International Journal of Pharmaceutics 487 (2015) 223–233
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
The antitumor activity of PNA modified vinblastine cationic liposomes on Lewis lung tumor cells: In vitro and in vivo evaluation Xue-tao Li a , Mei-li He a , Zhi-yan Zhou b , Ying Jiang a , Lan Cheng a, * a b
School of Pharmacy, Liaoning University of Traditional Chinese Medicine, Dalian 116600, China School of Stomatology, Jilin University, Changchun 130021, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 27 January 2015 Received in revised form 21 March 2015 Accepted 14 April 2015 Available online 17 April 2015
Non-small cell lung cancer (NSCLC) is one of the frequently-occurring disease in the world, and the treatment effects are usually unsatisfactory. Vinblastine is an anti-microtubule drug in clinic. In this study, a nanostructured liposome was designed and prepared for treating NSCLC. In the liposomes, peanut agglutinin (PNA) was modified on the liposomal surface, 3-(N-(N0 ,N0 -dimethylaminoethane) carbamoyl) cholesterol was used as cationic materials, and vinblastine was encapsulated in the aqueous core of liposomes, respectively. The PNA modified vinblastine cationic liposomes were approximately 100 nm in size with a positive potential. In vitro results showed that the targeting liposomes could significantly enhance cellular uptake, selectively accumulate in LLT cells, and dramatically initiate apoptosis via activating pro-apoptotic proteins and apoptotic enzymes, thus leading to the strongest antitumor efficacy to LLT cells. In vivo results demonstrated that the targeting liposomes could display a prolonged circulation time in the blood, accumulate more drug in tumor location, and induce most of tumor cells apoptosis. As a result, a robust overall antitumor efficacy in tumor-bearing mice was observed subsequently. In conclusion, the chemotherapy using the PNA modified vinblastine cationic liposomes could provide a potential strategy for treating non-small cell lung cancer. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Cationic liposomes Vinblastine Peanut agglutinin Non-small cell lung cancer
1. Introduction Lung cancer, the most common cause of cancer-related death in humans, is responsible for more than 1 million deaths worldwide annually (Cheng et al., 2014; Karachaliou and Rosell, 2014). There are two main types of lung cancer categorized by the size and appearance of the malignant cells, and the non-small cell lung cancer (NSCLC) is the most common type. Usually, treatment for NSCLC is a combined approach, including surgery, radiotherapy and chemotherapy, and it depends upon the histological type and the stage of cancer (Vasekar et al., 2014). However, surgery is only an option for patients in which the cancer cells have not spread beyond nearby lymph nodes. Furthermore, surgery and radiotherapy cannot eliminate all the cancer cells, thus leading to the fact that the five-year survival rate is less than 15% after comprehensive treatment (Vlahovic et al., 2007). Although chemotherapy is increasingly being used both pre-operatively and post-operatively,
* Corresponding author at: School of Pharmacy, Liaoning University of Traditional Chinese Medicine, Shengming 1 Road 77, Double D port, Dalian 116600, China. Tel.: +86 411 8589 0145; fax: +86 411 8589 0128. E-mail address: [email protected] (L. Cheng). http://dx.doi.org/10.1016/j.ijpharm.2015.04.035 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.
the treatment effects are usually unsatisfactory for the minimal accumulation in cancer cells and the serious side-effects. Therefore, developing a new chemotherapeutic strategy using nano-carriers is a critical issue for overcoming the limitation. Liposomes, an ideal drug carrier, are able to enhance the antitumor efficacy via improving the pharmaceutical properties of drugs and decreasing the systemic toxicity. Although liposomes have been successfully applied for several years, they are plagued by problems such as rapid uptake by the reticuloendothelial system (RES), poor active targeting, short half-life, easy leakage and main accumulation in liver or spleen (Andresen et al., 2005; Nag and Awasthi, 2013). PEGlyted liposomes could resolve these obstacles by providing a protective steric barrier against interaction with plasma proteins and cells of mononuclear phagocyte system (Sugiyama and Sadzuka, 2013). Cationic liposomes have been used as an excellent delivery carrier for antitumor drugs, and the cationic charge on the liposomal surface facilitates the intracellular uptake of the carrier via the electrostatic interaction with the anionic charge of cancer cell membrane (Han et al., 2014). Peanut agglutinin (PNA) is a plant lectin protein derived from the fruits of Arachis hypogaea, and is a common nutrient substance without obvious side-effects. PNA has good resistance to a variety of enzymes and exerts an ability of multivalent binding. Usually,
224
X.- Li et al. / International Journal of Pharmaceutics 487 (2015) 223–233
lectins recognize and bind particular sugar sequences in carbohydrates, and PNA is a targeting moiety that binds to b-D-galactosyl(1-3)-N-acetyl-D-galactosamine (Gal-b(1-3)GalNAc) specifically expressed on varying cancer cells (Cai and Zhang, 2005; Sakuma et al., 2011). Therefore, PNA is regularly used to be modified on the surface of nano-carriers to obtain a multiple active targeting effect. Vinblastine (VLB) is a vinca alkaloid and is used as an antimicrotubule drug in clinic. Vinblastine has been widely used to treat certain kinds of cancer, including non-small cell lung cancer, breast cancer, head and neck cancer, testicular cancer, and other solid tumors (Abbasov et al., 1972). Vinblastine binds tubulin, thereby inhibiting the assembly of microtubules (Kavallaris et al., 2001; Owellen et al., 1972). The treatment of vinblastine causes M phase specific cell cycle arrest by disrupting microtubule assembly and proper formation of the mitotic spindle and the kinetochore. In this study, we hypothesized that a kind of PNA modified vinblastine cationic liposomes would be able to target the cancer cells and treat NSCLC effectively. In the liposomes, PNA was modified on the surface of the liposomes for increasing intracellular uptake via the receptor-mediated endocytosis. 3-(N-(N0 ,N0 dimethylaminoethane)carbamoyl) cholesterol (DC-Chol) was used as cationic materials for targeting cancer cell membrane. Distearoyl phosphoethanolamine – PEG2000 (DSPE-PEG2000) was modified on the surface of liposomes for prolonging the circulation time. Vinblastine was encapsulated into the liposomal vesicles as an antitumor drug. The objectives of the present study were to develop the new type of targeting liposomes, to investigate their effects and action mechanism in vitro, and to assess their antitumor efficacies in tumor-bearing mice. 2. Materials and methods 2.1. Materials Vinblastine was purchased from Wuhan Jinnuo Chemicals (Wuhan, China). Egg phosphatidylcholine (EPC) and cholesterol were purchased from NOF Corporation (Tokyo, Japan). DSPEPEG2000 and N-hydroxysuccinimidyl-PEG2000-DSPE (DSPEPEG2000-NHS) were obtained from Nippon Fine Chemical Co., Ltd. (Osaka, Japan). DC-Chol was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USE). PNA was purchased from Shanghai BioSun Sci. & Tech. Co., Ltd. (Shanghai, China). Sephadex G-50 was purchased from Shanghai Chemical Technology (Shanghai, China). RPMI-1640 medium and fetal bovine serum (FBS) were purchased from Maichen Technology Co., Ltd. (Beijing, China). 2.2. Cells and animals Lewis lung tumor (LLT) cells were obtained from Cancer Institute & Hospital, Chinese Academy of Medical Sciences (Beijing, China), and were grown in RPMI-1640 supplemented with 10% FBS and antibiotics (penicillin 100 U/mL and streptomycin 100 mg/mL). Cells were cultured at 37 C with 5% CO2. C57BL/6 mice (16–18 g) were obtained from Dalian Medical University (Dalian, China). 2.3. Preparation of liposomes Blank PNA modified cationic liposomes were prepared using the film dispersion method (Du et al., 2009). Briefly, EPC, DC-Chol, DSPE-PEG2000 and DSPE-PEG2000-NHS (60:40:3:1, molar ratio) were dissolved in chloroform (10 mL), the solvent was evaporated using a rotary evaporator, and the lipid film was hydrated with 250 mM ammonium sulfate (5 mL) via sonication in a water bath at 40 C for 5 min. Subsequently, the suspensions were treated using an ultrasonic cell disruptor for 10 min (200 W). The obtained suspensions were extruded though polycarbonate membrane
using the pore sizes of 400 nm and 200 nm for three times, respectively. Then the blank liposomes were obtained. The drugloaded liposomes were prepared using an ammonium sulfate gradient loading method (Ju et al., 2014). In brief, the blank liposomes were further dialyzed (12,000–14,000, cut-off MW) in Hepes buffered saline (25 mM Hepes/150 mM NaCl) for 24 h. After dialysis, a certain amount of PNA (DSPE-PEG2000-NHS:PNA = 30:1, molar ratio) was added. The suspensions were stirred at room temperature for 10 h and then chromatography separation (Sephadex G-50) was performed to remove the unbound PNA. After the addition of free vinblastine (lipids:drug = 20:1, w/w), a volume of 5 mL blank liposomes was incubated in a water bath at 40 C with intermittent shaking for 30 min. Then the PNA modified vinblastine cationic liposomes were produced. Vinblastine cationic liposomes, PNA modified vinblastine liposomes, and vinblastine liposomes were prepared using the same procedures as described above, excluding the addition of DSPE-PEG2000-NHS and PNA, replacing DC-Chol with cholesterol or both. Besides, coumarin liposomes, coumarin cationic liposomes, PNA modified coumarin liposomes, PNA modified coumarin cationic liposomes (lipids:coumarin = 200:1, w/w), Cy7 (Invitrogen, Beijing, China) liposomes, and PNA modified Cy7 cationic liposomes (lipids:Cy7 = 300:1, w/w) were similarly prepared as the fluorescent probes for evaluating the targeting effects (Li et al., 2014b). 2.4. Characterization of the liposomes The particle size, polydispersity index (PDI) and zeta potential value were measured using a Nano Series Zen 4003 Zetasizer (Malvern Instruments Ltd., Malvern, UK). The morphology of PNA modified vinblastine cationic liposomes was observed using an atomic force microscopy (AFM; SPI3800N series SPA-400, NSK Ltd., Tokyo, Japan). Briefly, the PNA modified vinblastine cationic liposomes were diluted with distilled water and then placed on a silicon slice. The samples were air-dried and observed using AFM. The prepared liposomes were purified using a Sephadex G50 column equilibrated with PBS to remove the un-encapsulated vinblastine. The measurement of vinblastine was performed using a high-performance liquid chromatography (HPLC) system equipped with a UV detector (Agilent Technologies Inc., Cotati, CA, USA). The HPLC analysis was operated at 30 C with an ODS column (Diamonsil, 5 mm, 250 4.6 mm) at a wavelength of 264 nm. The mobile phase was consisted of acetonitrile, methanol, and 0.14 M diethylamine (pH 7.5) (14:54:32, v/v) with the flow rate of 1.0 mL/min (Volkov and Grodnitskaya, 1994; Zhou et al., 1990). Encapsulation efficiency (EE) of vinblastine was calculated using the formula: EE = (Wencap/Wtotal) 100%, where Wtotal and Wencap are the measured amount of vinblastine in the liposomal suspensions before and after passing over the Sephadex G-50 column, respectively (Li et al., 2011). PNA was measured by the BCA protein assay kit (Pierce Corporation, Beijing local agent, China). PNA coupling rate was calculated by the ratio of the PNA amount on the liposomes after separation to the added amount (Li et al., 2014a). 2.5. Release of vinblastine from liposomes The in vitro release of vinblastine from the drug-loaded liposomes was estimated as the previous reports (Guo et al., 2010). Briefly, the drug-loaded liposomes were dialyzed against normal saline or plasma-containing PBS (PBS containing 10% mouse plasma). A volume of 2.0 mL liposomes plus 2.0 mL release medium in dialysis tubing (12,000–14,000 molecular mass cutoff) was immersed in 20.0 mL of the release medium,
X.- Li et al. / International Journal of Pharmaceutics 487 (2015) 223–233
and oscillated with a shaker (100 times per minute) at 37 C. The release medium was sampled (0.5 mL) at 0, 4, 8, 12, 24 and 48 h, and was immediately replaced with an equal volume of fresh release medium after each sampling. The content of vinblastine in samples was measured using HPLC method as above described. The release rate (RR) was calculated with the formula: RR = (Wi/Wtotal) 100%, where Wi is the measured amount of vinblastine at the time-point of ith in release medium, and Wtotal is the total amount of vinblastine in the equal volume of liposomal suspensions prior to dialysis (Men et al., 2011). Each assay was repeated in triplicate.
2.6. Inhibitory effects of liposomes to LLT cells To evaluate the inhibitory effects of PNA modified vinblastine cationic liposomes, LLT cells were seeded in 96-well culture plates at a density of 5 103 cells/well, and cultured in serum-containing culture medium for 24 h. Then, the different concentrations of vinblastine were added into culture plates, respectively, including blank PNA modified cationic liposomes, vinblastine liposomes, vinblastine cationic liposomes, PNA modified vinblastine liposomes and PNA modified vinblastine cationic liposomes. The final concentration of vinblastine was 0.5, 1, 2, 4, 5, 8, 10 mM. Culture medium was used as blank control. After incubation for 48 h, the inhibitory effects were measured using sulforhodamine B (SRB) staining assay (Zhang et al., 2011). Briefly, the medium was removed, and the cells were fixed with 10% trichloroacetic aid (200 mL) for 1 h, washed for 5 times and stained with 0.4% SRB at room temperature. The absorbance was measured at 540 nm using a microplate reader (Tecan Infinite F50, Tecan Group Ltd., Shanghai, China). The survival rate was calculated using the following formula: survival% = (A540 nm for the treated cells/A540 nm for the control cells) 100%, where A540 nm is the absorbance value (Zhou et al., 2013). Each assay was repeated in triplicate. Finally, doseeffect curves were plotted.
2.7. Cellular uptake and targeting effects in LLT cells To evaluate the cellular uptake of LLT cells, LLT cells were seeded into 6-well culture plates at a density of 5 105 cells/well, and incubated for 24 h under the condition of 5% CO2 at 37 C. Then, the cells were treated with coumarin liposomes, coumarin cationic liposomes, PNA modified coumarin liposomes, and PNA modified coumarin cationic liposomes, respectively. Culture medium was used as blank control. Coumarin was used as a fluorescent probe (excitation at 488 nm, and emission at 530 nm), and the final concentration of coumarin was 1.0 mM. After incubation for another 3 h, the cells were washed 3 times with cold PBS, treated with 0.25% trypsin and harvested in 200 mL PBS. The fluorescence intensity of coumarin was analyzed using a FAScan flow cytometry (Becton Dickinson, San Jose, CA, USA) with 1 104 cells collected. To observe cellular distribution of PNA modified cationic liposomes, LLT cells were seeded into chambered coverslips at a density of 5 105 cells/well in 2 mL culture medium, and incubated for 24 h under the condition of 5% CO2 at 37 C. Subsequently, the cells were treated with coumarin liposomes, coumarin cationic liposomes, PNA modified coumarin liposomes and PNA modified coumarin cationic liposomes at a concentration of 0.5 mM coumarin for another 4 h. Control group was performed with culture medium. The cells were then washed 3 times with PBS, fixed with 4% paraformaldehyde for 10 min and stained with Hoechst 33258 (2 mg/mL) for 5 min. Finally, the samples were analyzed using a confocal laser scanning fluorescent microscopy (Leica, Heidelberg, Germany).
225
2.8. Apoptotic enzymes activity assays The regulating effects of PNA modified vinblastine cationic liposomes on pro-apoptotic proteins (Bak and Bok) and apoptotic enzymes (caspase 9 and caspase 3) in LLT cells were measured using an ELLSA kit (Cusabio Biotech Co., Ltd., Wuhan, China). Briefly, after incubation for 24 h under the condition of 5% CO2 at 37 C, the cells were treated with free vinblastine, vinblastine liposomes, vinblastine cationic liposomes, PNA modified vinblastine liposomes and PNA modified vinblastine cationic liposomes at a concentration of 10 mM vinblastine, respectively. Control experiment was performed with culture medium. After 12 h incubation, the cells were harvested and lysed, and the cell lysates were collected. The concentration of total proteins was measured with a bicinchoninic acid (BCA) kit at A562 nm according to manufacturer’s instructions of the kit. Samples were added to wells pre-coated with a monoclonal antibody, and incubated at 37 C for 2 h. Liquid samples were then removed and HRP-conjugate reagent was added into the wells, incubated for another 30 min at 37 C, followed by chromogen for 15 min. The reaction was terminated by addition of stop solution and immediately measured at 450 nm using a microplate reader (Tecan Infinite F50, Tecan Group Ltd., Shanghai, China). The protein ratio was calculated using the following formula: ratio% = (A450 nm for treated cells/A562 nm for treated cells)/(A450 nm for the control cells/A562 nm for the control cells), where A450 nm and A562 nm are absorbance values, respectively (Zhang et al., 2012). 2.9. In vivo imaging observation Non-invasive optical imaging systems were used to evaluate tumor accumulation ability of PNA modified cationic liposomes in tumor-bearing C57BL/6 mice, and Cy7 dye was used as the fluorescent probe. All procedures were preformed according to guidelines of the Institutional Authority for Laboratory Medicine. Briefly, approximately 1 107 LLT cells were re-suspended in 200 mL serum-free culture medium, and injected subcutaneously into the right flanks of the C57BL/6 mice. When the tumor reached approximately 400–500 mm3 in volume, the tumor-bearing mice were randomly divided into 4 groups (3 mice per group). Then, the tumor-bearing mice were administered physiological saline, free Cy7, Cy7 liposomes, and PNA modified Cy7 cationic liposomes via tail vein injection. The mice were anesthetized with isoflurane. Fluorescent images and X-ray images were captured at 1, 3, 6, 9, 12, 24 and 48 h using a Kodak multimodal imaging system (Carestream Health, Inc., USA) (Ma et al., 2013). 2.10. Antitumor efficacy in tumor-bearing mice Tumor-bearing C57BL/6 mice were used for investigating the antitumor efficacy in vivo. Briefly, the tumor-bearing mice were inoculated as above, and when the tumor reached about 300 mm3 in volume, the tumor-bearing mice were randomly divided into 6 groups (12 mice per group). At days 10, 13, 16, and 19 postinoculation, the tumor-bearing mice were treated with physiological saline, free vinblastine, vinblastine liposomes, vinblastine cationic liposomes, PNA modified vinblastine liposomes, and PNA modified vinblastine cationic liposomes via tail vein injection at a dosage of 0.5 mg/kg vinblastine, respectively. The tumor was measured every day with a caliper, and tumor volumes were calculated with the formula: V = length width2/2 (mm3) (n = 6). Tumor volume inhibitory rate = 100% (Vdrug/Vsaline) 100%, where Vdrug and Vsaline are the tumor volumes after treatment with drug and physiological saline, respectively. The mice monitored in tumor volume inhibitory were sacrificed at day 30 post-inoculation, and the tumors were removed and
226
X.- Li et al. / International Journal of Pharmaceutics 487 (2015) 223–233
Fig. 1. The schematic representation of the enhanced inhibiting effects.
photographed. Six tumor-bearing mice in each group were used for monitoring the survival curves, and the weights, behaviors and survivals of these mice were observed. The survival time was calculated from the day 0 since tumor inoculation to the day of death, and the Kaplan–Meier survival curves were made.
correction were used for comparisons between individual groups. A value of p < 0.05 was considered to be significant. 3. Results 3.1. Characterization of liposomes
2.11. In vivo TUNEL assay Apoptosis of tumor cells in vivo were identified by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) method (Wang et al., 2012; Zheng et al., 2012). Briefly, at days 10, 13, 16, and 19 post-inoculation, the tumorbearing mice were treated with physiological saline, vinblastine liposomes, vinblastine cationic liposomes, PNA modified vinblastine liposomes and PNA modified vinblastine cationic liposomes via tail vein injection at a dosage of 0.5 mg/kg vinblastine, respectively. At day 30 post-inoculation, three mice of each group were sacrificed by cervical dislocation. Tumor masses were carefully isolated, and tumor slices were prepared via frozen section technique (5 mm in thickness). Then, tumor slices were fixed in 4% paraformaldehyde for 20 min. After washing 3 times with PBS and incubated with 0.1% Triton X-100 for 2 min on ice, they were performed using the in situ cell death detection kit (Roche, Indianapolis, USA) according to the manufacturer’s protocol. The samples were treated with Hoechst 33342 (2 mg/ mL) for 10 min and then analyzed using a confocal laser scanning fluorescent microscopy (Leica, Heidelberg, Germany).
Fig. 1 shows the schematic representation of the enhanced inhibiting effects. Table 1 lists the measured encapsulation efficiency, particle size, polydensity index and zeta potential of the liposomes. In all liposomal formulations, the particle sizes were approximately 100 nm, and the encapsulation efficiencies of vinblastine were >91% with a narrow polydispersity index ( vinblastine cationic liposomes > PNA modified vinblastine liposomes > vinblastine liposomes > Blank PNA modified cationic liposomes. In addition, the blank PNA modified cationic liposomes exhibited minimal inhibitory effects to LLT cells. According to Fig. 4B, the
IC50 value of PNA modified vinblastine cationic liposomes was 1.43 0.16 mM, which was significantly lower than that of vinblastine liposomes (5.40 0.43 mM). 3.4. Cellular uptake and targeting effects in LLT cells Fig. 5A and B shows the cellular uptake in LLT cells after incubation with varying formulations using the fluorescent probe coumarin. According to Fig. 5B, the geometric mean intensity values in LLT cells after treatments with blank medium, coumarin liposomes, coumarin cationic liposomes, PNA modified coumarin liposomes and PNA modified coumarin cationic liposomes for 3 h were 5.23 0.01, 120.31 5.34, 198.25 8.34, 222.81 11.32 and 315.78 13.76, respectively. Fig. 5C illustrates the cellular targeting effects after treatments with varying formulations. Results showed that the fluorescence intensity rank was PNA modified coumarin cationic liposomes > PNA modified coumarin liposomes > coumarin cationic liposomes > coumarin liposomes. For
Fig. 3. Release rates of vinblastine in normal saline or in PBS solution containing 10% mouse plasma. Notes: A. Release rates of vinblastine from varying formulations in normal saline, B. release rates of vinblastine from varying formulations in PBS solution containing 10% mouse plasma. Data are presented as mean standard deviation (SD) (n = 3).
228
X.- Li et al. / International Journal of Pharmaceutics 487 (2015) 223–233
Fig. 4. Inhibitory effects and IC 50 values to LLT cells after treatments with varying formulations. Notes: A. Inhibitory effects to LLT cells, B. IC50 values. p < 0.05, a, vs. blank PNA modified cationic liposomes; b, vs. vinblastine liposomes; c, vs. vinblastine cationic liposomes; d, vs. PNA modified vinblastine liposomes. Data are presented as mean SD (n = 6).
all the liposomes, the highest fluorescence intensity in LLT cells after treatment with PNA modified coumarin cationic liposomes indicated that more drugs had been internalized in LLT cells. 3.5. Apoptotic enzymes activating assays Fig. 6 displays the regulating effects of pro-apoptotic proteins (Bak in Fig. 6A and Bok in Fig. 6B) and apoptotic enzymes (caspase 9 in Fig. 6C and caspase 3 in Fig. 6D) in LLT cells after treatments with varying formulations. After treatment with PNA modified vinblastine cationic liposomes, the activity ratios of Bak, Bok, caspase 9 and caspase 3 were 3.55 0.11, 2.65 0.21, 4.90 0.37 and 6.35 0.33, respectively. Results demonstrated that the PNA modified vinblastine cationic liposomes showed the strongest capability in activating both pro-apoptotic proteins and apoptotic enzymes in LLT cells. 3.6. Antitumor efficacy and in vivo imaging Fig. 7A indicates the antitumor efficacy in tumor-bearing mice after treatments with the experimental drug formulations. Compared with physiological saline, PNA modified vinblastine cationic liposomes showed significantly inhibition on tumor growth. Inhibitory ratios of tumor volumes at day 28 postincubation were 34.73 2.54% for free vinblastine, 52.66 4.34% for vinblastine liposomes, 60.55 7.83% for vinblastine cationic liposomes, 66.94 5.34% for PNA modified vinblastine liposomes and 79.98 6.55% for PNA modified vinblastine cationic liposomes, respectively. Fig. 7B shows the Kaplan–Meier survival curves of tumorbearing mice after treatments with the different types of liposomes. Results showed that the survival ranges were 25–37 days for physiological saline, 28–40 days for free vinblastine, 33–48 days for vinblastine liposomes, 39–54 days for vinblastine cationic liposomes, 38–58 days for PNA modified vinblastine liposomes and 43–67 days for PNA modified vinblastine cationic liposomes, respectively. The medium survival time of tumorbearing mice after treatment with PNA modified vinblastine cationic liposomes (57.33 days) was significantly longer than that in saline group (31.50 days, Supplementary Table S1). Fig. 7C shows the photographs of the tumor tissues isolated from tumor-bearing mice after treatments with physiological saline, free vinblastine, vinblastine liposomes, vinblastine cationic liposomes, PNA modified vinblastine liposomes and PNA modified vinblastine cationic liposomes, respectively. Fig. 7D shows the average weights of tumor tissues after treatments with the
experimental drug formulations. Results showed that the average weights of tumor tissues were 2.13 0.23 g for saline, 1.75 0.16 g for free vinblastine, 1.67 0.11 g for vinblastine liposomes, 0.94 0.02 g for vinblastine cationic liposomes, 0.87 0.08 g for PNA modified vinblastine liposomes and 0.53 0.05 g for PNA modified vinblastine cationic liposomes, respectively. Fig. 7E depicts the real-time distribution and accumulation ability of PNA modified Cy7 cationic liposomes in the xenografted tumors derived from LLT cells in C57BL/6 mice. After intravenous injection of PNA modified Cy7 cationic liposomes, a strong Cy7 fluorescent signal was observed in the whole blood circulatory system and the tumor location at early stage, and maintained up to 48 h in the tumor tissue. In contrast, after administration of free Cy7, the fluorescent signal was rapidly distributed in the liver at 3 h, and gradually weakened or disappeared by 12 h. Fig. 7F shows TUNEL assay for apoptotic cells in vivo after treatments with the experimental drug formulations. Results demonstrated that PNA modified vinblastine cationic liposomes induced most of apoptotic tumor cells, while only a few scattered TUNEL-positive cells were visible after treatment with vinblastine liposomes. The results were in accordance with the regulating effects to the apoptotic proteins in vitro. 4. Discussion Non-small cell lung cancer (NSCLC), the most common form of lung cancer, is one of the leading cause of cancer-related death in humans (Genestreti et al., 2014). Currently, chemotherapy has been considered to be the primary treatment strategy for patients with NSCLC. However, a regular chemotherapy is usually unsatisfactory for the serious side-effects and the limited ability to eradicate all the tumor cells. Consequently, a kind of PNA modified vinblastine cationic liposomes was developed in the present study for treating NSCLC. In the PNA modified vinblastine cationic liposomes, PNA was coupled on the surface of the liposomes by conjugating with DSPEPEG2000-NHS, and DC-Chol was used as cationic materials. Vinblastine was encapsulated in the aqueous core of the liposomes as an antitumor drug. According to Table 1, all the nanostructured liposomes showed ideal physicochemical characteristics by measuring mean particle size, zeta potential and encapsulation efficiency. The characterizations of the PNA modified vinblastine cationic liposomes were measured with the following aspects: smooth surface (Fig. 2B), small and well-distributed particle size (Fig. 2C), and positive potential (Fig. 2D). These suitable particle size and high encapsulation efficiency enable the PNA modified
X.- Li et al. / International Journal of Pharmaceutics 487 (2015) 223–233
229
Fig. 5. Intracellular uptake and targeting effects after incubation with varying formulations. (For interpretation of the references to color in the text, the reader is referred to the web version of this article.) Notes: A. Cellular uptake of LLT cells, B. fluorescent intensity of coumarin in LLT cells, C. laser scanning confocal microscopy images of LLT cells incubated with varying formulations. a. Blank control; b. coumarin liposomes; c. coumarin cationic liposomes; d. PNA modified coumarin liposomes; e. PNA modified coumarin cationic liposomes.
vinblastine cationic liposomes to spontaneously penetrate into interstitium of tumor via the enhanced permeability retention (EPR) effect (Maeda et al., 2000). The zeta potential value of PNA modified vinblastine liposomes was 0.08 0.01 mV, while that of PNA modified vinblastine cationic liposomes was increased to 38.42 2.54 mV as a result of the addition of DC-chol. The high zeta potential value is beneficial to transport the nanostructured liposomes across the cancer cells via electrostatic interaction (Jung et al., 2009). The in vitro release of vinblastine from the drug-loaded liposomes was measured at 37 C in normal saline and plasma-
containing PBS, respectively. For all the liposomal formulations, the in vitro release of vinblastine was
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
The antitumor activity of PNA modified vinblastine cationic liposomes on Lewis lung tumor cells: In vitro and in vivo evaluation Xue-tao Li a , Mei-li He a , Zhi-yan Zhou b , Ying Jiang a , Lan Cheng a, * a b
School of Pharmacy, Liaoning University of Traditional Chinese Medicine, Dalian 116600, China School of Stomatology, Jilin University, Changchun 130021, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 27 January 2015 Received in revised form 21 March 2015 Accepted 14 April 2015 Available online 17 April 2015
Non-small cell lung cancer (NSCLC) is one of the frequently-occurring disease in the world, and the treatment effects are usually unsatisfactory. Vinblastine is an anti-microtubule drug in clinic. In this study, a nanostructured liposome was designed and prepared for treating NSCLC. In the liposomes, peanut agglutinin (PNA) was modified on the liposomal surface, 3-(N-(N0 ,N0 -dimethylaminoethane) carbamoyl) cholesterol was used as cationic materials, and vinblastine was encapsulated in the aqueous core of liposomes, respectively. The PNA modified vinblastine cationic liposomes were approximately 100 nm in size with a positive potential. In vitro results showed that the targeting liposomes could significantly enhance cellular uptake, selectively accumulate in LLT cells, and dramatically initiate apoptosis via activating pro-apoptotic proteins and apoptotic enzymes, thus leading to the strongest antitumor efficacy to LLT cells. In vivo results demonstrated that the targeting liposomes could display a prolonged circulation time in the blood, accumulate more drug in tumor location, and induce most of tumor cells apoptosis. As a result, a robust overall antitumor efficacy in tumor-bearing mice was observed subsequently. In conclusion, the chemotherapy using the PNA modified vinblastine cationic liposomes could provide a potential strategy for treating non-small cell lung cancer. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Cationic liposomes Vinblastine Peanut agglutinin Non-small cell lung cancer
1. Introduction Lung cancer, the most common cause of cancer-related death in humans, is responsible for more than 1 million deaths worldwide annually (Cheng et al., 2014; Karachaliou and Rosell, 2014). There are two main types of lung cancer categorized by the size and appearance of the malignant cells, and the non-small cell lung cancer (NSCLC) is the most common type. Usually, treatment for NSCLC is a combined approach, including surgery, radiotherapy and chemotherapy, and it depends upon the histological type and the stage of cancer (Vasekar et al., 2014). However, surgery is only an option for patients in which the cancer cells have not spread beyond nearby lymph nodes. Furthermore, surgery and radiotherapy cannot eliminate all the cancer cells, thus leading to the fact that the five-year survival rate is less than 15% after comprehensive treatment (Vlahovic et al., 2007). Although chemotherapy is increasingly being used both pre-operatively and post-operatively,
* Corresponding author at: School of Pharmacy, Liaoning University of Traditional Chinese Medicine, Shengming 1 Road 77, Double D port, Dalian 116600, China. Tel.: +86 411 8589 0145; fax: +86 411 8589 0128. E-mail address: [email protected] (L. Cheng). http://dx.doi.org/10.1016/j.ijpharm.2015.04.035 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.
the treatment effects are usually unsatisfactory for the minimal accumulation in cancer cells and the serious side-effects. Therefore, developing a new chemotherapeutic strategy using nano-carriers is a critical issue for overcoming the limitation. Liposomes, an ideal drug carrier, are able to enhance the antitumor efficacy via improving the pharmaceutical properties of drugs and decreasing the systemic toxicity. Although liposomes have been successfully applied for several years, they are plagued by problems such as rapid uptake by the reticuloendothelial system (RES), poor active targeting, short half-life, easy leakage and main accumulation in liver or spleen (Andresen et al., 2005; Nag and Awasthi, 2013). PEGlyted liposomes could resolve these obstacles by providing a protective steric barrier against interaction with plasma proteins and cells of mononuclear phagocyte system (Sugiyama and Sadzuka, 2013). Cationic liposomes have been used as an excellent delivery carrier for antitumor drugs, and the cationic charge on the liposomal surface facilitates the intracellular uptake of the carrier via the electrostatic interaction with the anionic charge of cancer cell membrane (Han et al., 2014). Peanut agglutinin (PNA) is a plant lectin protein derived from the fruits of Arachis hypogaea, and is a common nutrient substance without obvious side-effects. PNA has good resistance to a variety of enzymes and exerts an ability of multivalent binding. Usually,
224
X.- Li et al. / International Journal of Pharmaceutics 487 (2015) 223–233
lectins recognize and bind particular sugar sequences in carbohydrates, and PNA is a targeting moiety that binds to b-D-galactosyl(1-3)-N-acetyl-D-galactosamine (Gal-b(1-3)GalNAc) specifically expressed on varying cancer cells (Cai and Zhang, 2005; Sakuma et al., 2011). Therefore, PNA is regularly used to be modified on the surface of nano-carriers to obtain a multiple active targeting effect. Vinblastine (VLB) is a vinca alkaloid and is used as an antimicrotubule drug in clinic. Vinblastine has been widely used to treat certain kinds of cancer, including non-small cell lung cancer, breast cancer, head and neck cancer, testicular cancer, and other solid tumors (Abbasov et al., 1972). Vinblastine binds tubulin, thereby inhibiting the assembly of microtubules (Kavallaris et al., 2001; Owellen et al., 1972). The treatment of vinblastine causes M phase specific cell cycle arrest by disrupting microtubule assembly and proper formation of the mitotic spindle and the kinetochore. In this study, we hypothesized that a kind of PNA modified vinblastine cationic liposomes would be able to target the cancer cells and treat NSCLC effectively. In the liposomes, PNA was modified on the surface of the liposomes for increasing intracellular uptake via the receptor-mediated endocytosis. 3-(N-(N0 ,N0 dimethylaminoethane)carbamoyl) cholesterol (DC-Chol) was used as cationic materials for targeting cancer cell membrane. Distearoyl phosphoethanolamine – PEG2000 (DSPE-PEG2000) was modified on the surface of liposomes for prolonging the circulation time. Vinblastine was encapsulated into the liposomal vesicles as an antitumor drug. The objectives of the present study were to develop the new type of targeting liposomes, to investigate their effects and action mechanism in vitro, and to assess their antitumor efficacies in tumor-bearing mice. 2. Materials and methods 2.1. Materials Vinblastine was purchased from Wuhan Jinnuo Chemicals (Wuhan, China). Egg phosphatidylcholine (EPC) and cholesterol were purchased from NOF Corporation (Tokyo, Japan). DSPEPEG2000 and N-hydroxysuccinimidyl-PEG2000-DSPE (DSPEPEG2000-NHS) were obtained from Nippon Fine Chemical Co., Ltd. (Osaka, Japan). DC-Chol was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USE). PNA was purchased from Shanghai BioSun Sci. & Tech. Co., Ltd. (Shanghai, China). Sephadex G-50 was purchased from Shanghai Chemical Technology (Shanghai, China). RPMI-1640 medium and fetal bovine serum (FBS) were purchased from Maichen Technology Co., Ltd. (Beijing, China). 2.2. Cells and animals Lewis lung tumor (LLT) cells were obtained from Cancer Institute & Hospital, Chinese Academy of Medical Sciences (Beijing, China), and were grown in RPMI-1640 supplemented with 10% FBS and antibiotics (penicillin 100 U/mL and streptomycin 100 mg/mL). Cells were cultured at 37 C with 5% CO2. C57BL/6 mice (16–18 g) were obtained from Dalian Medical University (Dalian, China). 2.3. Preparation of liposomes Blank PNA modified cationic liposomes were prepared using the film dispersion method (Du et al., 2009). Briefly, EPC, DC-Chol, DSPE-PEG2000 and DSPE-PEG2000-NHS (60:40:3:1, molar ratio) were dissolved in chloroform (10 mL), the solvent was evaporated using a rotary evaporator, and the lipid film was hydrated with 250 mM ammonium sulfate (5 mL) via sonication in a water bath at 40 C for 5 min. Subsequently, the suspensions were treated using an ultrasonic cell disruptor for 10 min (200 W). The obtained suspensions were extruded though polycarbonate membrane
using the pore sizes of 400 nm and 200 nm for three times, respectively. Then the blank liposomes were obtained. The drugloaded liposomes were prepared using an ammonium sulfate gradient loading method (Ju et al., 2014). In brief, the blank liposomes were further dialyzed (12,000–14,000, cut-off MW) in Hepes buffered saline (25 mM Hepes/150 mM NaCl) for 24 h. After dialysis, a certain amount of PNA (DSPE-PEG2000-NHS:PNA = 30:1, molar ratio) was added. The suspensions were stirred at room temperature for 10 h and then chromatography separation (Sephadex G-50) was performed to remove the unbound PNA. After the addition of free vinblastine (lipids:drug = 20:1, w/w), a volume of 5 mL blank liposomes was incubated in a water bath at 40 C with intermittent shaking for 30 min. Then the PNA modified vinblastine cationic liposomes were produced. Vinblastine cationic liposomes, PNA modified vinblastine liposomes, and vinblastine liposomes were prepared using the same procedures as described above, excluding the addition of DSPE-PEG2000-NHS and PNA, replacing DC-Chol with cholesterol or both. Besides, coumarin liposomes, coumarin cationic liposomes, PNA modified coumarin liposomes, PNA modified coumarin cationic liposomes (lipids:coumarin = 200:1, w/w), Cy7 (Invitrogen, Beijing, China) liposomes, and PNA modified Cy7 cationic liposomes (lipids:Cy7 = 300:1, w/w) were similarly prepared as the fluorescent probes for evaluating the targeting effects (Li et al., 2014b). 2.4. Characterization of the liposomes The particle size, polydispersity index (PDI) and zeta potential value were measured using a Nano Series Zen 4003 Zetasizer (Malvern Instruments Ltd., Malvern, UK). The morphology of PNA modified vinblastine cationic liposomes was observed using an atomic force microscopy (AFM; SPI3800N series SPA-400, NSK Ltd., Tokyo, Japan). Briefly, the PNA modified vinblastine cationic liposomes were diluted with distilled water and then placed on a silicon slice. The samples were air-dried and observed using AFM. The prepared liposomes were purified using a Sephadex G50 column equilibrated with PBS to remove the un-encapsulated vinblastine. The measurement of vinblastine was performed using a high-performance liquid chromatography (HPLC) system equipped with a UV detector (Agilent Technologies Inc., Cotati, CA, USA). The HPLC analysis was operated at 30 C with an ODS column (Diamonsil, 5 mm, 250 4.6 mm) at a wavelength of 264 nm. The mobile phase was consisted of acetonitrile, methanol, and 0.14 M diethylamine (pH 7.5) (14:54:32, v/v) with the flow rate of 1.0 mL/min (Volkov and Grodnitskaya, 1994; Zhou et al., 1990). Encapsulation efficiency (EE) of vinblastine was calculated using the formula: EE = (Wencap/Wtotal) 100%, where Wtotal and Wencap are the measured amount of vinblastine in the liposomal suspensions before and after passing over the Sephadex G-50 column, respectively (Li et al., 2011). PNA was measured by the BCA protein assay kit (Pierce Corporation, Beijing local agent, China). PNA coupling rate was calculated by the ratio of the PNA amount on the liposomes after separation to the added amount (Li et al., 2014a). 2.5. Release of vinblastine from liposomes The in vitro release of vinblastine from the drug-loaded liposomes was estimated as the previous reports (Guo et al., 2010). Briefly, the drug-loaded liposomes were dialyzed against normal saline or plasma-containing PBS (PBS containing 10% mouse plasma). A volume of 2.0 mL liposomes plus 2.0 mL release medium in dialysis tubing (12,000–14,000 molecular mass cutoff) was immersed in 20.0 mL of the release medium,
X.- Li et al. / International Journal of Pharmaceutics 487 (2015) 223–233
and oscillated with a shaker (100 times per minute) at 37 C. The release medium was sampled (0.5 mL) at 0, 4, 8, 12, 24 and 48 h, and was immediately replaced with an equal volume of fresh release medium after each sampling. The content of vinblastine in samples was measured using HPLC method as above described. The release rate (RR) was calculated with the formula: RR = (Wi/Wtotal) 100%, where Wi is the measured amount of vinblastine at the time-point of ith in release medium, and Wtotal is the total amount of vinblastine in the equal volume of liposomal suspensions prior to dialysis (Men et al., 2011). Each assay was repeated in triplicate.
2.6. Inhibitory effects of liposomes to LLT cells To evaluate the inhibitory effects of PNA modified vinblastine cationic liposomes, LLT cells were seeded in 96-well culture plates at a density of 5 103 cells/well, and cultured in serum-containing culture medium for 24 h. Then, the different concentrations of vinblastine were added into culture plates, respectively, including blank PNA modified cationic liposomes, vinblastine liposomes, vinblastine cationic liposomes, PNA modified vinblastine liposomes and PNA modified vinblastine cationic liposomes. The final concentration of vinblastine was 0.5, 1, 2, 4, 5, 8, 10 mM. Culture medium was used as blank control. After incubation for 48 h, the inhibitory effects were measured using sulforhodamine B (SRB) staining assay (Zhang et al., 2011). Briefly, the medium was removed, and the cells were fixed with 10% trichloroacetic aid (200 mL) for 1 h, washed for 5 times and stained with 0.4% SRB at room temperature. The absorbance was measured at 540 nm using a microplate reader (Tecan Infinite F50, Tecan Group Ltd., Shanghai, China). The survival rate was calculated using the following formula: survival% = (A540 nm for the treated cells/A540 nm for the control cells) 100%, where A540 nm is the absorbance value (Zhou et al., 2013). Each assay was repeated in triplicate. Finally, doseeffect curves were plotted.
2.7. Cellular uptake and targeting effects in LLT cells To evaluate the cellular uptake of LLT cells, LLT cells were seeded into 6-well culture plates at a density of 5 105 cells/well, and incubated for 24 h under the condition of 5% CO2 at 37 C. Then, the cells were treated with coumarin liposomes, coumarin cationic liposomes, PNA modified coumarin liposomes, and PNA modified coumarin cationic liposomes, respectively. Culture medium was used as blank control. Coumarin was used as a fluorescent probe (excitation at 488 nm, and emission at 530 nm), and the final concentration of coumarin was 1.0 mM. After incubation for another 3 h, the cells were washed 3 times with cold PBS, treated with 0.25% trypsin and harvested in 200 mL PBS. The fluorescence intensity of coumarin was analyzed using a FAScan flow cytometry (Becton Dickinson, San Jose, CA, USA) with 1 104 cells collected. To observe cellular distribution of PNA modified cationic liposomes, LLT cells were seeded into chambered coverslips at a density of 5 105 cells/well in 2 mL culture medium, and incubated for 24 h under the condition of 5% CO2 at 37 C. Subsequently, the cells were treated with coumarin liposomes, coumarin cationic liposomes, PNA modified coumarin liposomes and PNA modified coumarin cationic liposomes at a concentration of 0.5 mM coumarin for another 4 h. Control group was performed with culture medium. The cells were then washed 3 times with PBS, fixed with 4% paraformaldehyde for 10 min and stained with Hoechst 33258 (2 mg/mL) for 5 min. Finally, the samples were analyzed using a confocal laser scanning fluorescent microscopy (Leica, Heidelberg, Germany).
225
2.8. Apoptotic enzymes activity assays The regulating effects of PNA modified vinblastine cationic liposomes on pro-apoptotic proteins (Bak and Bok) and apoptotic enzymes (caspase 9 and caspase 3) in LLT cells were measured using an ELLSA kit (Cusabio Biotech Co., Ltd., Wuhan, China). Briefly, after incubation for 24 h under the condition of 5% CO2 at 37 C, the cells were treated with free vinblastine, vinblastine liposomes, vinblastine cationic liposomes, PNA modified vinblastine liposomes and PNA modified vinblastine cationic liposomes at a concentration of 10 mM vinblastine, respectively. Control experiment was performed with culture medium. After 12 h incubation, the cells were harvested and lysed, and the cell lysates were collected. The concentration of total proteins was measured with a bicinchoninic acid (BCA) kit at A562 nm according to manufacturer’s instructions of the kit. Samples were added to wells pre-coated with a monoclonal antibody, and incubated at 37 C for 2 h. Liquid samples were then removed and HRP-conjugate reagent was added into the wells, incubated for another 30 min at 37 C, followed by chromogen for 15 min. The reaction was terminated by addition of stop solution and immediately measured at 450 nm using a microplate reader (Tecan Infinite F50, Tecan Group Ltd., Shanghai, China). The protein ratio was calculated using the following formula: ratio% = (A450 nm for treated cells/A562 nm for treated cells)/(A450 nm for the control cells/A562 nm for the control cells), where A450 nm and A562 nm are absorbance values, respectively (Zhang et al., 2012). 2.9. In vivo imaging observation Non-invasive optical imaging systems were used to evaluate tumor accumulation ability of PNA modified cationic liposomes in tumor-bearing C57BL/6 mice, and Cy7 dye was used as the fluorescent probe. All procedures were preformed according to guidelines of the Institutional Authority for Laboratory Medicine. Briefly, approximately 1 107 LLT cells were re-suspended in 200 mL serum-free culture medium, and injected subcutaneously into the right flanks of the C57BL/6 mice. When the tumor reached approximately 400–500 mm3 in volume, the tumor-bearing mice were randomly divided into 4 groups (3 mice per group). Then, the tumor-bearing mice were administered physiological saline, free Cy7, Cy7 liposomes, and PNA modified Cy7 cationic liposomes via tail vein injection. The mice were anesthetized with isoflurane. Fluorescent images and X-ray images were captured at 1, 3, 6, 9, 12, 24 and 48 h using a Kodak multimodal imaging system (Carestream Health, Inc., USA) (Ma et al., 2013). 2.10. Antitumor efficacy in tumor-bearing mice Tumor-bearing C57BL/6 mice were used for investigating the antitumor efficacy in vivo. Briefly, the tumor-bearing mice were inoculated as above, and when the tumor reached about 300 mm3 in volume, the tumor-bearing mice were randomly divided into 6 groups (12 mice per group). At days 10, 13, 16, and 19 postinoculation, the tumor-bearing mice were treated with physiological saline, free vinblastine, vinblastine liposomes, vinblastine cationic liposomes, PNA modified vinblastine liposomes, and PNA modified vinblastine cationic liposomes via tail vein injection at a dosage of 0.5 mg/kg vinblastine, respectively. The tumor was measured every day with a caliper, and tumor volumes were calculated with the formula: V = length width2/2 (mm3) (n = 6). Tumor volume inhibitory rate = 100% (Vdrug/Vsaline) 100%, where Vdrug and Vsaline are the tumor volumes after treatment with drug and physiological saline, respectively. The mice monitored in tumor volume inhibitory were sacrificed at day 30 post-inoculation, and the tumors were removed and
226
X.- Li et al. / International Journal of Pharmaceutics 487 (2015) 223–233
Fig. 1. The schematic representation of the enhanced inhibiting effects.
photographed. Six tumor-bearing mice in each group were used for monitoring the survival curves, and the weights, behaviors and survivals of these mice were observed. The survival time was calculated from the day 0 since tumor inoculation to the day of death, and the Kaplan–Meier survival curves were made.
correction were used for comparisons between individual groups. A value of p < 0.05 was considered to be significant. 3. Results 3.1. Characterization of liposomes
2.11. In vivo TUNEL assay Apoptosis of tumor cells in vivo were identified by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) method (Wang et al., 2012; Zheng et al., 2012). Briefly, at days 10, 13, 16, and 19 post-inoculation, the tumorbearing mice were treated with physiological saline, vinblastine liposomes, vinblastine cationic liposomes, PNA modified vinblastine liposomes and PNA modified vinblastine cationic liposomes via tail vein injection at a dosage of 0.5 mg/kg vinblastine, respectively. At day 30 post-inoculation, three mice of each group were sacrificed by cervical dislocation. Tumor masses were carefully isolated, and tumor slices were prepared via frozen section technique (5 mm in thickness). Then, tumor slices were fixed in 4% paraformaldehyde for 20 min. After washing 3 times with PBS and incubated with 0.1% Triton X-100 for 2 min on ice, they were performed using the in situ cell death detection kit (Roche, Indianapolis, USA) according to the manufacturer’s protocol. The samples were treated with Hoechst 33342 (2 mg/ mL) for 10 min and then analyzed using a confocal laser scanning fluorescent microscopy (Leica, Heidelberg, Germany).
Fig. 1 shows the schematic representation of the enhanced inhibiting effects. Table 1 lists the measured encapsulation efficiency, particle size, polydensity index and zeta potential of the liposomes. In all liposomal formulations, the particle sizes were approximately 100 nm, and the encapsulation efficiencies of vinblastine were >91% with a narrow polydispersity index ( vinblastine cationic liposomes > PNA modified vinblastine liposomes > vinblastine liposomes > Blank PNA modified cationic liposomes. In addition, the blank PNA modified cationic liposomes exhibited minimal inhibitory effects to LLT cells. According to Fig. 4B, the
IC50 value of PNA modified vinblastine cationic liposomes was 1.43 0.16 mM, which was significantly lower than that of vinblastine liposomes (5.40 0.43 mM). 3.4. Cellular uptake and targeting effects in LLT cells Fig. 5A and B shows the cellular uptake in LLT cells after incubation with varying formulations using the fluorescent probe coumarin. According to Fig. 5B, the geometric mean intensity values in LLT cells after treatments with blank medium, coumarin liposomes, coumarin cationic liposomes, PNA modified coumarin liposomes and PNA modified coumarin cationic liposomes for 3 h were 5.23 0.01, 120.31 5.34, 198.25 8.34, 222.81 11.32 and 315.78 13.76, respectively. Fig. 5C illustrates the cellular targeting effects after treatments with varying formulations. Results showed that the fluorescence intensity rank was PNA modified coumarin cationic liposomes > PNA modified coumarin liposomes > coumarin cationic liposomes > coumarin liposomes. For
Fig. 3. Release rates of vinblastine in normal saline or in PBS solution containing 10% mouse plasma. Notes: A. Release rates of vinblastine from varying formulations in normal saline, B. release rates of vinblastine from varying formulations in PBS solution containing 10% mouse plasma. Data are presented as mean standard deviation (SD) (n = 3).
228
X.- Li et al. / International Journal of Pharmaceutics 487 (2015) 223–233
Fig. 4. Inhibitory effects and IC 50 values to LLT cells after treatments with varying formulations. Notes: A. Inhibitory effects to LLT cells, B. IC50 values. p < 0.05, a, vs. blank PNA modified cationic liposomes; b, vs. vinblastine liposomes; c, vs. vinblastine cationic liposomes; d, vs. PNA modified vinblastine liposomes. Data are presented as mean SD (n = 6).
all the liposomes, the highest fluorescence intensity in LLT cells after treatment with PNA modified coumarin cationic liposomes indicated that more drugs had been internalized in LLT cells. 3.5. Apoptotic enzymes activating assays Fig. 6 displays the regulating effects of pro-apoptotic proteins (Bak in Fig. 6A and Bok in Fig. 6B) and apoptotic enzymes (caspase 9 in Fig. 6C and caspase 3 in Fig. 6D) in LLT cells after treatments with varying formulations. After treatment with PNA modified vinblastine cationic liposomes, the activity ratios of Bak, Bok, caspase 9 and caspase 3 were 3.55 0.11, 2.65 0.21, 4.90 0.37 and 6.35 0.33, respectively. Results demonstrated that the PNA modified vinblastine cationic liposomes showed the strongest capability in activating both pro-apoptotic proteins and apoptotic enzymes in LLT cells. 3.6. Antitumor efficacy and in vivo imaging Fig. 7A indicates the antitumor efficacy in tumor-bearing mice after treatments with the experimental drug formulations. Compared with physiological saline, PNA modified vinblastine cationic liposomes showed significantly inhibition on tumor growth. Inhibitory ratios of tumor volumes at day 28 postincubation were 34.73 2.54% for free vinblastine, 52.66 4.34% for vinblastine liposomes, 60.55 7.83% for vinblastine cationic liposomes, 66.94 5.34% for PNA modified vinblastine liposomes and 79.98 6.55% for PNA modified vinblastine cationic liposomes, respectively. Fig. 7B shows the Kaplan–Meier survival curves of tumorbearing mice after treatments with the different types of liposomes. Results showed that the survival ranges were 25–37 days for physiological saline, 28–40 days for free vinblastine, 33–48 days for vinblastine liposomes, 39–54 days for vinblastine cationic liposomes, 38–58 days for PNA modified vinblastine liposomes and 43–67 days for PNA modified vinblastine cationic liposomes, respectively. The medium survival time of tumorbearing mice after treatment with PNA modified vinblastine cationic liposomes (57.33 days) was significantly longer than that in saline group (31.50 days, Supplementary Table S1). Fig. 7C shows the photographs of the tumor tissues isolated from tumor-bearing mice after treatments with physiological saline, free vinblastine, vinblastine liposomes, vinblastine cationic liposomes, PNA modified vinblastine liposomes and PNA modified vinblastine cationic liposomes, respectively. Fig. 7D shows the average weights of tumor tissues after treatments with the
experimental drug formulations. Results showed that the average weights of tumor tissues were 2.13 0.23 g for saline, 1.75 0.16 g for free vinblastine, 1.67 0.11 g for vinblastine liposomes, 0.94 0.02 g for vinblastine cationic liposomes, 0.87 0.08 g for PNA modified vinblastine liposomes and 0.53 0.05 g for PNA modified vinblastine cationic liposomes, respectively. Fig. 7E depicts the real-time distribution and accumulation ability of PNA modified Cy7 cationic liposomes in the xenografted tumors derived from LLT cells in C57BL/6 mice. After intravenous injection of PNA modified Cy7 cationic liposomes, a strong Cy7 fluorescent signal was observed in the whole blood circulatory system and the tumor location at early stage, and maintained up to 48 h in the tumor tissue. In contrast, after administration of free Cy7, the fluorescent signal was rapidly distributed in the liver at 3 h, and gradually weakened or disappeared by 12 h. Fig. 7F shows TUNEL assay for apoptotic cells in vivo after treatments with the experimental drug formulations. Results demonstrated that PNA modified vinblastine cationic liposomes induced most of apoptotic tumor cells, while only a few scattered TUNEL-positive cells were visible after treatment with vinblastine liposomes. The results were in accordance with the regulating effects to the apoptotic proteins in vitro. 4. Discussion Non-small cell lung cancer (NSCLC), the most common form of lung cancer, is one of the leading cause of cancer-related death in humans (Genestreti et al., 2014). Currently, chemotherapy has been considered to be the primary treatment strategy for patients with NSCLC. However, a regular chemotherapy is usually unsatisfactory for the serious side-effects and the limited ability to eradicate all the tumor cells. Consequently, a kind of PNA modified vinblastine cationic liposomes was developed in the present study for treating NSCLC. In the PNA modified vinblastine cationic liposomes, PNA was coupled on the surface of the liposomes by conjugating with DSPEPEG2000-NHS, and DC-Chol was used as cationic materials. Vinblastine was encapsulated in the aqueous core of the liposomes as an antitumor drug. According to Table 1, all the nanostructured liposomes showed ideal physicochemical characteristics by measuring mean particle size, zeta potential and encapsulation efficiency. The characterizations of the PNA modified vinblastine cationic liposomes were measured with the following aspects: smooth surface (Fig. 2B), small and well-distributed particle size (Fig. 2C), and positive potential (Fig. 2D). These suitable particle size and high encapsulation efficiency enable the PNA modified
X.- Li et al. / International Journal of Pharmaceutics 487 (2015) 223–233
229
Fig. 5. Intracellular uptake and targeting effects after incubation with varying formulations. (For interpretation of the references to color in the text, the reader is referred to the web version of this article.) Notes: A. Cellular uptake of LLT cells, B. fluorescent intensity of coumarin in LLT cells, C. laser scanning confocal microscopy images of LLT cells incubated with varying formulations. a. Blank control; b. coumarin liposomes; c. coumarin cationic liposomes; d. PNA modified coumarin liposomes; e. PNA modified coumarin cationic liposomes.
vinblastine cationic liposomes to spontaneously penetrate into interstitium of tumor via the enhanced permeability retention (EPR) effect (Maeda et al., 2000). The zeta potential value of PNA modified vinblastine liposomes was 0.08 0.01 mV, while that of PNA modified vinblastine cationic liposomes was increased to 38.42 2.54 mV as a result of the addition of DC-chol. The high zeta potential value is beneficial to transport the nanostructured liposomes across the cancer cells via electrostatic interaction (Jung et al., 2009). The in vitro release of vinblastine from the drug-loaded liposomes was measured at 37 C in normal saline and plasma-
containing PBS, respectively. For all the liposomal formulations, the in vitro release of vinblastine was
