–558.

21 G. Cavallaro, L. Maniscalco, P. Caliceti, S. Salmaso,A. Semenzato and G. Giammona, J. Drug Targeting, 2004,12, 593–605.

22 D. Paolino, M. Licciardi, C. Celia, G. Giammona, M. Frestaand G. Cavallaro, Eur. J. Pharm. Biopharm., 2012, 82, 94–102.

23 D. Paolino, D. Cosco, L. Racanicchi, E. Trapasso, C. Celia,M. Iannone, E. Puxeddu, G. Costante, S. Filetti, D. Russoand M. Fresta, J. Controlled Release, 2010, 144, 144–150.

24 R. Torres Martin de Rosales, C. Finucane, J. Foster,S. J. Mather and P. J. Blower, Bioconjugate Chem., 2010, 21,811–815.

25 K. Ogawa, H. Kawashima, K. Shiba, K. Washiyama,M. Yoshimoto, Y. Kiyono, M. Ueda, H. Mori and H. Saji,Nucl. Med. Biol., 2009, 36, 129–135.

26 H. Shmeeda, Y. Amitay, D. Tzemach, J. Gorin andA. Gabizon, J. Controlled Release, 2013, 167, 265–275.

27 M. Licciardi, D. Paolino, C. Celia, G. Giammona, G. Cavallaroand M. Fresta, Biomaterials, 2010, 31, 7340–7354.

28 D. Paolino, D. Cosco, L. Racanicchi, E. Trapasso, C. Celia,M. Iannone, E. Puxeddu, G. Costante, S. Filetti, D. Russoand M. Fresta, J. Controlled Release, 2010, 144, 144–150.

J. Mater. Chem. B, 2015, 3, 250–259 | 259

Bisphosphonate–polyaspartamide conjugates as bone targeted drug delivery systems

Documents

Transcript of Bisphosphonate–polyaspartamide conjugates as bone targeted drug delivery systems