Methods

Initially, 3 g of commercially available ACNPs (200 nm) were suspended in sterile distilled water (2 L), and the mixed ACNP suspension was precipitated at room temperature for 72 hours. The supernatant was collected and filtered successively with 0.25 and 0.1 μm microporous membranes, and ACNPs with a particle size of 100–150 nm were obtained. The purified ACNPs were lyophilized and stored at −20°C.

Purified ACNPs 100 mg were activated at 100°C for 2 hours and suspended in triple-distilled water. Then, 50 mg of DOX were added to the ACNP suspension using magnetic stirring for 30 minutes at room temperature in phosphate-buffered saline (PBS), pH 7.4. The solution was bathed in ice in an intermittent ultrasound field for 4 hours. The suspension was centrifuged at 16,000 rpm for 20 minutes. The ACNP-DOX precipitates were collected, resuspended, and centrifuged to remove unbound DOX. The supernatants were collected, and the DOX concentration was measured in an ultraviolet (UV) spectrometer. The DL coefficient and the encapsulation efficiency were calculated from the DOX concentration in the supernatant, using formulae (1) and (2), as given below.33 The ACNP-DOX was freeze-dried and stored at −20°C until required for further use.

DSPE-PEG2000 and DSPE-PEG2000-NH₂ (5:1, w/w) were mixed and dissolved in organic solvent (chloroform: methanol =4:1 v/v). The prepared ACNP-DOX/ACNP was added to the DSPE-PEG2000 organic solution and mixed ultrasonically (DSPE-PEG2000:DSPE-PEG2000-NH₂: ACNP/ACNP-DOX =5:1:2). The organic solvent was evaporated using a rotary evaporator at 30°C. Following evaporation, the walls of the container were lined by the phospholipid complex. The ACNP-DOX-DSPE-PEG2000-NH₂ was redispersed in PBS using ultrasonic mixing. The final concentration of ACNP-DOX in ACNP-DOX-DSPE-PEG2000-NH₂ was 1 mg/mL, and the corresponding final concentration of DOX was 250 μg/mL.

Preparation of ACNP-DOX-DSPE-PEG2000-NH₂ conjugated anti-CD20.

Abbreviations: ACNP, active carbon nanoparticles; DOX, doxorubicin; DSPE, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine; EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; NHS, N-hydroxysuccinimide; PEG, polyethylene glycol.

For the covalent attachment of anti-CD20 antibodies onto the nanoparticle surface, EDC and NHS reagents were used. EDC·HCl (1 mg/mL) was added to 5 mL of the prepared ACNP-DOX-DSPE-PEG2000-NH₂ or ACNP-DSPE-PEG2000-NH₂ and 200 μL of anti-CD20 antibody (10 mg/mL). Following gentle mixing for 30 minutes at room temperature, the pH of the reaction solution was adjusted to 8.5 with NaOH solution. Next, 1.7 mg of NHS was added to the reaction solution and the solution was stirred for 2 hours at room temperature. The solution was dialyzed for 12 hours in PBS (pH 7.4) using a 100 kDa dialysis bag for 12 hours, to obtain the final product, which was the monoclonal antibody targeted NDDS, ACNP-DOX-DSPE-PEG2000-anti-CD20.

For each complex, 5 mg of ACNP-DOX, ACNP-DOX-DSPE PEG2000 and ACNP-DOX-DSPE-PEG2000-anti-CD20 were each dispersed in 2 mL of PBS at pH 7.4 and pH 5.5 and transferred to disposable 3,500 kDa molecular weight cut-off dialysis bags, which were sealed and then immersed in 20 mL of release medium with vortex mixing at 100 rpm at 37°C. A 1 mL volume of release medium was taken at predetermined time intervals, followed by immediately supplementing an equal volume of fresh medium. The concentration of released DOX in the medium was determined by UV–visible spectroscopy, and the amount of released DOX (W) was calculated using the formula:

where V was the volume of medium and C was the DOX concentration in the medium.

The cumulative release rate (RR, %) was calculated using the formula:

where W_i is the measured amount of DOX in the release medium at the given time point, and W_t is the total DOX amount in the equal volume of ACNP suspensions before dialysis. Each experiment was repeated in triplicate.34

The particle size, polydispersity index, and zeta potential values were measured using Nano Series Zen 4003 Zetasizer (Malvern Instruments Ltd).33 The suspension stability of ACNP, ACNP-DOX, ACNP-DSPE-PEG2000, ACNP-DOX-DSPE-PEG2000 and the NDDS, ACNP-DOX-DSPE-PEG2000-anti-CD20 in PBS (10 mM, pH 7.4) were observed by mixing 1 mg of corresponding preparation in 2 mL of PBS, followed by sonication for 30 minutes at room temperature.

The stability of the compound was evaluated after 1 month. The size and morphology of the ACNP-DOX, ACNP-DSPE-PEG2000, ACNP-DOX-DSPE-PEG2000 and the NDDS, ACNP-DOX-DSPE-PEG2000-anti-CD20 were evaluated using transmission electron microscopy (TEM) (Hitachi H7650, Hitachi Ltd., Tokyo, Japan). Each 0.1 mg/mL of sample was resuspended in water and mixed by sonication for 30 seconds. One drop of this suspension was placed on a carbon-coated copper TEM grid (200 mesh, Sangerbio, People’s Republic of China) and allowed to dry at room temperature. The samples were imaged at 80 kV with TEM. The absorption spectra were recorded in the range 200–900 nm using a UV–visible absorption spectrometer. Fluorescence spectra were recorded by a FluoroLog steady-state spectrofluorometer (excitation at 488 nm) with the same DOX concentration.34

Raji cells, derived from human Burkitt’s lymphoma, were obtained from the Shanghai Institute of Cell Biology, Shanghai, People’s Republic of China, and were cultured in Roswell Park Memorial Institute 1640 medium (Gibco, Billings, MT, USA) supplemented with 10% heat-inactivated fetal bovine serum (Gibco) and antibiotics (penicillin 100 U/mL, and streptomycin 100 μg/mL).35 Cells were cultured in 5% CO₂ at 37°C. Cells were passaged every 2 days, and cells in logarithmic growth phase were used for experiments. The YTS cell line, derived from human natural killer/T-cell lymphoma (kindly donated by Scott Kaufmann, Mayo Clinic, Rochester, MN, USA), was cultured in the same way as for the Raji cells.

Following the addition of 50 μL of 1.55 mg/mL FITC-anti-CD20 to 100 μL of Raji or YTS cell suspension, the unstained cell suspension and FITC-anti-CD20-labeled cell suspension were incubated simultaneously in the dark for 2 hours at 37°C. The incubated cell suspensions were centrifuged at 900 rpm for 3 minutes, the supernatant was removed, the cells were washed twice with PBS, and the number of CD20-positive cells was measured using flow cytometry.

The coverslips were washed with 75% ethanol and soaked in 1 mg/mL L-poly-lysine PBS solution for 2 hours at room temperature. The coverslips were gently washed five times with triple-distilled water to remove excess L-poly-lysine and then dried. The 100 μL of cell suspension was placed on prepared L-poly-lysine-coated coverslips for 2 minutes, followed by removal of the excess PBS, and the cells were fixed with 100 μL of paraformaldehyde (4%) for 15 minutes, washed five times with PBS, and three times with triple-distilled water, and air-dried.

The cells were observed using high-resolution, tapping mode NanoWizard atomic force microscopy (AFM) (JPK Instruments AG, Berlin, Germany). RTESP silicon AFM probes with spring constants of 40 N/m (Veeco, Santa Barbara, CA, USA) were used for scanning in the air. The scan frequency was 1 Hz. The ambient temperature was kept at 25°C with humidity <30%. Images were processed using AFM offline data processing software.

To observe the morphological cell changes brought about by the use of the NDDS, ACNP-DOX-DSPE-PEG2000-anti-CD20, Raji and YTS cells were seeded into 6-well plates at a density of 2.5×10⁵ cells/well. Cells were cultured in an incubator at 37°C in the presence of 5% CO₂. When the cells achieved exponential cell proliferation, formulations of the NDDS, ACNP-DOX-DSPE-PEG2000-anti-CD20 were added to the cell cultures with a final DOX concentration of 1–10 μg/mL.

At 48 hours after dosing, the cultured cells were imaged and photographed using light microscopy. The survival rate of cells was assessed using the MTT assay and was calculated using the following formula:

where A₅₇₀ nm is the absorbance value. The dose–effect curves were plotted. All the experiments were performed in triplicate.

The intracellular targeting efficiency of the NDDS, ACNP-DOX-DSPE-PEG2000-anti-CD20 complex was assessed by flow cytometry. Raji or YTS cells were seeded into 6-well culture plates with a density of 2.5×10⁵ cells/well and cultured for 24 hours at 37°C. The cells were treated with ACNP-DOX or the NDDS, ACNP-DOX-DSPE-PEG2000-anti-CD20 at a concentration of 10 mM DOX. After incubation for 3 hours, the cells were resuspended in PBS.

The fluorescence intensities of DOX in cells were examined by FACScan (Becton Dickinson, San Jose, CA, USA) flow cytometry (excitation at 488 nm; emission at 590 nm). In a competition assay, the cells were preincubated with 5 mg/mL anti-CD20 antibodies for 30 minutes to saturate the cell surface CD20 antigens, before ACNP-DOX or the NDDS, ACNP-DOX-DSPE-PEG2000-anti-CD20 were added. For a qualitative analysis of the ability of the NDDS to target the B cells, CD20-positive Raji cells or CD20-negative YTS cells were seeded into 6-well culture plates and treated with ACNP-DOX or the NDDS, ACNP-DOX-DSPE-PEG2000-anti-CD20 as described earlier. Following 3 hours of incubation, the drug-treated cells were washed with PBS and fixed with 4% (v/v) paraformaldehyde, followed by (blue fluorescence) cell nuclear staining with Hoechst 33258. The cells were examined by confocal laser scanning microscopy (Leica, Heidelberg, Germany).34

Data were presented as mean ± standard deviation.