161

ReseaRch aRticle

ISSN 1743-588910.2217/17435889.4.2.161 © 2009 Future Medicine Ltd Nanomedicine (2009) 4(2), 161–175

A novel biomaterial for osteotropic drug nanocarriers: synthesis and biocompatibility evaluation of a PLGA–ALE conjugate

New approaches to therapy are offered from the use of multifunctional nanoscale devices, partic-ularly towards sites of the body that are difficult to access by drugs because of biological barriers. Nanoscale devices can, in fact, have multifunc-tional properties and can contain both target-ing agents and therapeutic payloads. Injectable nanoparticulate carriers have important poten-tial applications in site-specific drug delivery or medical imaging [1,2].

Poly(lactide-co-glycolide) (PLGA) copolymer is a biocompatible, biodegradable and nontoxic material used to obtain nano- and micropar-ticle drug-delivery systems (DDS) [3–6] and has received worldwide marketing approvals [7]. PLGA nanoparticles have been described recently as potential carriers for anticancer therapy, for example, to promote penetration of antitumor docetaxel in the CNS by overcoming the blood–brain barrier [8]. Using drug-loaded nanoparticles enables a passive targeting to some tissues, such as tumors, to be achieved by means of phenomena, such as the well-known enhanced permeation and retention effect [9]. However, active targeting should in some cases be more useful to reach effective drug concentra-tions in specific tissues and organs. Osteotropic DDS (ODDS) were proposed some years ago as

a possible means to provide drugs with affin-ity to bone tissues [10,11]. For instance, bispho-sphonates (BPs) have been conjugated to drug molecules [12–14], proteins [15,16] and radiophar-maceuticals to obtain novel agents for bone scintigraphy [17].

BPs are a class of synthetic compounds related structurally to pyrophosphate. Owing to their high affinity for hydroxyapatite (HA) crystals, they localize rapidly on the bone surface after intravenous or oral administration [18]. Because HA is only present in ‘hard’ tissues, such as bone and teeth, conjugation to BPs represents a valid strategy for targeting drugs selectively to the bone. Recently, cholesteryl-trisoxyethyl-ene-bisphosphonic acid, a new tailor-made BP derivative, was used as bone-targeting moiety for liposomes [19].

The starting aim of the present study was to merge these approaches, such as nanoparticle-mediated drug targeting and BP-prodrug strat-egy; therefore, we synthesized a polymer conju-gate by reacting the free carboxylic group of a biodegradable PLGA copolymer (Resomer® RG 502 H) with alendronate (4-amino-1-hydroxy-butyldiene-1,1-phosphoric acid [ALE]), a drug belonging to the class of amino-BP (Figure 1). The obtained polymer conjugate can be used

Background & aims: Osteotropic drug-delivery systems have been proposed as a means to provide drugs with affinity to bone tissues. Drugs or proteins have been linked chemically to bone-seeking agents, such as bisphosphonates (BPs); alternatively, drug-loaded nanoparticles have been used to target specific tissues, such as tumor areas. In our current research, these approaches were merged by synthesizing a novel bone-seeking polymer conjugate, from which targetable nanoparticles can be produced. Materials & methods: An amino-BP, alendronate (ALE) was bound covalently to a biodegradable polymer, poly(lactic-co-glycolide) (PLGA), containing a free end carboxylic group. Blood compatibility and cytotoxicity were assessed in vitro. Results & discussion: By a classical solvent-evaporation method, nanoparticles with a mean size of 200–300 nm were prepared from the conjugate; sterilization was achieved by g-irradiation, confirming their potential as injectable drug nanocarriers. Owing to the presence of the BP residue, PLGA–ALE nanoparticles were adsorbed onto hydroxyapatite to a higher extent than pure PLGA nanoparticles. The PLGA–ALE conjugate did not induce either hemolysis or alterations of the plasmatic phase of coagulation, or cytotoxic effects on endothelial cells and trabecular osteoblasts. Conclusion: The prepared conjugate represents a novel biomaterial that is able to provide nanoparticles, which can be further loaded with drugs, such as anticancer agents, and addressed to osteolytic or other bone diseases.

keywoRds: alendronate, biocompatibility, bisphosphonates, nanoparticles, odds, poly(lactide-co-glycolide), polymer conjugates, osteotropic drug-delivery systems

Rosario Pignatello1†, Elisabetta Cenni2, Dorotea Micieli3, Caterina Fotia2, Manuela Salerno2, Donatella Granchi2, Sofia Avnet2, Maria G Sarpietro3, Francesco Castelli3 & Nicola Baldini2,4

†Author for correspondence: 1Department of Pharmaceutical Sciences, Città Universitaria, Università degli Studi di Catania, Catania, Italy Tel.: +39 095 738 4021; E-mail: r.pignatello@unict.it 2Laboratory for Pathophysiology of Orthopedic Implants, Istituto Ortopedico Rizzoli, 40136 Bologna, Italy 3Department of Chemical Sciences, Università degli Studi di Catania, Catania, Italy4Department of Human Anatomy & Musculoskeletal Pathophysiology, University of Bologna, 40100 Bologna, Italy

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in a following step to obtain colloidal nanopar-ticle-delivery systems loaded with anticancer drugs and addressed to osteolytic diseases. This approach could ensure a more effective drug concentration at the site of the disease and avoid systemic side effects that, at present, hinder the use of potentially useful drugs.

When a polymer material is injected in blood, detrimental interactions with blood constituents must be avoided. The effects of PLGA polymers on blood coagulation and hemolysis are well known, as are their effects on endothelial cells. Also, when the polymer–BP conjugate reaches the bone tissues, it should inhibit osteoclast activity without altering osteoblasts. BPs may additionally promote bone marrow stromal cell proliferation and their differentiation into osteo-blasts [20]. However, the conjugation of the BP molecule with the biodegradable polymer may alter these properties and make it also cytotoxic for the osteoblasts. Hence, the blood compat-ibility and cytotoxicity for endothelial cells and osteoblasts of the prepared conjugate was assessed in vitro.

Materials & methodsMaterials��

Poly(d,l-lactide-co-glycolide), 50:50, contain-ing free carboxylic acid end-groups (Resomer RG 502 H; inherent viscosity: 0.16–0.24 dl/g [0.1% in chloroform at 25°C]) was purchased from Boehringer Ingelheim (Ingelheim am Rhein, Germany). Sodium ALE was purchased from Sigma-Aldrich Chimica Srl (Milan, Italy).

Commercial reactants and anhydrous solvents were used throughout the reactions without further purification.

Synthesis of the PLGA–ALE conjugate��Method ASodium ALE (0.25 mmol) was dissolved in 10% aqueous acetic acid and the solution was frozen and lyophilized. The free carboxylic group of Resomer RG 502 H was activated by dissolv-ing the polymer (0.25 mmol) in 4 ml of a 1:1 mixture of dimethylsulfoxide (DMSO) and dichloromethane (DCM). Then, 0.25 mmol of 1-hydroxybenzotriazole (HOBt), 0.37 mmol of N -́(3-dimethylaminopropyl)-N-ethyl carbodi-imide hydrochloride (EDAC) and 0.37 mmol of triethylamine were added. The solution was kept 2 h at 0°C, under stirring.

The previously lyophilized alendronic acid was dissolved in 1 ml of DMSO and added to the reaction mixture, which was stirred for 2 h at 2°C and then at room temperature for approximately 8 h. The reaction course was monitored by TLC (1:1 DCM/methanol, v/v). The solvent was partially removed under vac-uum and the remaining solution was dialyzed (CelluSep H1 MWCO 2000; M-Medical s.r.l., Cornaredo, Italy) for 48 h against 1 l water, which was changed every 12 h. The dialyzed sample was frozen in liquid nitrogen and lyophilized.

Method BResomer RG 502 H was activated previously with N-hydroxysuccinimide (NHS) according to the literature [21]. Dicyclohexylcarbodiimide (0.11 mmol) and NHS (0.11 mmol) were added to a polymer solution (0.1 mmol) in dioxane (25 ml) at 15°C under stirring for 3 h. The dicyclohexylurea formed was removed by fil-tration (0.45 µM syringe nylon-membrane fil-ter) and the solution was poured into diethyl ether (100 ml). The solvent was decanted and the oily residue was purified by dissolution in dioxane and precipitation with diethyl ether (three times) and finally dried under reduced pressure.

A solution of NHS–PLGA (0.075 mmol) in DMSO (4 ml) was treated with triethylamine (3.5 ml) and sodium ALE (0.070 mmol) under stirring at room temperature for 12 h. At the end of the reaction, as verified by TLC (see Method A), the solvent was partially removed under vacuum and the remaining solution was purified by dialysis as described earlier, being thereafter frozen and lyophilized.

HO

O

CH3O

O

O

H

P

OH

OH

O OH

(CH2)3 NH2

P

O

OH

OHC Alendronic acid

Resomer® RG 502 H[(C6H8O4)x(C4H4O4)y]n

x y

Figure 1. Chemical structure of the Resomer® RG 502 H copolymer and alendronic acid.

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Characterization of the ��PLGA–ALE conjugate1H-NMR ana lysisSpectra were registered in DMSO-d6 with a Varian Unity INOVA instrument operating at 500 MHz. Tetramethylsilane was used as the internal standard; chemical shifts are reported in ppm.

MALDI-TOF ana lysisAn Ultraflex II instrument (Bruker Daltonics, Bremen, Germany) equipped with a UV nitro-gen laser (337 nm) was used. Matrix solution was prepared by dissolving 2,5-dihydroxy-benzoic acid (DHB) in water/tetrahydrofuran (30:70, v/v). The matrix:sample molar ratio was 1000:1. Spectra were obtained in reflec-tion-positive ion mode and were the average from approximately 500 laser shots to improve the signal-to-noise level. Mass assignment was made using the calibration mixture 2 solution (PE Biosystems, Foster City, CA, USA) as an external standard.

Differential-scanning calorimetryDifferential-scanning calorimetry (DSC) mea-surements of glass-transition temperature (Tg) were performed in triplicate with a Mettler Toledo STARe system equipped with a DSC-822e cell and a Mettler TA-STARe software (version 8.10). For comparison purposes, a phys-ical mixture was prepared for these experiments: solid PLGA and ALE (free acid form) were tritu-rated at 20°C for 30 min in a 2:1 weight ratio. Samples were placed in a 40 µl aluminum DSC pan with the cover holed and were scanned at 5–65°C with a heating-scan rate of 5°C/min. An empty pan was used as a reference. The calori-metric system was calibrated, in transition tem-perature and enthalpy changes, using indium, stearic acid and cyclohexane by following the procedure of the DSC-822e Mettler TA-STARe instrument. All the DSC thermograms were obtained from the third heating cycle. Nitrogen was used as the gas.

In vitro�� hemolysis assayIn the biocompatibility tests, PLGA–ALE con-jugate was dissolved in DMSO at the concentra-tion of 52.45 mg/ml. From this solution, scalar dilutions in phosphate-buffered saline (PBS), plasma or culture medium were tested. As con-trols, DMSO alone, at the same dilutions, was assessed. All the experiments of blood compat-ibility were performed using human venous blood from healthy volunteer donors, which was

supplied by a Blood Bank. Hemolysis was evalu-ated on venous blood anticoagulated with 119 IU of lithium heparinate. Within 2 h, the erythro-cytes were washed and resuspended in PBS at a 1:10 w/v ratio. In this assay, as well as in the other biological tests, PLGA–ALE and DMSO were sterilized by filtration through sterile 0.22-µm polysulfone membrane filters (Whatman Inc., Florham Park, NJ, USA). PLGA–ALE and DMSO alone were tested at different dilutions in PBS. Saponin (Sigma) (25 mg/ml in PBS) was used as a positive control. After incubation at 37°C for 4 h, specimens were centrifuged at 1000 rpm for 15 min to remove the nonlysed erythrocytes. The supernatant was collected and analyzed for the released hemoglobin by spec-trophotometric determination at 540 nm. The samples were tested in triplicate.

To obtain 0 and 100% hemolysis, the erythro-cyte suspension was added to PBS and distilled water, respectively. The degree of hemolysis was determined by the following equation:

(%)

/

Hemolysis

Abs Abs Abs Abs x0 100 0 100

=

- -^ ^h h7 A

where Abs, Abs0 and Abs100 are the absor-bance of the test samples, a solution of 0% hemolysis and a solution of 100% hemolysis, respectively.

Evaluation of the effects of ��PLGA–ALE on the plasmatic phase of coagulationVenous blood from healthy donors was collected in siliconized tubes containing 3.8% sodium citrate at a blood:citrate ratio of 9:1. Platelet-rich plasma (PRP) was obtained by centrifuga-tion at 1000 rpm for 5 min. Scalar dilutions of PLGA–ALE and DMSO were tested. In siliconized tubes, 0.1 ml of each dilution were added to 0.9 ml of PRP. The negative control was obtained by adding 0.1 ml of PBS to 0.9 ml of PRP. All the tubes were rotated for 30 min at room temperature and were then centrifuged at 3000 rpm for 15 min. From the supernatants, prothrombin activity and activated partial-thromboplastin time (APTT) were determined by ACL 300 (Instrumentation Laboratory IL, Lexington, MA, USA) with commercial reagents (PT-Fibrinogen HS, Hemosil, IL, USA, for prothrombin activity and APTT-SP liquid, Hemosil, IL, USA, for APTT).

Prothrombin activity of the samples was extrapolated on a calibration curve obtained form scalar dilutions of a calibrator plasma (Calibration Plasma, Hemosil, IL, USA).

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Culture of endothelial cells��Human umbilical vein endothelial cells (HUVECs) were harvested according to the method of Jaffe et al. [22], after informed con-sensus. The cells were grown to confluence in a medium composed of an equal mixture of medium RPMI 1640 (Sigma) and medium 199 (Sigma), supplemented with 20% fetal bovine serum (FBS; Cambrex, Verviers, Belgium), 25 µg/ml endothelial cell-growth supplement (BD Biosciences, Bedford, MA, USA), 2 mM l-glutamine (Sigma), 100 IU/ml of penicil-lin (Invitrogen, Carlsbad, CA, USA) and 100 µg/ml of streptomycin (Invitrogen) under an atmosphere consisting of 95% O

2 and 5%

CO2. Initially, the cells were seeded on a 25 cm2

f lask coated with collagen I (Biocoat, BD Biosciences); after the first passage and during the experiments, the cells were cultured on cell culture-treated polystyrene coated with gelatin 0.2% from bovine skin (Sigma).

Trabecular osteoblast isolation ��& cultureThe protocol of the bone-sample collection was approved by the Institutional Ethical Committee on human research of the Rizzoli Orthopedic Institute and was performed in compliance with human studies with the Helsinki Declaration of 1975 as revised in 1996. Human primary osteoblasts were isolated from trabecular bone [23] from patients undergoing

O

CH3 H

O

O

CH3 H

O

O

H H

O

O

H H

OC

C

C

C

C

C

C

C* *

n

y

a b

c

c

c

b

b

a

a

5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 ppm 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 ppm

5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 ppm

H2PO3 PO3H2

OH

NH2

d

e

e

d

d

d

Figure 2. 1H-NMR spectra of (A) Resomer® RG 502 H, (B) ALe and (C) the PLGA–ALe conjugate prepared by method A. Analysis was performed in DMSO-d6 at 500 MHz. ALE: Alendronate; PLGA: Poly(lactide-co-glycolide).

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PLGA–ALE conjugate for osteotropic nanocarriers ReseaRch aRticle

Inte

nsi

ty (

a.u

.)In

ten

sity

(a.

u.)

0.2

0.4

0.6

0.8

1.0

1.2

m/z600 800 1000 1200 1400 1600 1800 2000

1935

.337

1905

.342

1877

.373

1847

.402

1805

.465

1759

.490

1731

.474

1689

.472

1659

.482

1629

.485

1603

.429

1557

.478

1515

.423

1471

.365

1441

.400

1399

.308

1369

.294

1341

.239

1299

.138

1253

.107

1210

.997

1182

.949

1152

.901

1124

.811

1080

.727

1052

.641

1008

.535

980.

446

950.

377

922.

278

892.

201

862.

104

834.

018

805.

907

775.

81974

3.69

371

5.57

7685.

219

657.

219

627.

09159

8.96

358

4.90

756

8.84

454

0.70

352

6.66

8

4

3

2

1

0

1961

.220

1933

.221

1903

.272

1861

.289

1831

.334

1801

.373

1773

.358

1745

.327

1715

.366

1687

.360

1659

.358

1629

.373

1585

.350

1557

.347

1529

.291

1499

.313

1455

.271

1427

.236

1399

.1951369

.180

1325

.126

1311

.096

1283

.040

1253

.007

1222

.953

1194

.902

1152

.796

1108

.703

1078

.64310

50.5

6510

22.4

8299

2.42

196

2.32

793

4.25

4906.

157

862.

02984

7.97

681

9.86

878

9.79

076

1.66

974

7.61

971

7.52

470

3.46

168

9.40

965

9.17

564

5.11

061

6.98

558

6.86

757

2.79

455

8.73

352

8.62

9

Figure 3. MALdI-ToF mass spectrum of (A) Resomer® RG 502 H and (B) PLGA–ALe conjugate. ALE: Alendronate; a.u.: Arbitrary units; PLGA: Poly(lactide-co-glycolide).

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total hip arthroplasty, after informed consent, and cultured in Dulbecco’s modified Eagle’s medium (Sigma) supplemented with 2 mM of l-glutamine, 100 IU/ml of penicillin, 0.1 µg/ml of streptomycin and 10% FBS. At least 60% of the cells in the cultures were for alkaline phosphatase staining evaluated by optical-microscopy examination, which is a marker of the osteoblast phenotype.

Neutral-red test��Cytotoxicity of PLGA–ALE for HUVECs and trabecular osteoblasts was evaluated by neutral-red test. A 0.4% solution of neutral-red dye tested for cell cultures (color index 50040) in distilled water was prepared and stored at 4°C (stock solu-tion). The working solution was prepared imme-diately before use by diluting the stock solution in complete medium to yield a final concentration of 50 ng/ml. The cells were suspended in complete medium and seeded into tissue culture-treated polystyrene 96-well microplates (3 × 104 cells/well). The cells were incubated at 37°C for 24 h. After incubation, the cells were washed with PBS, then 0.2 ml of each sample was added. Scalar dilu-tions of PLGA–ALE and DMSO were prepared. The negative control was the complete medium. The positive control was a 0.64% solution of phenol (Sigma) in complete medium, prepared immediately before use. The microplate was incu-bated for 24 h at 37°C, then, after discarding the supernatants, 0.2 ml of neutral-red dye solution

were added to the wells for 2 h at 37°C. The super-natant was removed, the wells washed with PBS and 0.1 ml of lysing solution (50% ethanol in 1% acetic acid) were added. The color intensity of each well was read at 540 nm, the optical densities of the replicates were averaged and the viability of the samples was calculated as a percentage of the nega-tive control. The sample was considered as toxic if the viability percentage was less than 80%.

PLGA–ALE nanoparticles��Two alternate methods were used to obtain nano-particles from the synthesized conjugate. In a solvent-displacement method, the PLGA–ALE conjugate (100 mg) was dissolved in 10 ml of acetone, DMSO or an acetone/DMSO 1:1 (v/v) mixture. The organic phase was added drop-wise to 25 ml of PBS (pH 7.4) containing 100 mg of Pluronic® F68. After stirring at room temperature for 10 min, acetone was removed at 30°C under reduced pressure and the suspension was purified by dialysis as described earlier. The final volume of the suspension was adjusted to 35 ml with PBS.

PLGA–ALE nanoparticles were also prepared by a dialysis method [24]. A total of 20 mg of the polymer conjugate were dissolved in 4 ml DMSO and the solution was dialyzed for 12 h in a dialysis tube (CelluSep H1 MWCO 2000; M-Medical s.r.l., Cornaredo, Italy) against 3 l of water, which was entirely changed every 4 h.

Characterization of nanoparticles��The mean particle size of nanoparticle prepara-tions was measured by photon-correlation spec-troscopy (PCS) with a Zetamaster (Malvern Instruments, UK), equipped with the Malvern PCS software. The dispersions were diluted appropriately with proinjection water before the ana lysis and the reading was carried out at a fixed angle of 90°. The average diameter and the polydispersity index of nanoparticles were determined; reported values are the mean of 30 measurements.

The electrophoretic mobility was obtained by a laser Doppler anemometer with the same instrument. A suitable amount of each sample (80 µl) was diluted with 20 ml of HPLC-grade water and injected in the electrophoretic cell of the instrument. The ζ-potential value was cal-culated by the instrument software, using the Smoluchowsky’s equation.

A morphological investigation of nanopar-ticles was performed using a Philips XL-20 scanning-electron microscope operating at 15 kV. The samples were sputter-coated with palladium-gold prior to examination.

10 15 20 25 30 35 40 45 50 55 60

°C

endo

0.2 mW

Figure 4. differential-scanning calorimetry curves of (A) PLGA, (B) PLGA–ALe conjugate, (C) PLGA/ALe physical mixture and (d) sodium ALe. Heating scan rate was 5 °C/min. ALE: Alendronate; PLGA: Poly(lactide-co-glycolide).

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Sterilization of nanoparticles��The nanoparticle suspensions were aliquoted into glass vials and submitted directly to g-irradia-tion, at a strength of 10 kGy by a 60 Co source (Gammatom srl, Guanzate, Como, Italy).

HA affinity assay of ��PLGA–ALE nanoparticlesPLGA–ALE or PLGA nanoparticles were prepared by the solvent-evaporation method described earlier, using an 1:1 acetone/DMSO mixture containing the 0.1% (w/w) of the lipo-philic probe Oil Red O (Sigma). An aliquot of 2 ml of each nanoparticle dispersion was mixed with 2 ml of a HA aqueous suspension at two different concentrations (1 or 5 mg/ml) and shaken gently for 15 or 30 min at room tempera-ture. Each experiment was made in quadrupli-cate. The whole suspension was filtered through a 1.2 µm pore-size nylon-syringe filter to sepa-rate the HA particles and the filtered liquid was ultracentrifuged (Beckman L8-M, type SW41 Rotor) at 15,000 rpm for 1 h, at 4°C. The pel-lets were freeze-dried and a known amount of each sample was dissolved completely in DMSO and the Oil Red O absorbance was measured at 523 nm by UV/VIS ana lysis versus a calibration curve of the same compound in DMSO.

The relative amount of nanoparticles linked to the HA was calculated by the decrease in absorbance of these samples, in respect to the initial absorbance of nanoparticles incubated without HA and treated under the same con-ditions. Similarly, Oil Red O-free PLGA and PLGA–ALE nanoparticles were processed in a similar way and assessed to compensate for the turbidity effect in the final UV ana lysis.

Statistics��Statistical ana lysis was performed with the StatViewTM 5.0.1 software for Windows. The results were expressed as the arithmetic mean ± standard error of the mean (mean ± SEM). The comparison among the groups was made by the Kruskal–Wallis test; the comparison between the pairs was made by the Mann Whitney test. Significance was set at p < 0.05.

ResultsConjugate synthesis ��

& characterizationThe synthesis of the polymeric conjugate was attempted using two alternative well-recognized methods, such as carbodiimide-assisted direct conjugation or preparation of an activated inter-mediate through n-hydroxysuccinimide [21]. However, both methods resulted in similar pro-duction yields (70–75%) of the final conjugate, although Method A was preferable because it is more direct and simple.

The basic chemical structure of the PLGA–ALE conjugate was confirmed by MALDI mass spectrometry (MS) and 1H-NMR. In the latter ana lysis, the pure copolymer showed the typical signals at approximately 1.55 ppm (overlapping doublets), owing to the methyl groups of the d- and l-lactic acid units, and multiplets at 4.8 ppm, corresponding to the methylene groups of glycolic acid, and at approximately 5.2 ppm, owing to the methine groups of lactic acid residues (Figure 2A)

(Boehringer Ingelheim Pharma GmbH & Co. KG, technical datasheet on Resomer). ALE gave a simple spectrum characterized mainly by two broad singlets centered at 1.86 and 2.71 ppm, respectively (Figure 2B). In the 1H-NMR spec-trum of the PLGA–ALE conjugate, a prevalence of the polymer signals were of course observed (Figure 2C), however, the presence of ALE was confirmed by a small broad peak at 1.79 ppm. Accumulation and enlargement of these diagnos-tic signals enabled calculation of the degree of substitution of ALE in the polymer backbone,

Table 1. Tg parameters of PLGA, PLGA–ALe conjugate and PLGA/ALe physical mixture*.

onset (°C) Mid point (°C) Inflection point (°C) ∆Cp (°C)

PLGA 38.82 ± 0.54 40.30 ± 1.31 42.30 ± 0.90 0.707 ± 0.011

PLGA–ALE 28.48 ± 0.49 32.66 ± 0.65 34.40 ± 0.31 0.449 ± 0.102

Physical mixture 38.63 ± 0.81 40.85 ± 0.56 42.23 ± 0.30 0.610 ± 0.006*The corresponding values for alendronate were not detectable.ALE: Alendronate; PLGA: Poly(lactide-co-glycolide).

Table 2. Arithmetic mean and standard error of the percentage of hemolysis induced by different concentrations of PLGA–ALe conjugate.

sample Concentration Hemolysis (%)

PLGA–ALE 3.14 mg/ml 0.01 ± 0.40

PLGA–ALE 314.00 µg/ml 0.01 ± 0.20

PLGA–ALE 31.40 µg/ml 0.01 ± 0.13

PLGA–ALE 3.14 µg/ml 0.17 ± 0.35

PLGA–ALE 314.00 ng/ml 0.01 ± 0.31

Saponin 25.00 mg/ml 100

Distilled water 100ALE: Alendronate; PLGA: Poly(lactide-co-glycolide).

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giving a mean conjugation yield of 38%, compat-ible with the value calculated by the mass ana lysis (vide infra).

The MALDI MS spectra of pure Resomer RG 502 H and the PLGA–ALE conjugate, prepared by Method A, are reported in Figure 3A

& B, respectively. The former shows the presence of oligomers in the m/z 500–2500 range that, because of the kind of ionization used, can be

considered a range of molecular weights. Clusters split by 14 amu were visible in this complex spec-trum, suggesting that the polymer was obtained by C

3H

4O

2 and/or C

2H

2O

2 monomer units.

The PLGA–ALE conjugate gave a different spectrum (Figure 3B). In fact, in the clusters of the various oligomers, a marked increase of abun-dance of the M-2 and M-4 species was registered, suggesting that a reaction between the polymer

Pro

thro

mb

in a

ctiv

ity

(%)

AP

TT

(s)

150

120

90

60

30

0

Basal PBS 31.4 µg/mlPLGA–ALE

3.14 µg/mlPLGA–ALE

314 ng/mlPLGA–ALE

Basal PBS 31.4 µg/mlPLGA–ALE

3.14 µg/mlPLGA–ALE

314 ng/mlPLGA–ALE

0

10

20

30

40

Figure 5. Arithmetic mean ± standard error of (A) prothrombin activity and (B) APTT of human plasma incubated with different concentrations of the PLGA–ALe conjugate. ALE: Alendronate; APTT: Activated partial thromboplastin time; PBS: Phosphate-buffered saline; PLGA: Poly(lactide-co-glycolide).

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PLGA–ALE conjugate for osteotropic nanocarriers ReseaRch aRticle

and ALE had occurred. For instance, the reac-tion between H

3C([C

6H

8O

4]

5[C

4H

4O

4]

5)OH

(1332 Da; or m/z 1355 in the sodium salt form) and alendronic acid would lead to the species at m/z 1563 (or m/z 1585 in the form of sodium salt). By comparing the mass spectra of Resomer

and PLGA–ALE (Figure 3A & B), the increment of abundance of the ion at m/z 1585 was clearly vis-ible, thus proving the reaction between the two molecules. Such behavior was observed for all the oligomer species.

A semi-quantitative estimation, based on the increase of abundance of the species at m/z 1585, indicated a conjugation yield of 30–35%. In a sample of PLGA–ALE conjugate obtained by Method B, a conjugation yield of 35–40% was obtained, confirming that the stoichiometry of this conjugation was independent on the reaction pathway.

DSC thermograms of PLGA, PLGA–ALE conjugate, a physical mixture of PLGA and sodium ALE and pure sodium ALE are reported in Figure 4. PLGA (A) showed a glass transition temperature (mid point) at 40.84°C and a ∆Cp = 0.705 J/g K (TABle 1). A well-shaped endo-thermic peak (relaxation peak) was visible on the first heating (not reported). This peak, known as ‘kinetic overshoot’, is a quantitative measure of the relaxation enthalpy (∆H) that occurs in a polymer during sub-Tg annealing [25,26]. Glasses exist in a non-equilibrium state and relax towards a lower-energy and meta-stable state on anneal-ing [27] and this relaxation brings about the endothermic peak in the DSC heating scans.

The PLGA–ALE conjugate (Figure 4B) showed a glass transition at a lower temperature, with a mid point at 33.72°C and a ∆Cp =0.547 J/g K (TABle 1). By comparing the Tg observed with the PLGA–ALE physical mixture (Figure 4C) and the pure ALE (Figure 4D), the PLGA physicochemical structure appeared changed by the bonding to ALE. In addition, a sample of PLGA submit-ted to the same preparative conditions used to prepare the PLGA–ALE conjugate, but with-out the addition of the drug, showed a similar thermogram to that of pure PLGA (not shown), confirming that the different glass transition observed in the PLGA–ALE calorimetric ther-mograms was due to the presence of chemically linked ALE.

Biocompatibility studies��The effects of PLGA–ALE on erythrocytes were evaluated in two experiments, carried out in trip-licate. Hemolytic activity of PLGA–ALE, as well the hemolysis induced by DMSO, was less than

1%. However, saponin solution (i.e., the positive control) induced a complete hemolysis in all the experimental sessions (TABle 2).

The effects of PLGA–ALE on the plasmatic phase of coagulation were evaluated in two exper-iments, carried out in duplicate. In basal condi-tions, prothrombin activity was 140.2 ± 2.5 and APTT was 35.6 ± 0.3. In plasma incubated with PLGA–ALE at different dilutions, prothrombin activity and APTT was not significantly different from the plasma incubated with PBS (Figure 5). DMSO at the same dilutions did not affect either prothrombin activity or APTT.

In the neutral-red test, neither PLGA–ALE nor DMSO at the tested dilutions affected endothelial cell viability significantly (Kruskal–Wallis test, p = 0.6058) (Figure 6). PLGA–ALE determined a viability percentage varying from 121.3 ± 10.3% when tested at 314 µg/ml to 108.3 ± 5.6% when tested at 314 ng/ml. Also, DMSO had no effects on cell viability at the tested dilutions. However, phenol (positive con-trol) reduced viability to 14.5 ± 1.3% and was therefore highly cytotoxic (Figure 6).

Via

bili

ty (

%)

150

120

90

60

30

0Medium 314 µg/ml

PLGA–ALE31.4 µg/mlPLGA–ALE

3.14 µg/mlPLGA–ALE

314 ng/mlPLGA–ALE

Phenol(positivecontrol)

Figure 6. Arithmetic mean ± standard error of the viability of human umbilical vein endothelial cells after incubation with the PLGA–ALe conjugate. ALE: Alendronate; PLGA: Poly(lactide-co-glycolide).

Via

bili

ty (

%)

200

160

120

80

40

0Medium 314 µg/ml

PLGA–ALE31.4 µg/mlPLGA–ALE

3.14 µg/mlPLGA–ALE

314 ng/mlPLGA–ALE

Phenol(positivecontrol)

Figure 7. Arithmetic mean ± standard error of the viability of human primary osteoblasts, from trabecular bone, after incubation with the PLGA–ALe conjugate. ALE: Alendronate; PLGA: Poly(lactide-co-glycolide).

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Similarly, neither PLGA–ALE nor DMSO at the tested dilutions affected osteoblast viability significantly (Kruskal–Wallis test, p = 0.9781) (Figure 7). PLGA–ALE determined a viability per-centage varying from 151.0 ± 22.1% when tested at 314 µg/ml to 134.5 ± 13.94% when tested at

314 ng/ml. Also, DMSO had no effect on cell viability at any of the tested dilutions. The posi-tive control phenol was highly cytotoxic, with a viability reduction to 19.0 ± 2.4%.

Characterization of nanoparticles��PLGA and PLGA–ALE nanoparticles made by solvent evaporation and submitted to micro-scopy and dynamic light-scattering dimensional analyses appear to be spherical (Figure 8), with a mean size of approximately 200–300 nm and a relatively homogeneous distribution, as con-firmed by the relatively low polydispersity-index values (TABle 3). Owing to the low solubility of the PLGA–ALE conjugate in acetone, DMSO was tested as the organic phase in the nano-particle production. However, using DMSO alone to dissolve the conjugate resulted in larger nanoparticles than those obtained when a 1:1 (v/v) acetone/DMSO mixture was used (Figure 9

& TABle 3). Therefore, the latter solvent mixture was selected for the forthcoming studies.

The sterilization of the nanoparticle suspen-sions by g-irradiation (10 kGy) only caused a lim-ited size reduction of nanoparticles, from approxi-mately 200 to 186 nm, prospecting the possibility of using such a technique for the further in vivo studies on this system. More detailed studies on the effect of such treatment – although well-standardized for polymeric nanoparticles – on the conjugate structure and potential molecular weight changes are, however, in progress.

The dialysis method described in the litera-ture for analogous systems [24] produced larger nanoparticles (Figure 9), although with a homo-geneous population, as confirmed by the low polydispersion-index values (TABle 3).

The PLGA–ALE nanoparticles displayed a net-negative surface charge (ζ-potential of ~ –38 mV) (TABle 3), close to that measured for the nanoparticles prepared from pure PLGA (-41.80 ± 3.92 mV).

Incubation of Oil Red O-loaded PLGA–ALE nanoparticles with HA indicated a rela-tive increase of affinity towards the phosphate salt compared with pure PLGA nanoparticles (Figure 10). Such affinity increased with the length of incubation and was proportional to the concentration of HA in the suspension. In particular, while at a HA concentration of 1 mg/ml, the two kinds of nanoparticles did not show remarkable differences in terms of reduction of Oil Red O absorbance and, thus, of adsorption to HA. At a higher salt concentration (5 mg/ml), PLGA–ALE-based nanoparticles remained linked to HA to a higher extent than PLGA

1 mg 15 0Kv ×8400 1 µm

Figure 8. scanning-electron microscopy of PLGA–ALe nanoparticles prepared by the solvent evaporation method from a 1:1 (v/v) acetone/dimethylsulfoxide solution. ALE: Alendronate; PLGA: Poly(lactide-co-glycolide).

Inte

nsi

ty

Size (nm)

12

10

8

6

4

2

00 1200200 400 600 800 1000

Acetone

1:1 acetone/DMSO

DMSO

Dialysis

Figure 9. Gaussian fits of PLGA–ALe nanoparticle size distribution prepared by either the solvent evaporation method, using different solvents as the organic phase (unbroken line), or the dialysis method (broken line). ALE: Alendronate; DMSO: Dimethylsulfoxide; PLGA: Poly(lactide-co-glycolide).

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nanoparticles, showing a 43 and 50% increase of affinity after 15 and 30 min of incubation, respectively (Figure 10).

discussionA polymeric material (Resomer RG 502 H) (Figure 1) made of 50:50 polylactide co-glycolide was used because of its biodegradability and ability to form nanoparticle structures applying simple and biocompatible techniques [24,28–30]. Its physicochemical properties, such as swelling and biodegradation kinetics, or molecular inter-action potential with carried drugs, offer several opportunities for the design of controlled release systems. These properties are strongly defined by structural features, such as copolymer composi-tion, molecular weight and nature of the chain end groups.

ALE (Figure 1) was selected as a targeting mole-cule in the prepared conjugate for different rea-sons. First of all, it bears a free amine group that enabled an easy bond to the copolymer free car-boxylic group using standard peptide synthesis wet methods.

Moreover, the amide bond between the target-ing drug and the polymer should display a higher resistance to enzymatic hydrolysis in plasma, for instance, in respect to an ester bond, thus enabling a longer circulation time of the intact conjugate. In the meantime, the monomer composition of the used polymer (50:50, lactide to glycolide) would ensure a relatively long circulation time in the human body, up to 60 days [31], enough to enable the bound BP moiety to recognize the bone tissues.

Finally, the free amino group of ALE is not involved in the binding to HA, the main target of BP drugs. BP binding to bones is, in fact, strictly dependent on the presence of the phosphonate group and is reinforced by a free hydroxyl group [32]. Therefore, the osteotropicity of ALE could be transferred to the whole polymer conjugate, as demonstrated for other conjugates [33].

Nanoparticles were obtained from this conjugate through two alternate methods. However, although the dialysis method gave a uniform population of larger nanoparticles, the solvent-displacement technique provided smaller particles with a good polydispersity index. Therefore, the latter procedure is planned to be used for our forthcoming studies on drug delivery with this nanocarrier.

A PLGA copolymer was conjugated with ALE to target osteolytic areas of bone. The conjugation of water-soluble polymers to drugs with affinity for the bone hydroxyapatite, for example, BPs or

glutamic/aspartic acid peptides, is one of the most used chemical approaches [10,16,34,35]. These conju-gates are able to target the whole delivery system to the bones and improve the pharmaco kinetics of the conjugated drugs. Recently, new BPs have been synthesized and incorporated in DDS based on calcium-phosphate supports and acrylic for-mulations, for the treatment of osteoporosis and osteolytic diseases [36].

Incubation of PLGA–ALE as well as pure PLGA nanoparticles with HA was performed to assess the increased affinity induced by the

Table 3. Properties of PLGA–ALe nanoparticles prepared by solvent evaporation from different solvents and by the dialysis method.

Method Zeta potential Mean size (nm) PdI

Solvent evaporation (organic phase)

Acetone -37.6 ± 4.1 188.0 ± 21.3 0.258

Acetone/DMSO (1:1, v/v) -37.2 ± 5.0 198.7 ± 17.3 0.348

DMSO -38.9 ± 3.7 286.9 ± 9.6 0.156

Dialysis

-39.3 ± 4.7 438.6 ± 11.1 0.033

ALE: Alendronate; PLGA: Poly(lactide-co-glycolide).

Red

uct

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of

Oil

Red

O a

bso

rban

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%)

100

80

60

40

20

0

1 mg/ml HA 5 mg/ml HA

PLGA: 15 min

PLGA–ALE: 15 min

PLGA: 30 min

PLGA–ALE: 30 min

Figure 10. evaluation of the affinity of PLGA–ALe and PLGA nanoparticles for hydroxyapatite. Oil Red O-loaded nanoparticle suspensions were incubated at room temperature for either 15 or 30 min with an aqueous suspension containing 1 or 5 mg/ml of the phosphate salt. Results are expressed as the percentage decrease of absorbance at 523 nm with respect to nanoparticles incubated without HA. ALE: Alendronate; HA: Hydroxyapatite; PLGA: Poly(lactide-co-glycolide).

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presence of ALE on the particle surface [24,33]. Two different HA concentrations and two incu-bation times were used as variables that could evidence the different link to HA, which could be due to the chemical reaction with BP but also to the mere mechanical adsorption of the salt onto the colloidal-particle surface [24]. The finding that PLGA–ALE nanoparticles showed a higher affinity to HA as a function of the salt concentration and incubation time would sug-gest that association of PLGA–ALE nanopar-ticles to the phosphate salt was owing to the development of a chemical interaction between HA and the BP moiety, besides a mere physical absorption of HA onto the nanoparticle surface that would also explain the affinity observed for PLGA nanoparticles.

In the biocompatibility assays, the prepared conjugate was evaluated for blood compatibil-ity and cytotoxicity with preliminary screening tests that were able to highlight harmful effects, which may preclude any further biological appli-cation. The conjugate was tested as a solution in DMSO. Obviously, in the following steps, when nanoparticles will be prepared from the conjugate, they will be assayed as a suspension in an aqueous buffer solution.

The effect of ALE on cell viability was not assayed because the aim of the research was not to compare the cytocompatibility of the new conjugate with that one of single components but rather to ascertain whether this conjugate was suitable for generating nanoparticles for bone therapy. On the contrary, in viability tests, we compared the PLGA–ALE conjugate with phenol, which has a well-known cytotoxicity, and with the culture medium, which was an index of the basal condition of cells.

Hemolysis was evaluated because erythrocytes are among the first cells that come into contact with foreign materials in the blood vessels. The results did not demonstrate any hemolytic effects of PLGA–ALE. The percentage of hemolysis was similar to the control, such as the erythrocytes incubated with PBS.

The plasmatic phase of coagulation is usually assessed through global tests, such as prothrom-bin activity and APTT. APTT evaluates the intrinsic phase and prothrombin activity evalu-ates the extrinsic phase. Both have a higher sen-sitivity in demonstrating coagulation defects but a lower sensitivity for a thrombophylic state. The latter is demonstrated through multiple and more complex tests. In this preliminary phase, our aim was to screen for coagulation inhibition, whereas coagulation activation will

be evaluated directly on nanoparticles. The conjugate did not determine any alteration of the plasmatic phase of coagulation, at the tested dilutions.

In the bloodstream, nanoparticles come into contact with the endothelium before passing through the vessel wall and reaching tissues. Therefore, the effect of PLGA–ALE on endothe-lial cells was tested to demonstrate absence of cytotoxicity, which could affect the use of this conjugate for the preparation of nanoparticles. PLGA–ALE was not cytotoxic to endothelial cells, as demonstrated by the neutral-red test. This test is based on the principle that living cells absorb the dye and accumulate it in the lysosomes. Cytotoxic agents can disrupt the metabolic energy required for dye accumulation and thus decrease dye uptake: such a change may be quantified by a simple method [37]. Therefore, the neutral-red test, which measures the active cellular uptake, may be considered as a good alternate to the MTT test, which rather measures mitochondrial-related reduction capacity. For this reason, the neutral-red test has been used for cytotoxicity screening of several biomaterials [38,39].

Bone-targeted nanoparticles should not dam-age normal osteoblasts. Therefore, PLGA–ALE cytotoxicity was evaluated on trabecular osteo-blasts but no effect was observed. Therefore, the conjugate PLGA–ALE did not induce either hemolysis or alterations of the plasmatic phase of coagulation or cytotoxic effects on endothelial cells and trabecular osteoblasts.

ConclusionDuring research aimed at realizing osteotropic polymeric carriers for targeting drugs to bone diseases, a conjugate between 50:50 PLGA and the amino-BP ALE has been synthesized and characterized. From this material, nanoparticles with a mean size of 200–300 nm were obtained easily by classical production methods. The PLGA–ALE conjugate did not cause hemolysis or alterations of the plasmatic phase of coagula-tion in vitro or cytotoxic effects on endothelial cells and trabecular osteoblasts, thus becoming a promising biomaterial for the forthcoming preparation of drug-loaded nanoparticles.

Future perspectiveAt the beginning of this decade, the studies of Hirabayashi and colleagues led to the proposal of BP prodrugs of bioactive molecules to produce bone-seeking or ‘osteotropic’ compounds [10,14]. This strategy has been seldom taken on by other authors who described other prodrugs in which

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anticancer or anti-inflammatory drugs, as well as peptides and proteins, have been covalently linked to BPs or other compounds with bone affinity.

In general, the literature has revealed that, in many cases, the efficacy of the prodrug approach is hampered by the low solubility of drugs, which makes the circulation in the bloodstream and an efficient distribution to the sites of action difficult. Also, BP-drug conjugates might not have the optimal physicochemical properties to achieve a valid pharmacokinetic profile.

Recently, Liu et al. published and patented a β-cyclodextrin–ALE derivative with a high-bind-ing affinity to HA. Drugs, such as dexametha-sone and prostaglandin E

1, were included in the

targeted cyclodextrin–ALE system for the treat-ment of mouth and skeleton diseases, respectively [33,40]. Such a soluble carrier has the potential to become a valid means to improve the efficacy of drugs at the level of biomineral tissues.

The encapsulation of drugs in colloidal trans-port systems, such as liposomes and nanopar-ticles, is an alternate widely explored strategy to bypass the adverse physicochemical nature of drugs and take advantage of abnormal distribu-tion phenomena, such as the enhanced perme-ation and retention effect that often characterize inflamed and neoplastic areas. Several nanoparti-cle systems have been investigated for the targeted delivery of cytotoxic drugs to cancer, however, very few have been investigated regarding bone tissues [41–43].

We believe that the merged approach described in this study, such as loading anticancer drugs into nanoparticles made by a novel osteotropic

biopolymer, might deserve attention and further investigation. Preliminary in vitro and in vivo biological studies with PLGA–ALE nanopar-ticles, loaded with cytotoxic drugs and still in progress at present, provide, in fact, positive out-comes. This will prompt us to a wider exploita-tion of the potential ability of this nanocarrier for the targeted delivery of drugs and other bio-active molecules in the treatment of bone cancer or other bone disorder-related conditions.

AcknowledgementsB Bracci and M Gazzano, from the ISOF-CNR of Bologna, Italy, are gratefully acknowledged for SEM ana lysis. V Pistarà, at the Department of Chemical Sciences, University of Catania, Italy, is gratefully acknowledged for assistance in NMR ana lysis.

Financial & competing interests disclosureThis research was supported by a grant from the Associazione Italiana per la Ricerca sul Cancro (AIRC): ‘Targeting skel-etal metastases by nanoscale multifunctional bone-seeking agents’. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

ethical conduct of research The authors state that they have obtained appropriate insti-tutional review board approval or have followed the princi-ples outlined in the Declaration of Helsinki for all human or animal experimental investigations.

executive summary

Osteotropic drug-delivery systems can represent a strategy of imparting affinity to bone tissues to �drugs. Usually, osteotropic prodrugs have been obtained by linking the active molecules to bone-seeking agents, such as bisphosphonates.In this study, we merged the approaches of osteotropic prodrugs and nanoparticle drug delivery by �producing a bone-seeking polymer conjugate of poly(lactic-co-glycolide) (PLGA) with alendronate (ALE).The copolymer showed complete biocompatibility � in vitro and was not cytotoxic to endothelial cells and trabecular osteoblasts.Small nanoparticles were produced easily from the PLGA–ALE conjugate. These nanoparticles �showed affinity in vitro towards hydroxyapatite.The overall experimental results prompt us to further investigate this novel biomaterial, by producing �drug-loaded nanoparticles with a potential use in the targeted therapy of bone disorder-related diseases and cancer.

nanoparticles allow gene delivery into brain tumors via intravenous administration. Cancer Res. 66, 11878–11887 (2006).

Mitra A, Nan A, Line BR, Ghandehari H: 2

Nanocarriers for nuclear imaging radiotherapy of cancer. Curr. Pharm. Des. 12, 4729–4749 (1995).

Gref R, Minamitake Y, Peracchia MT, 3

Trubetskoy V, Torchilin V, Langer R: Biodegradable long-circulating polymeric nanospheres. Science 263, 1600–1603 (1994).

Zimmer A, Kreuter J: Microspheres and 4

nanoparticles used in ocular delivery systems. Adv. Drug Deliv. Rev. 16, 61–73 (1995).

BibliographyPapers of special note have been highlighted as:� of interest�� of considerable interest

Lu W, Sun Q, Wan J, She Z, Jiang XG: 1

Cationic albumin-conjugated pegylated

Nanomedicine (2009) 4(2)174 future science group

ReseaRch aRticle Pignatello, Cenni, Micieli et al.

Langer R: Drug delivery and targeting. 5

Nature 392, 5–10 (1998).

Bala I, Hariharan S, Ravi Kumar MNV: 6

PLGA nanoparticles in drug delivery: the state of the art. Crit. Rev. Ther. Drug Carrier Syst. 2, 387–422 (2004).

Okada H: One and three-month release 7

injectable microspheres of LH-RH superagonist leuprorelin acetate. Adv. Drug Deliv. Rev. 28, 43–70 (1997).

Crivellari D, Pagani O, Veronesi A 8 et al.: International Breast Cancer Study Group. High incidence of central nervous system involvement in patients with metastatic or locally advanced breast cancer treated with epirubicin and docetaxel. Ann. Oncol. 12, 353–356 (2001).

Greish K: Enhanced permeability and 9

retention of macromolecular drugs in solid tumors: a royal gate for targeted anticancer nanomedicines. J. Drug Target. 15, 457–464 (2007).

Hirabayashi H, Fujisaki J: Bone-specific drug 10

delivery systems: approaches via chemical modification of bone-seeking agents. Clin. Pharmacokinet. 42, 1319–1330 (2003).

Represents one of the first descriptions ���

of an osteotropic drug-delivery system approach.

Wang D, Miller SC, Kopeckova P, Kopecek J: 11

Bone-targeting macromolecular therapeutics. Adv. Drug Del. Rev. 57, 1049–1076 (2005).

Describes the initial approaches to ���

multifunctional osteotropic polymers, a research line still investigated by this research group.

Niemi R, Vepsalainen J, Taipale H, 12

Jarvinen T: Bisphosphonate prodrugs: synthesis and in vitro evaluation of novel acyloxyalkyl esters of clodronic acid. J. Med. Chem. 42, 5053–5058 (1999).

Ezra A, Hoffman A, Breuer E 13 et al.: A peptide prodrug approach for improving bisphosphonate oral absorption. J. Med. Chem. 43, 3641–3652 (2000).

Hirabayashi H, Takahashi T, Fujisaki J 14

et al.: Bone-specific delivery and sustained release of diclofenac, a non-steroidal anti-inflammatory drug, via bisphosphonic prodrug based on the Osteotropic Drug Delivery System (ODDS). J. Control. Rel. 70, 183–191 (2001).Represents one of the first descriptions ���

of an osteotropic drug-delivery system approach.

Wright JE, Gittens SA, Bansal G 15 et al.: A comparison of mineral affinity of bisphosphonate–protein conjugates

constructed with disulfide and thioether linkages. Biomaterials 27, 769–784 (2006).

Gittens SA, Bansal G, Zernicke RF, 16

Uludag H: Designing proteins for bone targeting. Adv. Drug Deliv. Rev. 57, 1011–1036 (2005).

Ogawa K, Mukai T, Inoue Y, Ono M, Saji H: 17

Development of a novel 99mTc-chelate conjugated bisphosphonate with high affinity for bone as a bone scintigraphic agent. J. Nucl. Med. 47, 2042–2047 (2006).

Barrett J, Worth E, Bauss F, Epstein S: 18

Ibandronate: a clinical pharmacological and pharmacokinetic update. J. Clin. Pharmacol. 44, 951–965 (2004).

Hengst V, Oussoren C, Kissel T, Storm G: 19

Bone targeting potential of bisphosphonate targeted liposomes. Preparation, characterization and hydroxyapatite binding in vitro. Int. J. Pharm. 331, 224–227 (2007).

One of the more recent reports on ��

osteotropic drug-delivery systems.

Von Knoch F, Jaquiery C, Kowalsky M 20 et al.: Effects of bisphosphonates on proliferation and osteoblast differentiation of human bone marrow stromal cells. Biomaterials 26, 6941–6949 (2005).

Tosi G, Rivasi F, Gandolfi F, Costantino L, 21

Vandelli MA, Forni F: Conjugated poly(d,l-co-glycolide) for the preparation of in vivo detectable nanoparticles. Biomaterials 26, 4189–4195 (2005).

Jaffe EA, Nachman RL, Becker CG, 22

Minick CR: Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunological criteria. J. Clin. Invest. 52, 2745–2756 (1973).

Di Silvio L, Gurav N: 23 Osteoblasts. Human Cell Culture (Volume V, Chapter 11). Kluwer, UK (2001).

Choi SW, Kim JH: Design of surface-24

modified poly(d,l-lactide-co-glycolide) nanoparticles for targeted drug delivery to bone. J. Control. Rel. 122, 24–30 (2007).

Noel TR, Parker R, Ring SG, Tatham AS: 25

The glass-transition behaviour of wheat gluten proteins. Int. J. Biol. Macromol. 17, 81–85 (1995).

Petrie SEB: Thermal behavior of annealed 26

glasses. J. Polym. Sci. 10, 1255–1272 (1972).

McKenna GB: 27 Comprehensive Polymer Science (Volume 1). Pergamon Press, Oxford, UK (1988).

Astete CE, Sabliov CM: Synthesis and 28

characterization of PLGA nanoparticles. J. Biomater. Sci. Polym. Ed. 17, 247–289 (2006).

Bala I, Hariharan S, Kumar MN: PLGA 29

nanoparticles in drug delivery: the state of the art. Crit. Rev. Ther. Drug Carrier Syst. 21, 387–422 (2004).

Costantino L, Gandolfi F, Tosi G, Rivasi F, 30

Vandelli MA, Forni F: Peptide-derivatized biodegradable nanoparticles able to cross the blood-brain barrier. J. Control. Rel. 108, 84–96 (2005).

Cutright DE, Perez B, Beasley JD 3rd, 31

Larson WJ, Posey WR: Degradation rates of polymers and copolymers of polylactic and polyglycolic acids. Oral Surg. Oral Med. Oral Pathol. 37, 142–152 (1974).

Lin JH: Bisphosphonates: a review of their 32

pharmacokinetic properties. Bone 18, 75–85 (1996).

Liu X-M, Wiswall AT, Rutledge JE 33 et al.: Osteotropic β-cyclodextrin for local bone regeneration. Biomaterials 29, 1686–1692 (2008).

One of the more recent reports on ��

osteotropic drug-delivery systems.

Blanco-Prieto MJ, Campanero MA, 34

Besseghir K, Heimgatner F, Gander B: Importance of single or blended polymer types for controlled in vitro release and plasma levels of a somatostatin analogue entrapped in PLA/PLGA microspheres. J. Control. Rel. 96, 437–448 (2004).

Wang D, Miller S, Sima M, Kopeckova P, 35

Kopecek J: Synthesis and evaluation of water soluble polymeric bone-targeted drug delivery systems. Bioconj. Chem. 14, 853–859 (2003).

Describes the initial approaches to ��

multifunctional osteotropic polymers, a research line still investigated by this research group.

Rodriguez-Lorenzo V, Fernandez M, Parra J, 36

Vasquez B, Lopez-Bravo A, San Roman J: Novel N-bisphosphonate based drug delivery systems: synthesis and preparation of targeting vehicles. Presented at: 20th European Conference on Biomaterials. Nantes, France, 27 September–1 October 2006.

Hansen MB, Nielsen SE, Berg K: 37

Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. J. Immunol. Methods 119, 203–210 (1989).

Low SP, Williams KA, Canham LT, 38

Voelcker NH: Evaluation of mammalian cell adhesion on surface-modified porous silicon. Biomaterials 27, 4538–4546 (2006).

www.futuremedicine.com 175future science group

PLGA–ALE conjugate for osteotropic nanocarriers ReseaRch aRticle

Sousa SR, Lamghari M, Sampaio P, 39

Moradas-Ferreira P, Barbosa MA: Osteoblast adhesion and morphology on TiO2 depends on the competitive preadsorption of albumin and fibronectin. J. Biomed. Mater. Res. A 84, 281–290 (2008).

Liu XM, Lee HT, Reinhardt RA, Marky LA, 40

Wang D: Novel biomineral-binding cyclodextrins for controlled drug delivery in the oral cavity. J. Control. Release 122, 54–62 (2007).

One of the more recent reports on ��

osteotropic drug delivery systems.

Nalwa HS, Webster T (Eds): 41 Cancer nanotechnology – Nanomaterials for cancer diagnosis and therapy. American Scientific Publishers, Valencia, CA, USA (2006).

Bondì ML, Craparo EF, Giammona G 42 et al.: Nanostructured lipid carriers-containing anticancer compounds: preparation, characterization, and cytotoxicity studies. Drug Deliv. 14, 61–67 (2007).

Pridgen EM, Langer R, Farokhzad OC: 43

Biodegradable, polymeric nanoparticle delivery systems for cancer therapy. Nanomed. 2, 669–680 (2007).

Interesting review on innovative anticancer ��

therapy strategies.