Materials
Soybean lecithin consisting of 90–95% phosphatidylcholine and mPEG2000-DSPE, and Mal-PEG2000-DSPE were purchased from Avanti Lipid (Alabaster, AL, USA). Cholesterol (CHO) was purchased from Chengdu Kelong Chemical Company (Chengdu, China). Rhodamine-PE was purchased from Avanti Lipid. T7 peptide with terminal cysteine (Cys-HAIYPRH) and TAT peptide with terminal cysteine (Cys-AYGRKKRRQRRR) were produced according to the standard solid phase peptide synthesis by Shanghai Jier Bio-Pharmaceutical Co., Ltd. (Shanghai, China). Cell culture plates were purchased from Wuxi NEST Biotechnology Co., Ltd. (Wuxi, China). Other chemicals and reagents were of analytical grade and obtained commercially.
BALB/c male athymic nude mice (~20 g) were purchased from the Experiment Animal Center of Shandong University (Jinan, China).
Synthesis of TAT-PEG<sub>2000</sub>-DSPE and T7-PEG<sub>2000</sub>-DSPE
TAT-PEG2000-DSPE was synthesized as described previously (25–27), DSPE-PEG2000-Mal and Cys-TAT (molar ratio, 1:1.5) were reacted in the mixture of CHCl3/MeOH (v:v, 2:1) with gentle stirring at room temperature overnight. After thin layer chromatography (TLC) showed the disappearance of DSPE-PEG2000-Mal, the mixture was evaporated under vacuum, the residue was redissolved by CHCl3, and the insoluble material was filtered, the supernatant (DSPE-PEG2000-TAT) was evaporated again under vacuum and stored at −20°C until use.
The T7-PEG2000-DSPE was synthesized according to literature with a modest modification (16,28). T7 was conjugated with DSPE-PEG2000-BTC in 0.01 M isotonic HEPES buffer (pH 7.5) under the reaction conditions of 4 h at 4°C, gentle stirring and 1:2 molar ratio of peptides to DSPE-PEG2000-BTC. The reaction was traced by TLC until the peptide was completely consumed. The mixture was then dialyzed against water, and lyophilized. The resulting conjugate DSPE-PEG2000-T7 was then used to prepare liposomes without further purification.
Preparation of liposomes
TAT and T7 co-modified PTX-loaded liposomes (T7/TAT-LP-PTX) were prepared by thin film hydration methods (29,30). Briefly, SPC, CHO, PTX (10% of the SPC + CHO weight), DSPE-PEG2000, DSPE-PEG2000-TAT and DSPE-PEG2000-T7 were dissolved in chloroform (total molar ratio of phospholipid and CHO derivatives was 3:2, molar ratio of DSPE-PEG2000, DSPE-PEG2000-TAT and DSPE-PEG2000-T7 was 9.5:4.5:0.5). Chloroform was then evaporated by rotary evaporation and residual organic solvent was removed in vacuum overnight. The thin film was hydrated in phosphate-buffered saline (PBS, pH 7.4) for 1 h at 37°C, followed by an intermittent probe sonication for 50 sec at 100 W.
Rhodamine-labeled liposomes were prepared as the T7/TAT-LP-PTX with the SPC being replaced by the rhodamine-PE. The final concentration of rhodamine-PE was 10 μg/ml.
Characterization of liposomes
Size and ζ potential measurements
The size and zeta potential of the liposomes were measured by a dynamic light scattering detector (Zetasizer Nano ZS90; Malvern Instruments, Ltd., UK).
Drug encapsulation efficiency (EE) and drug-loading efficient
The free PTX was removed by passing through a Sephadex G-50 column. The amount of PTX encapsulated in the liposome was measured by high-performance liquid chromatography (HPLC, Agilent LC1100). A reversed phase Inertsil® ODS-3 column (150–4.6 mm, pore size 5 mm; GL Sciences Inc., Tokyo, Japan) was used. Liposomes were dissolved in 1 ml DCM. After evaporating DCM, 3 ml mobile phase (50:50 v/v acetonitrile/water solutions) was added to dissolve the drugs. The solution was then filtered by 0.45 mm PVDF syringe filter for HPLC analysis. The column effluent was detected at 227 nm with a UV/VIS detector. EE (%) was calculated as: (amount of drug encapsulated in LP/initial amount of drug used in the fabrication of LP) ×100%.
In vitro stability of liposomes in serum
To demonstrate the serum stability of liposomes, particle sizes and turbidity variations were monitored in the presence of fetal bovine serum (FBS) (31,32). Briefly, liposomes were mixed with an equal volume of FBS at 37°C with gentle agitation at 30 rpm. At predetermined time-points (1, 2, 4, 8 and 24 h), 200 ml of the sample was pipetted out and onto a 96-well plate to measure the transmittance at 750 nm using a microplate reader (Varioskan Flash; Thermo Scientific, Waltham, MA, USA) and another 200 ml was diluted to 1 ml with 5% glucose solution for the particle size measurements by Malvern Zetasizer Nano ZS90 Instrument (Malvern Instruments Ltd.).
In vitro drug release
In vitro PTX release study was conducted using dialysis method under sink conditions (33). An aliquot of each PTX-loaded liposome (0.1 ml) or free PTX was placed into the dialysis tube (MWCO 8000) and tightly sealed. The dialysis tubes were immersed into 100 ml PBS (pH 7.4) containing 0.1% (v/v) Tween-80 and were incubated at 37°C for 24 h with mild oscillating at 50 rpm. At predetermined time-points, 0.1 ml release medium was sampled and replaced with equal volume of fresh release medium. The samples were diluted with acetonitrile and the concentrations of PTX were determined by HPLC.
In vitro cellular uptake
A549 cells were grown in RPMI-1640 medium (Gibco-Life Technologies, Carlsbad, CA, USA) contains 10% FBS, 100 μg/ml streptomycin, and 100 U/ml penicillin. The cells were maintained at 37°C in a humidified incubator with 5% CO2.
For quantitative study, A549 cells a (American Type Culture Collection; Manassas, VA, USA) were harvested with 0.125% Gibco Trypsin-EDTA solution (Invitrogen-Life Technologies) and seeded into 6-well assay plates (Corning Inc., Corning, NY, USA) at 5×105 viable cells/well. After 24 h, it was replaced with fresh serum-free medium, and then the prepared rhodamine-labeled liposomes were added to the cells. The final lipid concentration was 0.2 mg/ml. After incubation for 4 h, the medium was discarded and the cells were washed three times with cold PBS. The cells were harvested and washed with cold PBS, centrifuged at 4,000 rpm and re-suspended three times. The cellular uptake efficiency was determined by FACS.
For the qualitative study, A549 cells were harvested with 0.125% Gibco Trypsin-EDTA solution (Invitrogen) and seeded in Lab-Tek coverglass chambers (Nagle Nunc, IL, USA) with RPMI-1640 at a concentration of 5×103 viable cells/chamber. The cells were incubated overnight and were subsequently incubated with rhodamine-labeled liposomes in the RPMI-1640 (concentration of 10 μg/ml) at 37°C. After 4 h, the cells were washed three times with cold PBS and fixed with 4% paraformaldehyde for 20 min. The cells were washed twice with cold PBS. The nuclei were stained by incubating with DAPI (Roche, Mannheim, Germany) for another 10 min. The cell monolayer was washed three times with PBS and observed by confocal laser scanning microscopy (Leica, Munich, Germany).
Identification of cellular uptake pathways
To study the effect of different inhibitors on the cellular uptake of T7/TAT-LP, the cells were pre-incubated with different inhibitors for 30 min at 37°C. Poly-lysine (800 mg/ml), amiloride (1.48 mg/ml), chlorpromazine (20 mg/ml), filipin (5 mg/ml), free T7 peptide (5 mg/ml) and free TAT peptide (5 mg/ml), respectively, were added. To study the effect of temperature on the cellular uptake, the cells were incubated at 37 and 4°C. The inhibitor-containing culture media were discarded and rhodamine-labeled liposome, containing culture media, was applied for 4 h incubation. The cells were treated as described in the quantitative study, and the fluorescence intensity was determined by flow cytometer (Cytomics™ FC 500; Beckman Coulter, Miami, FL, USA).
In vitro cytotoxicity and anti-proliferation assay
Comparison of in vitro cytotoxicity and tumor cell proliferation assay of various formulations was performed on A549 cells using SRB colorimetric assay. Briefly, A549 cells (4×103 cells) were seeded in 96-well plates and incubated overnight. The cells were exposed to serial concentrations of different PTX formulations in the culture medium for 48 h at 37°C. Subsequently, cells were fixed with trichloroacetic acid, washed, and stained with SRB. Absorbance was measured at 540 nm using a 96-well plate reader (Bio-Rad Laboratories, Hercules, CA, USA). Dose-response curves were generated, and the concentrations of drug resulting in 50% cell killing (IC50) were calculated by Origin 7.0 (OriginLab, Northampton, MA, USA).
Evaluation of tumor spheroid penetration
To prepare the three-dimensional tumor spheroids, A549 cells were seeded at a density of 2×103 cells/200 μl per well in 96-well plates coated with 80 μl of a 2% low-melting-temperature agarose. Seven days after the cells were seeded, tumor spheroids were treated with 10 μg/ml rhodamine-labeled liposome. After 4 h of incubation, the spheroids were rinsed three times with ice-cold PBS and fixed with 4% paraformaldehyde for 30 min. The spheroids were transferred to glass slides and covered by glycerophosphate. Fluorescent intensity was observed by laser scanning confocal microscopy (Leica).
In vivo tumor growth inhibition study
The lung cancer nude mouse xenograft models were established by injecting A549 cells (1×107 cells/animal, subcutaneous injection) into the back of 4- to 6-week-old BALB/c male athymic nude mice. Tumor volume (mm3) was measured with vernier caliper. Fifty nude mice with lung cancer xenograft models were divided into five groups. When the tumors reached 100–200 mm3, the mice were administrated with PBS, free PTX, TAT-LP-PTX, T7-LP-PTX and T7/TAT-LP-PTX, respectively. The drugs were administered once every other day (totally 10 mg/kg) and the tumor volumes were measured. The tumor inhibition rate was calculated using the formula: Tumor inhibition rate = (1-Vt/V0) × 100%, where Vt was the tumor volume of the treated mice and V0 was the initial tumor volume of the untreated mice.
In vivo imaging
The DIR-loaded liposomes were utilized, as previously described, to investigate the distribution of liposomes in nude mouse-bearing A549 cells. The DIR-loaded liposomes were injected into nude mouse-bearing A549 cells via intravenous administration, and then the in vivo fluorescence imaging was performed by IVIS Spectrum system (Caliper Life Sciences, Hopkington, MA, USA).
Statistical analysis
Data are presented as means ± SD. Analysis of variance (ANOVA) was used to determine the variance of the whole values in each group. Statistical significance was evaluated using the Student’s t-test for comparisons of the experimental groups.