Article pubs.acs.org/molecularpharmaceutics
Synergistic Dual-Ligand Doxorubicin Liposomes Improve Targeting and Therapeutic Efficacy of Brain Glioma in Animals Taili Zong,†,‡ Ling Mei,† Huile Gao,† Wei Cai,§ Pengjin Zhu,§ Kairong Shi,† Jiantao Chen,† Yang Wang,† Fabao Gao,*,§ and Qin He*,† †
Key Laboratory of Drug Targeting and Drug Delivery Systems, West China School of Pharmacy, Sichuan University, No. 17, Block 3, Southern Renmin Road, Chengdu 610041, P. R. China § Molecular Imaging Center, Department of Radiology, West China Hospital, Sichuan University, No. 37 Guo Xue Xiang, Chengdu, Sichuan 610041, China ‡ Chongqing Pharmaceutical Research Institute Co., Ltd, No. 565, Tushan Road, Nan’an District, Chongqing 400061, P. R. China S Supporting Information *
ABSTRACT: Therapeutic outcome for the treatment of glioma was often limited due to low permeability of delivery systems across the blood−brain barrier (BBB) and poor penetration into the tumor tissue. In order to overcome these hurdles, we developed the dual-targeting doxorubicin liposomes conjugated with cell-penetrating peptide (TAT) and transferrin (T7) (DOX-T7-TAT-LIP) for transporting drugs across the BBB, then targeting brain glioma, and penetrating into the tumor. The dual-targeting effects were evaluated by both in vitro and in vivo experiments. In vitro cellular uptake and three-dimensional tumor spheroid penetration studies demonstrated that the system could not only target endothelial and tumor monolayer cells but also penetrate tumor to reach the core of the tumor spheroids and inhibit the growth of the tumor spheroids. In vivo imaging further demonstrated that T7-TAT-LIP provided the highest tumor distribution. The median survival time of tumor-bearing mice after administering DOX-T7-TAT-LIP was significantly longer than those of the single-ligand doxorubicin liposomes and free doxorubicin. In conclusion, the dual-ligand liposomes comodified with T7 and TAT possessed strong capability of synergistic targeted delivery of payload into tumor cells both in vitro and in vivo, and they were able to improve the therapeutic efficacy of brain glioma in animals. KEYWORDS: dual-targeting, synergistic effect, brain glioma, glioma penetration, glioma-bearing survival
1. INTRODUCTION
The reported dual-targeting delivery included three different strategies. The first strategy of dual-targeting was to construct the nanocarriers modified with one kind of ligand, such as transferrin (Tf) and angiopep,3,6 the corresponding receptor of which is overexpressed on both the BBB and tumor cells. The second strategy was to fabricate the nanocarriers modified with two kinds of ligands, one of which could target the BBB, and the other could target tumor cells. The third strategy was to build the nanocarriers still modified with two kinds of ligands, one of which could target the BBB and tumor cells, and the other could promote the targeting effect.1 Transferrin receptors (TfR) are highly expressed on both brain capillary endothelial cells (BCECs)7,8 and brain glioma cells.9 Accordingly, the drug-loaded liposomes modified with Tf may contribute to enhancing both the transportation of drug across the BBB and the targeting ability to brain glioma.10 The
Glioma has been considered as one of the most devastating malignant primary brain tumors because it is very difficult to treat and cure.1 Due to the infiltrate growth of glioma, it is hard to completely remove the tumor through surgery.2 Chemotherapy is the most common method for the treatment of glioma.3 Unfortunately, the clinical therapeutic effect of glioma by drug treatment is very unsatisfying because of the existence of the blood−brain barrier (BBB), which excludes more than 98% of small molecule drugs and almost 100% of large molecule drugs.3,4 Another obstacle in chemotherapy is to maintain a higher concentration of therapeutic agents at the tumor site and prevent their spreading into healthy tissues.5 Therefore, it is apparently clear that there is an urgent need for effective brain glioma targeting systems with high BBB penetrating abilities, specific glioma targeting, and glioma penetrating abilities. In this regard, we designed the dualtargeting liposomes modified with ligands which could transfer therapeutic agent across the BBB, then target brain glioma, and penetrate into the tumor. © 2014 American Chemical Society
Received: Revised: Accepted: Published: 2346
January 19, 2014 March 12, 2014 June 3, 2014 June 3, 2014 dx.doi.org/10.1021/mp500057n | Mol. Pharmaceutics 2014, 11, 2346−2357
Molecular Pharmaceutics
Article
feasibility of Tf as a targeting ligand had been demonstrated.11−13 However, the concentration of endogenous Tf in blood (25 μM) is very high, compared with the Kd of Tf binding to TfR. The endogenous Tf may competitively inhibit the binding of Tf-modified drug delivery systems to TfR; in addition, the relatively high molar weight of Tf, about 80 kDa, makes it troublesome for the construction of drug delivery systems.10 A unique targeting agent, HAIYPRH (T7), was screened by a phage display system on the cells expressing human TfR.14 Besides the high affinity for TfR (Kd of ∼10 nM), the binding site of T7 to TfR is different from that of Tf to TfR.15 Thus, endogenous Tf will not inhibit the uptake of T7-modified drug delivery systems; on the contrary, endogenous Tf in vivo can promote the uptake of T7.15 In short, T7, as a ligand targeting TfR, is more advantageous than Tf. Although single peptide could enhance the targeting effect, the presence of receptor-targeting moiety alone on liposomes limited the enhanced uptake of liposomes due to the receptor saturation.16,17 Considering the fact that an ideal tumor targeted drug delivery system should not only selectively deliver drugs to targeted tumor but also deliver the drugs into the center of the tumor with high efficacy, the receptor saturation needs to be overcome. TAT, one of the cellpenetrating peptides (CPPs), can facilitate the intracellular delivery of cargoes with various sizes and physicochemical properties.18−20 Liposomes modified with TAT can deliver the cargoes into cells with high efficiency via an unsaturated and receptor/transporter independent pathway.21 Doxorubicin (DOX) is an anthracycline antibiotic that possesses broad spectrum antineoplastic activity and is one of the most important anticancer agents in use.22 However, clinical utility is hampered by cumulative, dose-limiting cardiotoxicity, myelosuppression, and the developmental drug resistance.23 To avoid such complications, the use of liposomes as carriers for DOX has been recently explored in both animal and human trials.24 In this study, we established a dual-targeting liposomal system modified with TAT and T7, in which specific ligand T7 could target the BBB and brain glioma tumor and nonspecific ligand TAT could enhance the effect of passing through the BBB, and elevate the penetration into the tumor (as illustrated in Figure 1). DiR, DOX, and CFPE were utilized to track the behavior of liposomes in vitro and in vivo. To identify the targeting effect, in vitro cellular uptake was performed. Tumor spheroid penetration was performed to evaluate the penetration characteristics of the dual-targeting liposome. In vivo imaging, tissue distribution, and tumor slice were further utilized to evaluate the glioma targeting efficiency of the dual-targeting liposome and to elucidate the potential targeting pathways. To demonstrate the chemotherapy effect of the dual-targeting liposome, a survival study of brain glioma-bearing mice was performed. Since T7 is more advantageous than Tf, the delivery system modified with CPP and T7 is more advantageous than that with CPP and Tf.
Figure 1. Schematic illustration of DOX-loaded T7 and TAT comodified liposomes (DOX-T7-TAT-LIP). (A) Basic structure of DOX-T7-TAT-LIP. (B) Liposomes could specifically bind to transferrin receptors expressed on BCECs and transport across the BBB through a synergetic effect. Then the liposomes could accumulate in the glioma selectively, penetrate into the core region of the tumor, and release drugs.
Shanghai Advanced Vehicle Technology L.T.D. Co. (Shanghai, China), and cholesterol (CHO) was purchased from Chengdu Kelong Chemical Company (Chengdu, China). DSPE, DSPEPEG2000, DSPE-PEG2000-Mal, and 1, 2-dioleoyl-sn-glycero-3phosphoethanolamine-N-(carboxyfluorescein) (CFPE) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). NHS-PEG1000-Mal was obtained from Jenkem Technology (Beijing, China). 4′,6-Diamidino-2-phenylindole (DAPI) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Beyotime Institute Biotechnology (Haimen, China). 1,10-Dioctadecyl-3,3,30,30-tetramethylindotricarbocyanine iodide (DiR) was purchased from Biotium (Hayward, CA, USA). Annexin V-FITC/PI apoptosis detection kit was obtained from KeyGEN Biotech Co., Ltd. (China). Plastic cell culture dishes and plates were purchased from Wuxi NEST Biotechnology Co. (Wuxi, China). Other chemicals and reagents were of analytical grade. BALB/c mice were purchased from the experiment animal center of Sichuan University (P. R. China). All animal experiments were performed in accordance with the principles of care and use of laboratory animals and were approved by the experiment animal administrative committee of Sichuan University. 2.2. Synthesis of DSPE-PEG1000-TAT and DSPE-PEG2000T7. 2.2.1. Synthesis of DSPE-PEG1000-Mal. DSPE-PEG1000-Mal was synthesized by conjugating NHS-PEG1000-Mal with DSPE. NHS-PEG1000-Mal (3 μmol) and DSPE (4.5 μmol) were dissolved in dry chloroform containing triethylamine (6 μmol) in darkness at room temperature under argon for about 5 h. After thin layer chromatography showed the disappearance of NHS-PEG1000-Mal, the excessive DSPE was separated from the product by adding an appropriate amount of acetonitrile to precipitate it. After that, the supernatant was collected and evaporated. The obtained DSPE-PEG1000-Mal was used for the next reaction.
2. MATERIALS AND METHODS 2.1. Materials. DOX was a gift from Haizheng Co. Ltd. T7 with a cysteine on the N-terminal (cys-T7) was synthesized by ChinaPeptides Co., Ltd. (Shanghai, China). TAT peptide with terminal cysteine (Cys-AYGRKKRRQRRR) was synthesized by Chengdu KaiJie Biopharmaceutical Co., Ltd. (Chengdu, China). Soybean phospholipid (SPC) was purchased from 2347
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2.2.2. Synthesis of DSPE-PEG1000-TAT and DSPE-PEG2000T7. DSPE-PEG2000-Mal and the obtained DSPE-PEG1000-Mal were respectively reacted with 1.5-fold molar excess of Cys-T7 and Cys-TAT in the mixed solvent of CHCl3/MeOH (V:V = 2:1) containing 3-fold molar excess of triethylamine in darkness at room temperature with stirring for about 30 h, and the reaction was traced by TLC until DSPE-PEG1000-Mal or DSPEPEG2000-Mal was completely consumed. The organic solvent was filtered, and the filtrates were evaporated by rotary evaporation. The residues were redissolved by chloroform and filtered again to purify the production. The supernatants were evaporated by rotary evaporation and stored at −20 °C until used. The existence of the yielded products was confirmed by MALDI-TOF mass spectrometry (MALDI-TOF MS). 2.3. Preparation and Characterization of Liposomes. DOX-loaded nonligand liposome (DOX-LIP), single-ligand liposomes (DOX-T7-LIP and DOX-TAT-LIP), and dual-ligand liposome (DOX-T7-TAT-LIP) were prepared by remote loading using an ammonium sulfate gradient.25,26 Briefly, various amounts of lipid materials (see Table 1) were dissolved
The DiR-loaded and CFPE-loaded liposomes were stored at 4 °C for later use. The mean particle sizes and zeta potentials of the liposomes were measured by Malvern Zetasizer Nano ZS90 instrument (Malvern Instruments Ltd., U.K.). Particle morphology was detected by a transmission electron microscope (TEM) (100CX, JEOL, Japan) followed by negative staining procedure using phosphotungstic acid. 2.4. In Vitro Stability of Liposomes in Serum. In order to demonstrate the serum stability of liposomes, particle sizes and turbidity variations were monitored in the presence of fetal bovine serum (FBS).27 Briefly, 50 μL of different formulations of liposomes was added to 1 mL of culture medium containing 10% FBS and incubated at 37 °C with gentle shaking at 50 rpm. At predetermined time points, the transmittance was measured at 750 nm by a microplate reader (Thermo Scientific Varioskan Flash, USA) and the size change was measured by Malvern Zetasizer Nano ZS90 instrument (Malvern Instruments Ltd., U.K.). 2.5. In Vitro Release of DOX from Liposomes. In vitro release of DOX from liposomes was investigated in PBS (pH 7.4) containing 10% FBS. An aliquot of each DOX-loaded liposome (0.3 mL) or free DOX was respectively mixed with the same volume of PBS (pH 7.4) containing 10% FBS, placed into a dialysis tube (MW = 8000−14,000), and tightly sealed. Then the dialysis tubes were immersed into 30 mL of PBS (pH 7.4) containing 10% FBS in an incubator at 37 °C for 48 h with mild oscillation at 50 rpm. At predetermined time points, 0.3 mL of release medium was sampled and replaced with an equal volume of fresh release medium. DOX concentration was measured by a microplate reader (Thermo Scientific Varioskan Flash, USA) at Ex = 470 nm, Em = 590 nm.28 2.6. In Vitro Cellular Uptake Study. 2.6.1. Confocal Laser Scanning Microscopy (CLSM). C6 and bEnd.3 cells were plated at a density of 1 × 105 cells/well on coverslip in 6-well plates. After 24 h incubation, different groups of DOX liposomes and free DOX were added to the plates with a final DOX concentration of 12.5 μg/mL. After incubation at 37 °C in 5% CO2 for 4 h, the cells were washed three times with cold PBS and fixed with 4% paraformaldehyde for 30 min, following by nucleus staining with DAPI for 5 min. Finally, cells were imaged by a laser scanning confocal microscope (TCS SP5 AOBS confocal microscopy system, Leica, Germany). 2.6.2. Quantitative Analysis of Cellular Uptake. C6 and bEnd.3 cells were plated in 6-well culture plates at a density of 1 × 106 cells/well and allowed to attach to the plate for 24 h. Different groups of DOX liposomes and free DOX were added to the plates with a final DOX concentration of 12.5 μg/mL. After incubation at 37 °C in 5% CO2 for 4 h, the cells were washed with cold PBS three times and were trypsinized and resuspended in 0.4 mL of PBS. The fluorescent intensity of cells was measured by a flow cytometer (Cytomics FC 500, Beckman Coulter, Miami, FL, USA). 2.6.3. Uptake Mechanism Study. In order to study the uptake mechanism of T7-TAT-LIP, C6 cells were preincubated with different endocytosis inhibitors for 30 min at 37 °C. Sodium azide (6.51 mg/mL), amiloride (1.48 mg/mL), chlorpromazine (10 μg/mL), filipin (10 μg/mL), and free T7 (1 mg/mL) peptide were added, respectively. To study the effect of temperature on the cellular uptake, the cells were incubated at both 37 and 4 °C. After 30 min preincubation with the inhibitors above, CFPE-T7-TAT-LIP was added for a further 4 h incubation. Then the cells were treated as described
Table 1. Composition of Liposomes (mol %) corresponding compositions abbreviations
SPC (%)
CHO (%)
T7-TAT-LIP
57
33
TAT- LIP
57
33
T7-LIP PEG-LIP
57 57
33 33
functional lipid composition 6% DSPE-PEG2000-T7/0.5% DSPE-PEG1000TAT/3.5% DSPE-PEG2000 0.5% DSPE-PEG1000-TAT/9.5% DSPEPEG2000 6%DSPE-PEG2000-T7/4% DSPE-PEG2000 10% DSPE-PEG2000
in the mixed solvent of chloroform and methanol (V:V = 2:1). Then the organic solvent was removed by rotary evaporation. The obtained film was kept in vacuum for over 6 h to completely remove the residual organic solvent. The thin film was hydrated in 300 mM of ammonium sulfate solution for 1 h at 37 °C, followed by a 2 min bath-type sonication. Then it was further intermittently sonicated by a probe sonicator at 80 W for 75 s. The free ammonium sulfate was removed by passing through a Sephadex G-50 column in PBS (pH 7.4) solution. DOX (doxorubicin hydrochloride:phospholipids = 1:20, w/w) was added, mixed, and then incubated at 45 °C for 20 min. The DOX-loaded liposomes were stored at 4 °C for later use. Free DOX was removed by passing through a Sephadex G-50 column. The envelopment efficiency of the liposomes was measured at Ex = 470 nm and Em = 590 nm, respectively, on a spectrofluorimeter (Shimadzu, Japan). DiR-loaded and CFPE-loaded nonligand liposomes (DiRLIP and CFPE-LIP), single-ligand liposomes (DiR-T7-LIP and DiR-TAT-LIP, CFPE-T7-LIP, and CFPE-TAT-LIP), and dualligand liposomes (DiR-T7-TAT-LIP and CFPE-T7-TAT-LIP) were prepared to investigate the cells’ uptake mechanism (CFPE-loaded liposomes) and distribution of each liposome in tissues of brain glioma-bearing mice (DiR-loaded liposomes). Various amounts of lipid materials (see Table 1) and DiR or CFPE were dissolved in mixed solvent of chloroform and methanol (V:V = 2:1). The solvent was then removed by rotary evaporation. The obtained thin film was kept in vacuum for over 6 h to completely remove the residual organic solvent. The thin film was hydrated in PBS (pH 7.4) for 1 h at 37 °C followed by a 2 min bath-type sonication. Then it was further intermittently sonicated by a probe sonicator at 80 W for 75 s. 2348
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in section 2.6.2 and the fluorescent intensity was determined by a flow cytometer (Cytomics FC 500, Beckman Coulter, USA). 2.6.4. Colocalization of Liposomes and Lysosome. In order to determine the intracellular location of different liposomes, lysosome staining was performed on C6 cells. After preincubation with CFPE-loaded liposomes for 4 h, cells were treated with Lyso-Tracker RED (50 nM) for 1 h. Then the cells were treated as described in section 2.6.1 and observed by confocal microscope (FV1000, Olympus, USA). 2.7. In Vitro Cytotoxicity. C6 cells were seeded into 96well plates at a density of 1 × 104 cells/well. After 24 h incubation, different groups of DOX liposomes and free DOX were diluted with PBS (pH 7.4) and added to 96-well cultured plates, respectively. The final concentrations of DOX were in the range of 0−2.5 μg/mL. 40 h later, 20 μL of MTT solution (5 mg/mL in PBS) was added into each well and cells were further incubated for 4 h at 37 °C. Then the cells were dissolved by 150 μL of dimethyl sulfoxide. The absorbance was measured by a microplate reader (Thermo Scientific Varioskan Flash, USA) at 570 nm. Cell viability (%) was calculated as Atest/Acontrol × 100%, where Atest and Acontrol represented the absorbance of cells treated with different test solutions and blank culture media, respectively. 2.8. In Vitro Apoptosis Assay. The quantitative analysis of apoptosis induced by different DOX liposomes and free DOX was performed by Annexin V-FITC/PI double staining. C6 cells were treated with different DOX liposomes and free DOX for 11 h at 37 °C. The final concentration of DOX was 12.5 μg/ mL. At the end of the treatment, cells were harvested, washed with cold PBS, suspended in 500 μL of binding buffer, and stained by 5 μL of Annexin V-FITC and 5 μL of PI. The cells were incubated in the darkness for 15 min and measured by flow cytometer (Cytomics FC 500, Beckman Coulter, Miami, FL, USA). 2.9. Tumor Spheroid Uptake. To prepare the threedimensional tumor spheroids, C6 cells were seeded at a density of 1 × 104 cells per well in 96-well plates coated by 80 μL of 2% low melting point agarose.29,30 5 days after the cells were seeded, tumor spheroids were treated with different groups of DOX liposomes and free DOX for 4 h at 37 °C. Then the spheroids were rinsed with cold PBS and fixed with 4% paraformaldehyde for 30 min. Finally the spheroids were subjected to confocal microscopy (TCS SP5 AOBS confocal microscopy system, Leica, Germany). As for the quantitative study, ten spheroids were collected for each group after incubation with different groups of DOX liposomes and free DOX and then incubated with 100 μL of Accumax for 30 min. Cell suspensions were analyzed by flow cytometry (Cytomics FC 500, Beckman Coulter, Miami, FL, USA). 2.10. Growth Inhibition of Tumor Spheroid. C6 glioma spheroids were prepared as described in section 2.9. Afterward, glioma spheroids were incubated with 1640 culture medium, free DOX, DOX-LIP, DOX-T7-LIP, DOX-TAT-LIP, and DOX-T7-TAT-LIP respectively. The final concentration of DOX was 12.5 μg/mL. Growth inhibition was monitored by measuring the size of C6 glioma spheroids using an inverted microscope (XD-RFL, Ningbo Sunny Instruments Co., LTD, Ningbo, China) at days 0, 1, 2, 3, 4, 5, and 6, respectively. The major (dmax) and minor (dmin) diameters of each spheroid were measured, and the spheroid volume was calculated using the following formula: V = (π × dmax × dmin)/6. The C6 glioma spheroid volume ratio was estimated with the following formula: R = (Vday i/Vday 0) × 100%, where Vday i was the C6
glioma spheroid volume at the ith day after applying the drug, and Vday 0 was the C6 glioma spheroid volume prior to administration, as described previously.31 2.11. In Vivo Imaging. BALB/c mice were anesthetized with 5% chloral hydrate and individually placed in a stereotaxic apparatus. The C6 cells (5 × 105 cells/7.5 μL of pH 7.4 PBS) were injected into the right brain of each BALB/c mouse (1.8 mm lateral to bregma and 3.0 mm deep from the dura) at a rate of 3.0 μL/min. Eight days after the injection, the DiR-loaded liposomes were injected via the tail vein into the BALB/c mice. Four hours after the injection, the in vivo fluorescence imaging was performed with an IVIS Spectrum system (Caliper, Hopkington, MA). Then the mice were sacrificed after heart perfusion with saline and 4% paraformaldehyde. The fluorescence intensity of various organs was measured. Brains were collected, and the glioma domains were sectioned at a thickness of 10 μm. Then the sections were incubated with DAPI to stain nuclei. Finally, the sections were imaged by a laser scanning confocal microscope (FV1000, Olympus, USA). 2.12. In Vivo Antiglioma Effect. The orthotropic glioma model was established as described in section 2.11. Eight days following the cell injection, the BALB/c mice were randomly divided into 6 groups (10 mice per group): saline group, DOX solution group, DOX-LIP group, DOX-T7-LIP group, DOXTAT-LIP group, and DOX-T7-TAT-LIP group. Each mouse received a dose of 2.5 mg/kg DOX by tail vein injection every 3 days, three times. Survival time was recorded and analyzed using GraphPad Prism 5 (GraphPad Software Inc., California, USA). 2.13. Statistical Analysis. Statistical comparisons were performed by Student’s t test, and p value
