Materials and Methods
Poly(D,L-lactide-co-glycolide), 50:50, containing 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 A. Sodium 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 dissolving 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 carbodiimide 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 vacuum 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 B. Resomer RG 502 H was activated previously with N-hydroxysuccinimide (NHS) according to the literature. 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 filtration (0.45 µM syringe nylon-membrane filter) 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.
Characterization of the PLGA-ALE Conjugate
1H-NMR analysis. Spectra 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 Analysis. An Ultraflex II instrument (Bruker Daltonics, Bremen, Germany) equipped with a UV nitrogen laser (337 nm) was used. Matrix solution was prepared by dissolving 2,5-dihydroxybenzoic acid (DHB) in water/tetrahydrofuran (30:70, v/v). The matrix:sample molar ratio was 1000:1. Spectra were obtained in reflection-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 Calorimetry. Differential-scanning calorimetry (DSC) measurements 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 physical mixture was prepared for these experiments: solid PLGA and ALE (free acid form) were triturated 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 calorimetric system was calibrated, in transition temperature 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 Assay
In the biocompatibility tests, PLGA-ALE conjugate was dissolved in DMSO at the concentration of 52.45 mg/ml. From this solution, scalar dilutions in phosphate-buffered saline (PBS), plasma or culture medium were tested. As controls, DMSO alone, at the same dilutions, was assessed. All the experiments of blood compatibility were performed using human venous blood from healthy volunteer donors, which was supplied by a Blood Bank. Hemolysis was evaluated on venous blood anticoagulated with 119 IU of lithium heparinate. Within 2 h, the erythrocytes 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 spectrophotometric determination at 540 nm. The samples were tested in triplicate.
To obtain 0 and 100% hemolysis, the erythrocyte suspension was added to PBS and distilled water, respectively. The degree of hemolysis was determined by the following equation:
where Abs, Abs0 and Abs100 are the absorbance 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 Coagulation
Venous 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 centrifugation 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).
Culture of Endothelial Cells
Human umbilical vein endothelial cells (HUVECs) were harvested according to the method of Jaffe et al., after informed consensus. 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 penicillin (Invitrogen, Carlsbad, CA, USA) and 100 µg/ml of streptomycin (Invitrogen) under an atmosphere consisting of 95% O2 and 5% CO2. Initially, the cells were seeded on a 25 cm2 flask 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 and Culture
The 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 from patients undergoing 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.
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 solution). The working solution was prepared immediately 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 dilutions 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 incubated 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 supernatant 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 negative control. The sample was considered as toxic if the viability percentage was less than 80%.
Two alternate methods were used to obtain nanoparticles 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. 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 preparations was measured by photon-correlation spectroscopy (PCS) with a Zetamaster (Malvern Instruments, UK), equipped with the Malvern PCS software. The dispersions were diluted appropriately with proinjection water before the analysis 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 calculated by the instrument software, using the Smoluchowsky's equation.
A morphological investigation of nanoparticles 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.
Sterilization of Nanoparticles
The nanoparticle suspensions were aliquoted into glass vials and submitted directly to γ-irradiation, at a strength of 10 kGy by a 60 Co source (Gammatom srl, Guanzate, Como, Italy).
HA Affinity Assay of PLGA-ALE Nanoparticles
PLGA-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 lipophilic 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 temperature. Each experiment was made in quadruplicate. The whole suspension was filtered through a 1.2 µm pore-size nylon-syringe filter to separate 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 pellets 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 analysis 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 conditions. 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 analysis.
Statistical analysis was performed with the StatView™ 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.
Nanomedicine. 2009;4(2):161-175. © 2009 Future Medicine Ltd.
Cite this: A Novel Biomaterial for Osteotropic Drug Nanocarriers: Synthesis and Biocompatibility Evaluation of a PLGA--ALE Conjugate - Medscape - Feb 01, 2009.