4.1 Materials
Amine-terminated G3 PAMAM dendrimer (G3-NH2 1) was synthesized and characterized at the Michigan Nanotechnology Institute for Medicine and Biological Science at the University of Michigan. All other chemicals were purchased from Sigma-Aldrich and used as received, unless otherwise specified. Water used in all experiments was purified by a Milli-Q Plus 185 water purification system (Millipore, Bedford, MA). The Spectra Por dialysis membranes (MWCO 3500) and phosphate buffer saline were acquired from Fisher.
4.2 Nuclear Magnetic Resonance Spectrometry (NMR)
1H NMR spectra were recorded on a 400 MHz Varian Inova 400 nuclear magnetic spectrometer in dimethyl sulfoxide (DMSO-d6).
4.3 Matrix-Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) Mass Spectrometry
MALDI-TOF mass spectra were recorded on a Waters TofSpec-2E spectrometer (Beverly, MA, USA), running in linear mode with the high mass PAD detector, and 2,5-dihydroxybenzoic acid (DHB) in acetonitrile/water (50:50, v/v) was used as the matrix. The instrument was calibrated with bovine serum albumin (BSA, Mw = 66.43 × 103) in sinapic acid. The data were acquired and processed by MassLynx 3.5 software.
4.4 High-Performance Liquid Chromatography (HPLC)
A reverse-phase HPLC instrument consisting of a System GOLD 126 solvent module, a Model 507 auto-sampler equipped with a 100 μL loop and a Model 166 UV detector (Beckman Coulter, Fullerton, CA, USA), and a Phenomenex (Torrance, CA, USA) Jupiter C5 silica-based HPLC column (250 × 4.6 mm, 300 Å) were used for analysis of the products in this work. The mobile phase for elution of PAMAM dendrimer products was a linear gradient beginning with 100:0 water/acetonitrile (ACN) (both containing 0.14 wt% TFA) at a flow rate of 1 mL/min., reaching 20:80 (or 50:50) within 35 min. Trifluoroacetic acid (TFA) (0.14 wt% in both water and ACN) was used as a counterion to make the dendrimer-conjugate surface hydrophobic. All samples were dissolved in the aqueous mobile phase (water containing 0.14% trifluoroacetic acid (TFA)) at a flow rate of 1 mL/min, reaching 20:80 (or 50:50) within 35 min. The injection volume in each case was 35 μL with a sample concentration of 1 mg/mL, and the detection wavelength was 210 or 285 nm. The analysis was performed using Beckman’s System GOLD Nouveau software.
4.5 Gel Permeation Chromatography (GPC)
GPC was used to evaluate the molecular weights and molecular weight distribution of the G3-PAMAM dendrimers and conjugates. GPC experiments were performed on an Alliance Waters 2690 separation module (waters Corp., Milford, MA) equipped with a Waters 2487 dual wavelength UV absorbance detector (Waters Corp.), a Wyatt Dawn HELEOS light scattering detector (Wyatt Technology Corp., Santa Barbara, CA), an Optilab rEX differential refractometer (Wyatt Technology Corp.), and TosoHaas TSK-Gel Guard PHW 06762 (75 × 7.5 mm, 12 μm), G 2000 PW 05761 (300 × 7.5 mm, 10 μm), G 3000 PW 05762 (300 × 7.5 mm, 10 μm), and G 4000 PW (300 × 7.5 mm, 17 μm) columns. Column temperature was maintained at 25 ± 0.2°C with a Waters temperature control module. Citric acid buffer (0.1 M) with 0.025% sodium azide in water, pH 2.74, was used as a mobile phase at a flow rate of 1 mL/min. The sample concentration was 2 mg/mL, with an injection volume of 100 μL. The number average of molecular weight, Mn, weight average molecular weight Mw and polydispersity index (PDI) were calculated using ASTRA 5.34.14 software (Wyatt Technology Corp.).
4.6 Synthesis of D-Glucoheptononyl-terminated G3 PAMAM Dendrimer (G3-OH) 3
G3-NH2 1 (691 mg, 0.10 mmol) was dissolved in 10 mL of DMSO in a 25 mL flask. To the solution was added D-glucoheptono-1,4-lactone (799 mg, 3.84 mmol). The mixture was stirred at room temperature under nitrogen overnight and then 40 mL of cold water was added. The product was purified by dialysis against isotonic phosphate-buffered saline (PBS) buffer (3 × 4L) and then water (3 × 4L) with a cellulose dialysis membrane (MWCO = 3500) over 48 hours. The product was dried by lyophilization to yield 3 as a white solid (1.30 g, 96%).
4.7 Synthesis of Conjugate of MTX and D-Glucoheptononyl-terminated G3 PAMAM Dendrimer (G3-MTX) 4
A solution of G3-OH 3 (100 mg, 7.37 μmol) and MTX (33.5 mg, 73.7 μmol) in DMSO (15 mL) was stirred at room temperature under nitrogen. To this solution were added 2-chloro-1-methylpyridinium iodide (23.3 mg, 88.4 μmol) and 4-(dimethylamino)pyridine (21.6 mg, 177 μmol). The mixture was further stirred overnight and then diluted with cold water (55 mL). The product was purified by dialysis against PBS buffer (3 × 4L) and then water (3 × 4L) with a cellulose dialysis membrane (MWCO = 3500) over 48 hours. The final product was dried by lyophilization to yield 4 as a yellow solid (110 mg, 82%).
4.8 Synthesis of Conjugate of FI and D-Glucoheptononyl-terminated G3 PAMAM Dendrimer (G3-FI) 5
A solution of G3-OH 3 (100 mg, 7.37 μmol) and FI (14.4 mg, 29.4 μmol) in DMSO (15 mL) was stirred at room temperature under nitrogen. To the solution was added 2-chloro-1-methylpyridinium iodide (11.3 mg, 44.1 μmol) and 4-(dimethylamino)pyridine (10.8 mg, 88.2 μmol). The mixture was further stirred overnight and then diluted with cold water (65 mL). The product was purified by dialysis against PBS buffer (3 × 4L) and then water (3 × 4L) with cellulose dialysis membrane (MWCO = 3500) over 48 hours. The final product was dried by lyophilization to yield 5 as an orange solid (88 mg, 77%).
4.9 Synthesis of Conjugate of FI, MTX, and D-Glucoheptononyl-terminated G3 PAMAM Dendrimer (G3-MTX-FI) 6
A solution of G3-MTX 4 (50 mg, 3.5 μmol) and FI (5.1 mg, 10.5 μmol) in DMSO (5 mL) was stirred at room temperature under nitrogen. To the solution was added 2-chloro-1-methylpyridinium iodide (3.2 mg, 12.6 μmol) and 4-(dimethylamino)pyridine (3.1 mg, 25.2 μmol). The mixture was further stirred overnight and then diluted with cold water (20 mL). The product was purified by dialysis against PBS buffer (3 × 4L) and then water (3 × 4L) with a cellulose dialysis membrane (MWCO = 10000) over 48 hours. The final product was dried by lyophilization to yield 6 as an orange solid (46 mg, 84%).
4.10 Molecular Dynamics (MD) Simulations
All the PAMAM dendrimers were built using the Insight II software package (Accelrys Inc., San Diego, CA). All the primary amines of G3 PAMAM dendrimer were protonated to simulate the dendrimer at pH 7. The simulation of G3 PAMAM dendrimer in implicit solvent was previously published.
32 MD simulations on the dendrimers were carried out using the CHARMM program Version 35
33 and the CHARMM22 all-atom topology and parameters.
34 After steepest descent minimization for 10,000 steps followed by adopted basis Newton-Raphson minimization process for 10,000 steps to obtain lower energy configurations, we further annealed the minimized dendrimer structures at very high temperature and cooled down to room temperature. We carried out equilibration on the systems for 200 ps and generated dynamics rum for 1 ns at 300 K. The total potential energy function (U
total) for MD simulations is described as
where ε is the minimum energy of the Lennard-Jones potential, σ the distance yielding a minimum Lennard-Jones potential,
q the partial charge on the atom, D the dielectric constant, r the distance between
i and
j, and
i,
j are nonbonded atom pairs. We used the dielectric constant D = r without a long-range nonbonded cut-off in the simulations.
The sizes of the simulated dendrimers have been estimated from the radius of gyration, Rg (Å), which is defined as
for a dendrimer composed of
N atoms where
M is the dendrimer’s total mass,
mi and
xi are the mass and position of the
ith atom, and
x0 is center-of-mass of the dendrimer.
4.11 Surface Plasmon Resonance (SPR) Measurements
SPR experiments were performed in Biacore® X (GE healthcare, Piscataway, USA) using a method similar to that which has been previously reported.
10 Folate-binding protein (FBP, bovine milk) was immobilized on the surface of a CM5 sensor chip via protein conjugation chemistry to a carboxymethylated dextran-coated layer by a standard amide coupling protocol. Briefly, the process of chip preparation included EDC/NHS activation at 0.4M/0.4M for 10 min., followed by 10 min. of protein immobilization and 10 min. of deactivation by 1M ethanolamine. The immobilization process of FBP on channel 2 resulted in a 13,000 response unit (RU) equivalent to 13 ng/mm
2. SPR signals for FBP binding were obtained by injection of each ligand dissolved in HBS-EP buffer at a flow rate of 30 μL/min. After each measurement, the surface of the chip was regenerated by injection of 5 μL of 10 mM glycine-HCl (pH 2.5).
The measured SPR sensorgrams were obtained by subtracting SPR signals from channel 2, the FBP immobilized flow channel, from SPR signals collected from channel 1, the reference flow channel. (ΔRU = RU2 − RU1). Kinetic binding parameters, the rate of association (
kon), and the rate of dissociation (
koff) were extracted by fitting each binding curve separately using BIA evaluation software with the Langmuir kinetic model and analyzed (dR/dt vs. R) following the method described by Glaser.
35 Dissociation constants (
KD =
koff/
kon) for each ligand were the mean of
KD at three different concentration of measurement in which chi square (χ
2) value of each fitting is lower than 3.
4.12 Measurement of Cellular Binding and Cytotoxicity
KB cells, a sub-line of the cervical carcinoma HeLa cells (ATCC, Manassas, VA, USA), were grown as a monolayer cell culture at 37°C and 5% CO
2 in FA-deficient RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS). The 10% FBS provided an FA concentration equivalent to that present in the human serum (~20 nM). For assessment of the cellular binding, KB cells plated in 24-well plates were treated with different concentrations of the conjugates for the indicated time periods on FA-free medium in the absence of serum. The cells were incubated at 37°C, rinsed, and the mean FL1 fluorescence of 10,000 cells was determined by flow cytometry, as described previously.
17 For the cytotoxicity experiments, the cells were seeded in 96-well microtiter plates (3000 cells/well) in medium containing dialyzed serum. Two days after plating, the cells were treated with the dendrimer conjugates in tissue culture medium under the indicated conditions. A colorimetric ‘XTT’ (sodium 3-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate) assay (Roche Molecular Biochemicals, Indianapolis, IN) was performed following the vendor’s protocol. After incubation with the XTT labeling mixture, the microtiter plates were read on an ELISA reader (Synergy HT, BioTek) at 492 nm with the reference wavelength at 690 nm.
11
The publisher's final edited version of this article is available at 
![[diamond]](/corehtml/pmc/pmcents/x25C6.gif)
) and FA + G3-FI-MTX 6 (□) on KB cells. The cells were maintained in FA-free medium and incubated with different concentrations of the indicated conjugates for 2 hours. The cells
![[diamond with plus]](/corehtml/pmc/pmcents/x25C7.gif)
), G3-MTX3.4 (■), G3-MTX5.2 (![[big up triangle, open]](/corehtml/pmc/pmcents/x25B3.gif)
), and G5-FA5-MTX7 (●), and MTX (○). KB cells plated in 96-well plates were incubated with different concentrations of the indicated dendrimers and