Patient selection and purification of lymphocytes
Blood was obtained from healthy donors or CLL patients who had provided written informed consent. The laboratory study was approved by the Mayo Clinic Institutional Review Board according to the regulations of the Declaration of Helsinki. All CLL patients had a confirmed diagnosis using the NCI working group definition [36
]. Patients in this cohort were from all Rai stages and had not been treated for at least 6 weeks prior to blood processing for this study. Peripheral blood mononuclear cells (PBMC) were separated from heparinized venous blood by density gradient centrifugation. To remove adherent cells, PBMC were suspended in RPMI 1640 supplemented with 10% fetal calf serum (FCS) and incubated in plastic dishes at 37.1°C for 1 h prior to collection of non-adherent cells. To obtain at least 95% purity of CLL B cells, non-adherent cells were depleted of T cells by incubation with sheep erythrocytes. In addition, we also purified CLL B cells from magnetic bead columns. To do this, highly purified CD19+
B cells (≥95%) were obtained from PBMC by standard negative selection using a cocktail of subset-specific antibodies conjugated with magnetic beads (Miltenyi Biotech, Auburn, CA, USA). These purified CLL B cells were then either used immediately for the laboratory studies described below or cryopreserved in RPMI 1640, 20% FCS, and 10% DMSO and stored in liquid nitrogen until use.
Primary CLL B cells and human splenic B cells obtained from controls were sorted by CD 19 antibody conjugated to magnetic beads (Miltenyi Biotec, Auburn, CA, USA) and were then cultured in serumfree AIM-V (Gibco BRL, USA) and RPMI (Biomol, USA) supplemented with 10% FCS, respectively. Cells were maintained at 37°C in an atmosphere containing 95% air-5% CO2 (v/v).
Immunological reagents that recognize the following antigens were purchased from the indicated suppliers: Mcl-1 from BD Pharmingen (San Diego, CA, USA); Bcl-2 from Dako Corp (Carpinteria, CA, USA); XIAP from R&D Systems, Inc. (Minneapolis, MN, USA); β-actin from Novus Biologicals (Littleton, CO, USA). VEGF neutralizing antibody (2C3) was a kind gift from Pergerine Pharmaceuticals. The antibody (2C3) specifically blocks the interaction between VEGF and VEGFR-2 [37
Synthesis and characterization of gold nanoparticles and its nanoconjugates
Gold nanoparticles were synthesized according to standard wet chemical methods using sodium borohydride as a reducing agent [11
]. Characteristic surface plasmon resonance (SPR) band of gold nanoparticles was observed in the UV-Visible spectrum, confirming the presence of spherical gold nanoparticles (Fig. ). TEM micrographs showed spherical gold nanoparticles of approximately 5 nm were formed by this method (Fig. ). The size distribution analysis of gold nanoparticles (after counting 500 individual particles) clearly shows that most of the gold nanoparticles (~90%) are in 4–5 nm range (Fig ).
Binding of VEGF antibody to gold nanoparticles was done according to our previously published report [13
]. In brief, 150 ml of gold nanoparticles were incubated with 600 μg of AbVF for 1 h at room temperature under stirring. After 1 h, gold conjugates thus obtained were ultracentrifuged at 25000 rpm at 10°C for 1 h. The loose pellet obtained at the bottom was collected and UV-irradiated for 20–30 minutes before use in apoptosis assays. The binding was monitored using UV-Visible spectroscopy. UV-Visible spectrum was recorded on a Shimadzu model system (UV2401 PC) and the saturation concentration was determined.
Quantitation of AbVF in the nanoconjugates using thermogravimetric analysis (TGA)
For TGA analysis, 150 ml of gold nanoparticles were incubated with 600 μg of AbVF for 1 h at room temperature under stirring. After 1 h, gold conjugates were centrifuged at 25000 rpm at 10°C for 1 h. Both the supernatant and the loose pellet at the bottom were collected. The loose pellet obtained after ultracentrifugation was used for the quantification of AbVF attached on gold nanoparticles and also for apoptosis studies with CLL B cells described below. For TGA analysis, the pellet obtained after ultracentrifugation was lyophilized to obtain gold nanoconjugates in powder form. The lyophilized powder of nanoconjugates was used for TGA analysis. TGA was done using a TA Instruments Q500 thermal analyst. The TGA data were obtained in flowing nitrogen at a heating rate of 20°C/min. Amount of drugs attached onto gold nanoparticles were obtained from weight loss from the TGA curve as previously described [10
Studying nature of bonding by X-ray photoelectron spectroscopy
X-ray photoelectron spectroscopy (XPS) was performed to find out the nature of bonding between gold nanoparticles and the antibody. XPS was obtained on a PHI 5400 instrument using a Mg Kα Xray (1253.6 eV) anode source operated at 250 W, pressure was below 2 × 10-9
torr. The electron pass energy on the hemispherical analyzer was set at 89.45 eV for survey scans and 17.9 eV for highresolution scans. The binding energy scale was referenced to that of C1s (285.0 eV). Samples were prepared by drop-coating anti-VEGF165 antibody conjugated gold solution on a clean silicon wafer and the drops were allowed to air dry [38
Studying apoptosis in CLL-B cells isolated from patients
To compare gold nanoconjugates containing AbVF (Au-AbVF) to AbVF alone, a humanized anti-VEGF-A monoclonal antibody 2C3 (Pergerine Pharma) was co-cultured with purified CLL B primary cells. In brief, CLL B cells (1 × 106
) were co-cultured with increasing concentrations of 2C3 conjugated to gold as well as 2C3 and gold (1 μg–25 μg/ml) alone as controls for 24–72 hours. Annexin/PI flow cytometry was done on those cells according to our prior procedures [3
]. We wish to indicate here that all the experiments reported here were repeated in triplicate.
To detect alterations of apoptotic proteins and confirm apoptosis we used immunoblot analysis. Primary CLL B cells were cultured in six-well tissue culture dishes and treated with either vehicle (control) or Au alone, AbVF, Au-AbVF for 24 h. The treated cells were then washed three times with calcium, magnesium-free Dulbecco's phosphate-buffered saline (PBS) and solubilized in alkylation buffer (6 M guanidine hydrochloride, 250 mM Tris-HCl, pH 7.4 at 21.1°C, and 10 mM EDTA supplemented immediately before use with 150 mM 2-mecaptoethanol and 1 mM phenylmethylsulfonyl fluoride). Samples were then dialyzed into 4 M urea and then 0.1% SDS. Protein extracts were then separated on 5–15% SDS-PAGE acrylamide gels and transferred to nitrocellulose membrane and blocked with 10% Tris-saline milk (TSM). Relevant primary antibodies were diluted in the 10% TSM milk and incubated overnight on the shaker at room temperature. After three extensive washing with 1 × PBS containing 0.05% Tween 20 for 15 min, the blot was then incubated with horseradish peroxidase-conjugated secondary antibody (KPL Inc., Gaithersburg, MD, USA) diluted 1:8000 in 10% TSM for 1 h at room temperature. After washing with 1× PBS containing 0.05% Tween 20 for another 30 min, bound antibodies were detected by ECL chemiluminescence (Amersham Pharmacia, Piscataway, NJ, USA).
Transmission electron microscopy
TEM sample preparation involving cells, however, was performed by treating cells with gold nanoparticles and gold nanoconjugates for 1 h with under serum-free conditions. After the incubation, CLL-B cells were centrifuged initially at 2500 rpm for 6 min. The resultant cell pellets were then washed thrice with PBS, and fixed in Trump's fixative (1% glutaraldehyde and 4% formaldehyde in 0.1 M phosphate buffer, pH 7.2). Both cell types were then rinsed for 30 min in 3 changes of 0.1 M phosphate buffer, pH 7.2, followed by a 1 hr postfix in phosphate-buffered 1% OsO4. After rinsing in 3 changes of distilled water for 30 min, the tissue was en bloc stained with 2% uranyl acetate for 30 min at 60°C. The cell was then rinsed in three changes of distilled water, dehydrated in progressively higher concentrations of ethanol and 100% propylene oxide, and embedded in Spurr's resin. Thin (90 nm) sections were cut on a Reichert Ultracut E ultramicrotome, placed on 200 mesh copper grids, and stained with lead citrate. Micrographs were taken on a TECNAI 12 operating at 120 KV.