International Journal of Biological Macromolecules 69 (2014) 532–541
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Chitosan cross-linked docetaxel loaded EGF receptor targeted nanoparticles for lung cancer cells S. Maya a , Bruno Sarmento b,c , Vinoth-Kumar Lakshmanan a , Deepthy Menon a , Vitor Seabra b , R. Jayakumar a,∗ a Amrita Centre for Nanosciences and Molecular Medicine, Amrita Institute of Medical Sciences and Research Centre, Amrita Vishwa Vidyapeetham University, Kochi 682041, India b IINFACTS, Department of Pharmaceutical Sciences, Instituto Superior de Ciências da Saúde, CESPU, Rua Central de Gandra, 1317, 4585-116 Gandra, Portugal c INEB, Institute of Biomedical Engineering, University of Porto, Rua do Campo Alegre, Porto, Portugal
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Article history: Received 24 March 2014 Received in revised form 16 May 2014 Accepted 5 June 2014 Available online 17 June 2014 Keywords: Targeted nanoparticles Poly(␥-glutamic acid) nanoparticles Chitosan Cetuximab Epidermal growth factor receptor
a b s t r a c t Lung cancer, associated with the up-regulated epidermal growth factor receptor (EGFR) led to the development of EGFR targeted anticancer therapeutics. The biopolymeric nanoparticles form an outstanding system for the targeted delivery of therapeutic agents. The present work evaluated the in vitro effects of chitosan cross-linked ␥-poly(glutamic acid) (␥-PGA) nanoparticles (Nps) loaded with docetaxel (DTXL) and decorated with Cetuximab (CET), targeted to EGFR over-expressing non-small-cell-lungcancer (NSCLC) cells (A549). CET-DTXL-␥-PGA Nps was prepared by ionic gelation and CET conjugation via EDC/NHS chemistry. EGFR specificity of targeted Nps was confirmed by the higher uptake rates of EGFR +ve A549 cells compared to that of EGFR −ve cells (NIH3T3). The cytotoxicity of Nps quantified using cell based (MTT/LDH) and flowcytometry (Cell-cycle analysis, Annexin V/PI and JC-1) assays showed superior antiproliferative activity of CET-DTXL-␥-PGA Nps over DTXL-␥-PGA Nps. The A549 cells treated with CET-DTXL-␥-PGA NPs underwent a G2/M phase cell cycle arrest followed by reduction in mitochondrial membrane potential of A549 cells, inducing apoptosis and necrosis resulting in enhanced cancer cell death. CET-DTXL-␥-PGA Nps exhibited enhanced cellular internalization and therapeutic activity, by actively targeting EGFR on NSCLC cells and hence could be an effective alternative to non-specific, conventional chemotherapy by increasing its efficiency by many folds. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Heavy toxicity burden and drug resistance from conventional chemotherapeutics and late stage diagnosis have made NSCLC a disease of high mortality with a median survival of less than a year [1–5]. The overall survival in lung cancer patients was improved with the introduction of third-generation cytotoxic drugs such as gemcitabine, vinorelbine, docetaxel and paclitaxel [6]. The disadvantage and serious adverse effects of conventional anticancer drug application lies in the fact that a fraction of the administered drug accumulates in normal tissue. Now the efficiency of the treatment modalities for NSCLC has reached a therapeutic plateau which urges for improvements in diagnostics and treatment of NSCLC [7–10].
∗ Corresponding author. Tel.: +91 484 2801234; fax: +91 484 2802020. E-mail addresses: [email protected], [email protected] (R. Jayakumar). http://dx.doi.org/10.1016/j.ijbiomac.2014.06.009 0141-8130/© 2014 Elsevier B.V. All rights reserved.
Currently targeted therapies inhibiting growth factor receptor systems, angiogenesis or both, are the focus of interest to improve the outcome of systemic therapy in patients with NSCLC [11–13]. The development of targeted therapies requires the identification of aberrant biochemical and molecular pathways which makes the cells metastatic. The genetic profiling showed that NSCLC is associated with 85% epidermal growth factor receptor (EGFR) expression and mutations in EGFR play a major role in the growth, invasion, metastasis and poor prognosis of the disease. Hence the identification of driver mutations as the primary oncogenic event led to the identification of EGFR as a target for therapeutic intervention in NSCLC treatment [14–16]. Anti-EGFR-targeted therapies based on monoclonal antibodies and tyrosine kinase inhibitors have improved the efficacy of conventional chemotherapy in both preclinical and clinical studies [17,18]. Cetuximab (Erbitux) is an immunoglobulin G1 mouse–human chimeric monoclonal antibody that targets human EGFR with high affinity and abrogates ligand-induced EGFR phosphorylation, thereby blocking EGFR signaling cascades. Cetuximab has been evaluated as a single agent
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or in combination for patients with advanced NSCLC in several phase II and two phase III trials [19,20]. Nanotechnology inculcates innovative strategies to overcome the limitations of conventional chemotherapeutics by entrapping the anticancer agents in biocompatible and biodegradable nanocarrier systems with varying architecture resulting in its controlled and specific release to the target cancer tissue [21]. ␥Poly(glutamic acid) (␥-PGA) are naturally occurring polyamides composed of amide linkages formed between the ␣-amino group and the ␥-carboxyl group in the polymer backbone [22,23]. The biodegradability and non-toxicity (as its breakdown product, glutamic acid, can enter normal cellular metabolism) of ␥-PGA made it a good candidate for various biological applications including sustained release material, drug carrier, curable biological adhesive, biodegradable fibers etc. It has carboxyl groups on the side chains that offer attachment points for the conjugation of chemotherapeutic agents, thereby rendering the drug more soluble and easier to administer [24,25]. Similarly chitosan in one among the very popular biopolymer which have been exploited as an efficient drug delivery carrier owing to its biocompatibility and biodegradability. This polycationic polysaccharide has been utilized in our study as a cross-linker wherein nanoparticles were formed by the electrostatic interaction between anionic ␥-PGA and cationic chitosan [26]. Our approach is to exploit nanotechnology in developing nanomedicines where ␥-PGA nanocarriers deliver the potent cytotoxic agent docetaxel (DTXL) in a targeted manner with the help of Cetuximab antibody conjugated to the Nps. The present work aims in studying the in vitro effects of targeted Nps (CET-DTXL-␥-PGA Nps) toward EGFR over expressing NSCLC cells compared to that of non-targeted Nps (DTXL-␥-PGA Nps). The potential of CET-DTXL-␥PGA Nps in actively targeting and inducing toxicity in A549 cells in receptor mediated fashion was evaluated and compared with EGFR −ve normal cells. Thus CET-DTXL-␥-PGA Nps has been proved to be advantageous as a targeted therapeutic nanomedicine for EGFR +ve NSCLC.
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FACS Aria II (Beckton and Dickinson, Sanjose, CA). Later the cells were analyzed under confocal microscopy for confirming the receptor expression. 2.3. In vitro targeting efficiency Uptake of targeted Nps (CET-␥-PGA Nps) by A549 (EGFR +ve) cells were quantified using flow cytometry and compared to that of NIH3T3 (EGFR −ve) cells by labeling the Nps with Rhodamine123 (Rho-123). 50,000 cells/well were seeded in a 24 well plate and treated with 0.1 mg/mL Rho-123-CET-␥-PGA Nps for 1 and 4 h. The flow cytometer measured the fluorescence intensity after excitation with a 488 nm argon laser using FACS Aria II (Beckton and Dickinson, Sanjose, CA) [27]. The efficiency of targeting EGFR by CET-␥-PGA Nps was further confirmed by analyzing the cellular uptake of these CET-␥-PGA Nps after pretreating with free cetuximab. After reaching confluence, cells were treated with 50 g of free CET for 2 h and then the media were removed followed by nontargeted and targeted Nps treatment for 1 and 4 h. The media were removed, washed well and the cells were trypsinized to measure the fluorescence intensity using flow cytometer. Similarly a qualitative analysis was performed to confirm the targeting efficiency of the CET conjugated Nps with confocal microscopy of A549 cells treated with AF-647-anti-EGFR antibody (633 laser excitation) and Rho-123-CET-␥-PGA Nps (488 laser excitation) simultaneously and also with individual pretreatments. After reaching confluence, one set of A549 cells were treated with a mixture of 20 g AF-647-anti-EGFR antibody and 100 g Rho-123CET-␥-PGA Nps for 4 h, second set of cells pretreated with 20 g AF-647-anti-EGFR antibody for 2 h followed by 100 g Rho-123CET-␥-PGA Nps for 4 h and third set of cells pretreated with 100 g Rho-123-CET-␥-PGA Nps for 2 h followed by 20 g AF-647-antiEGFR antibody for 4 h. Following sample treatments, the coverslips were taken out washed well with PBS to remove unbound particles and analyzed using confocal microscopy. 2.4. In vitro qualitative cellular uptake study
2. Materials and methods 2.1. Materials ␥-Poly glutamic acid (Mw —60 kDa) was purchased from Vedan (Taichung, Taiwan), Chitosan (Molecular Weight 100–150 kDa) from Koyo Chemical Co, Ltd., Japan and Docetaxel from AK Scientific, Inc, USA. Propidium iodide and RNAase for cell cycle analysis and Rhodamine-123 were purchased from Sigma Aldrich. Cetuximab (Erbitux) was kindly provided by Dr. Bruno Sarmento, University of Porto, Portugal. Alexa fluor-647 conjugated anti EGFR antibody (AF-647-anti EGFR antibody) was purchased from Santa Cruz, USA. The cell lines used for the study, viz A549 (NSCLC cells) and NIH3T3 (mouse fibroblast cells) were obtained from NCCS, Pune. Minimal Essential Medium (MEM) for culturing A549 and NIH3T3 cells were purchased from Sigma Aldrich. 2.2. EGFR expression by the cells Expression of EGFR by A549 and NIH3T3 cells were analyzed with AF-647-anti-EGFR antibody (Santa Cruz, USA) using flow cytometer and confocal microscopy. 105 cells were seeded in a 6well plate and after reaching confluence; cells were trypsinized, and re-suspended in 1% FBS (fetal bovine serum) containing phosphatebuffered saline (PBS; pH 7.4). Then 1 g of AF-647-anti EGFR antibody was added to the cell suspension and incubated at 37 ◦ C for 2 h. The cells were washed three times with PBS and analyzed using flow cytometer after excitation with a 633 nm laser using
Confocal microscopy qualitatively analyzed the internalization of Nps within EGFR +ve A549 cells. Cells were seeded onto coverslips and once confluent, cells were treated with 0.1 mg/mL Rho-123-CET-␥-PGA Nps for 6 h. Following sample incubation, cells were washed with PBS, fixed with PFA and mounted using DPX. Similarly Actin/DAPI staining of Rho-123-CET-␥-PGA Nps treated A549 cells were performed and imaged using confocal microscope. The cells were washed and permeabilized using 1% triton after treating with samples for 6 h. The fluorochrome for staining actin was added and incubated for 1 h followed by incubating the cells with DAPI. The cells were washed with PBS, dried and mounted using DPX. The slides were then viewed under the confocal microscope (Leica SP 5 II). 2.5. In vitro cytotoxicity assays 2.5.1. Live dead staining assay Live and dead A549 cells after treatment with Nps was qualitatively distinguished by sequential staining with acridine orange (AO) and ethidium bromide (EtBr). 25,000 A549 cells/coverslip were seeded onto the coverslips in 24 well plates followed by 24 h incubation with DTXL, DTXL-␥-PGA Nps and CET-DTXL-␥-PGA Nps. The cells were washed with PBS and stained with AO/EtBr (15 g/mL AO and 50 g/mL EtBr) for 20 min at room temperature and viewed under fluorescence microscope (Olympus). AO stains the live cells green whereas the EtBr labels the nuclei of dead cells red [28].
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2.5.2. MTT and LDH assays The cytotoxicity of CET-DTXL-␥-PGA Nps against A549 cells and NIH3T3 cells was evaluated by MTT assay and compared with that of DTXL-␥-PGA Nps. 104 cells/well were incubated with different concentrations of Nps (based on 20, 50, 100, 250 and 500 g/mL of DTXL) in media for 24 h and the cell viability was calculated according to standard MTT protocol [29]. The treated cells were incubated with MTT for 4 h, solubilized the formazan crystals and then absorbance were measured at a wavelength of 570 nm using a Beckmann Coulter Elisa plate reader (BioTek Power Wave XS). The toxicity induced by the targeted and non-targeted Nps was reconfirmed by LDH assay, where 104 A549 cells/well were incubated with Nps for 24 h followed by standard LDH protocol [30]. The 96 well plate was centrifuged at 300 × g for 10 min, 100 L supernatant was collected and placed on to another well plate containing 100 L of LDH reaction mixture for 30 min at 37 ◦ C. The plate was read at 490 nm using a micro plate reader. Triplicate samples were analyzed for each experiment. Cell viability was expressed as the percentage of the negative control calculated as Viability (%) =
N t
Nc
× 100;
Nt is the absorbance of cells treated with sample and Nc is the absorbance of the untreated cells.
glass slide and viewed under a fluorescent microscope for qualitative confirmation of cell death. 2.8. Mitochondrial membrane potential assay A549 cells were seeded at density of 105 per well in 6 well plates and treated with 0.25 mg/mL CET-DTXL-␥-PGA Nps and DTXL-␥-PGA Nps for 24 h after reaching 80% confluence. Mitochondrial membrane potential ( m) was determined based on JC-1 (5,5 ,6,6 -tetrachloro-1,1 ,3,3 tetraethylbenzimidazolylcarbocyanine iodide) assay according to the manufacturer’s instructions (BD Biosciences, San Diego, CA). The trypsinized cells after sample treatment were re-suspended in 0.5 mL of freshly prepared JC-1 working solution and incubated in a CO2 incubator at 37 ◦ C for 15 min followed by washing with assay buffer. Cells were resuspended in 0.5 mL assay buffer and analyzed by flow cytometer using green and red channel [33]. 2.9. Statistics analysis The experiments were carried out in triplicates and values were expressed as mean ± standard deviation (SD). A Student’s t-test was conducted to determine the significance. A probability level of p < 0.05 was considered to be statistically significant.
2.6. Cell cycle analysis 3. Results and discussions A549 cells (seeding density: 1 × 106 cells/well) were seeded in a 6 well plate until 80% confluence, followed by sample treatment (DTXL, DTXL-␥-PGA Nps and CET-DTXL-␥-PGA Nps) at a concentration of 0.25 mg/mL Nps for 12 h. After the required incubation time, cells were trypsinized and washed twice with cold PBS. The cells were fixed with 70% ice cold ethanol and stored at 4 ◦ C for 1 h. Centrifuge them at 1200 rpm for 5 min followed by 1 h incubation with 0.2 mg/mL of RNAase at 37 ◦ C. 10 g/mL of PI was added and the percentage of cells in each phase of the cell cycle were evaluated by a flow cytometer [31]. 2.7. Apoptosis assay The apoptotic or necrotic death induced by the Nps in A549 cells was measured using Annexin V-FITC/PI Vybrant apoptosis assay kit (Molecular probes, Eugene, OR). After reaching 80% confluence, the cells were treated with 0.25 mg/mL of CET-DTXL-␥-PGA Nps for 24 h. Cells were trypsinized and washed with ice-cold PBS and pelleted at 500 g for 5 min at 4 ◦ C. The samples were processed as described previously and the fluorescence intensity was analyzed by flow cytometry after excitation with a 488 nm argon laser [32]. Immediately after analysis, cell suspension was dropped on to a
Selectivity toward tumor tissue has become an important requirement for designing drug delivery systems wherein the ability of nanoparticles to passively (EPR effect) and actively (by specific ligands like folic acid, transferring, monoclonal antibodies etc.) target the tumor tissue are exploited to enhance the availability of anticancer drugs at the target site minimizing non-specific toxicity. The present work evaluated the in vitro targeting efficiency of DTXL loaded ␥-PGA Nps via CET conjugation toward EGFR over-expressing NSCLC cells (A549). The CET-DTXL-␥-PGA Nps was prepared by the simple technique of ionic gelation using chitosan as a cross-linker. Polycationic chitosan posses free NH2 group which is being utilized to cross-link with the carboxylic groups of ␥-PGA resulting in the formation of stable nanoparticles. The Nps was further surface conjugated with CET antibody exploiting the EDC/NHS chemistry. The synthesis and characterization of CET-DTXL-␥-PGA Nps has been recently reported by our group [27]. Non targeted DTXL-␥-PGA Nps (110 ± 40 nm) and targeted CET-DTXL-␥-PGA Nps (200 ± 20 nm) with −28 and −17 mV zeta potential, respectively, and with controlled release of DTXL was developed. CET was conjugated on to the DTXL-␥-PGA Nps with 42% efficiency [25]. Fig. 1 represents the SEM image of CET-DTXL-␥-PGA Nps.
Fig. 1. (A) SEM image of CET-DTXL-␥-PGA Nps showing 200–250 nm size Nps and (B) schematic representation of targeted CET-DTXL-␥-PGA Nps.
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Fig. 4. In vitro targeting (A) A549 cells treated with mixture of AF-647-anti-EGFR antibody and Rho123-CET-␥-PGA Nps (B) A549 cells pretreated with AF-647-anti-EGFR antibody followed by Rho123-CET-␥-PGA Nps treatment (C) A549 cells pretreated with Rho123-CET-␥-PGA Nps followed by AF-647-anti-EGFR antibody treatment.
the live and dead cells. Fig. 6A(a–d) shows the fluorescent microscopic images of control A549 cells, A549 cells treated with DTXL, non-targeted (DTXL-␥-PGA Nps) and targeted (CET-DTXL-␥-PGA Nps) Nps followed by AO/EtBr staining. The control cells appear green because at normal pH, AO fluoresce green, indicating live cells. Cells treated with DTXL appear red indicating dead cells where EtBr labels its nuclei red. Comparing the targeted and non-targeted Nps, cells treated with CET-DTXL-␥-PGA Nps showed enhanced cell death than with DTXL-␥-PGA Nps. Here AO shift its fluorescence from green at normal toward orange–red at acidic pH during apoptosis followed by EtBr labeling the nuclei of dead cells red [28].
Thus the cell viability assays supported the fact that enhanced cytotoxicity of CET-DTXL-␥-PGA Nps in EGFR +ve NSCLC cells was due to its targeted delivery through cellular binding and eventual uptake. Docetaxel is a well established potential antimicrotubule agent, which inhibit microtubule dynamics by stabilizing the tubulin polymers against depolymerization. Blocking microtubule dynamics impair mitotic progression, arresting the cell cycle, which prevents the cells to pass through the natural cell division process finally inhibiting cell proliferation. Here CETDTXL-␥-PGA Nps enhance the availability of DTXL to A549 cells more than that of non-targeted Nps, resulting in enhanced cell death [37,38].
3.4.2. MTT and LDH assays The cytotoxicity effect of free bare ␥-PGA Nps, DTXL-␥-PGA Nps and CET-␥-DTXL-␥-PGA Nps were evaluated using MTT assay (Fig. 6B(a and b)) for 24 h. The cell viability profiles of A549 (Fig. 6B(a)) and NIH3T3 (Fig. 6B(b)) exhibited dose-dependent cytotoxic effect by the targeted and non-targeted Nps. The doses for cytotoxic effect were determined based on IC50 values of these Nps. The CET-DTXL-␥-PGA Nps showed significant advantages in inhibiting cancer cell growth compared to DTXL-␥-PGA Nps. The IC50 value of CET-DTXL-␥-PGA Nps (50 g/mL) was 5 times less than that of DTXL-␥-PGA Nps (250 g/mL) for A549 cells (EGFR +ve). But there was no significant difference in the IC50 values of both targeted and non-targeted Nps toward normal NIH3T3 cells (250 g/mL). The carrier Nps (␥-PGA Nps) exhibited no toxicity in both cancer and normal cells. The cell viability based on membrane integrity can be primarily assessed by Lactate dehydrogenase (LDH) assay. The number of lysed cells is proportional to the amount of red formazan dye with absorption around 490 nm. The cytotoxicity based on the cell membrane integrity was further confirmed by the leakage of lactate dehydrogenase in the culture medium. The cell viability profile (Fig. 6B(c)) obtained from LDH assay also correlated MTT assay showing more cell death in cancer cells treated with the targeted Nps.
3.5. Flow cytomeric evaluation of cell death 3.5.1. Cell cycle analysis Flow cytometry determined the effect of targeted and nontargeted Nps on the cell cycle progression of A549 cells exposed to CET-DTXL-␥-PGA Nps and DTXL-␥-PGA Nps for 12 h. Fig. 7 represents the flow cytometry profile of cells in different phases of the cell cycle. Treatment with both CET-DTXL-␥-PGA Nps and DTXL␥-PGA Nps resulted in an arrest in G2/M phase followed by cancer cell death. The flow cytometry profile is divided into four quadrants four phases: P3 (G0/G1), P4 (S), P5 (G2/M) and P6 (dead cells). Compared to control cells, there was a reduction in the percentage of cells in the G0/G1 phase and an increase in the G2/M phase, when treated with Nps, indicating a mitotic arrest by the released DTXL which produces unstable microtubule, interfering with the mitotic spindle function, thereby arresting the cells in the G2/M phase of mitosis [39]. G2/M population (P5: 36%) and dead cells (P6: 23%) of A549 cells was higher when treated with CET-DTXL-␥-PGA Nps than that for cells treated with DTXL-␥-PGA Nps (P5: 29% and P6: 12%), indicating that targeted Nps exhibited higher arrest in the G2-M phase, followed by enhanced cancer cell death. The result indicated that EGFR +ve cells take up the targeted Nps (CET-DTXL␥-PGA Nps) more efficiently than the non-targeted (DTXL-␥-PGA)
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Fig. 5. (A) Qualitative analysis of cellular uptake by confocal microscopy (a) Control A549 cells alone (b) A549 cells treated with Rho123-CET-␥-PGA Nps (c) Representative Z-stacked images showing the section wise imaging of cellular localization of the Rho123-CET-␥-PGA Nps after 6 h treatment. Z stacking was performed for a depth of 16 m and 25 slices were obtained. (B) Confocal microscopic images of A549 cells stained with actin and DAPI. (a) Control A549 cells alone (b) A549 cells treated with Rho123-CET-␥-PGA Nps for 6 h.
Nps, causing G2/M phase arrest, inhibiting the cell division and further progressing into the death phase. 3.5.2. Annexin V-PI binding assay The ability of A549 cells to bind Annexin V/PI measured by flow cytometer correlated to the apoptotic or necrotic stage of A549 cells treated with CET-DTXL-␥-PGA Nps and DTXL-␥-PGA Nps. The scatter plot obtained provided four quadrants indicating different phenotypes: viable cells (Annexin −ve, PI −ve in the lower left quadrant Q3), early apoptotic cells (Annexin +ve, PI −ve in the lower right quadrant Q4), late apoptotic cells (Annexin +ve, PI +ve in the upper right quadrant Q2) and necrotic cells (PI +ve in the upper left quadrant Q1). Fig. 8A represents the scatter plot which showed 30% early apoptotic, 32% late apoptotic and 15% of necrotic cells when treated with CET-DTXL-␥-PGA Nps whereas only 22% early apoptotic, 3% late apoptotic and 5% of necrotic cells when treated with DTXL-␥-PGA Nps. The study showed that both CET-DTXL-␥PGA Nps and DTXL-␥-PGA Nps induced both necrotic and apoptotic cell death in A549 cells. But comparing targeted and non-targeted Nps, CET-DTXL-␥-PGA Nps induced significant enhancement in cancer cell death (Fig. 8B). From the fluorescent microscopic images (Fig. 8C), the early apoptotic cells clearly showed annexin V binding at the outer membrane which were stained green and late apoptotic or necrotic cells stained red due to PI.
Antimicrotubule agents are believed to phosphorylate Bcl-2, an important apoptosis regulator. The DTXL delivered by the targeted Nps to the EGFR +ve A549 cells causes taxane induced mitotic arrest in the G2/M cell cycle phase of the cells and also induces phosphorylation of genes regulating apoptosis (p21, p53, bcl-2) and these cascade of events resulted in apoptotic cell death. Our study via Annexin V/PI binding revealed co-existence of apoptotic and necrotic cell death of A549 cells after Nps treatment. Docetaxel is also reported to induce non-apoptotic cancer cell death (mitotic catastrophe, senescence and lytic necrosis) [40,41]. Taxanes are observed to induce dose- and cell line-specific mixtures of apoptotic and mitotic cell death [41]. At low concentrations, mitotic catastrophe and apoptosis are observed, whereas at high concentrations terminal mitotic arrest and necrosis are observed. So, here the Nps were taken up by A549 cells in EGFR specific (targeted Nps) pattern and the DTXL available to the cells might be in a concentration range inducing apoptotic as well as non apoptotic cell death. However, targeted Nps induce more cell death in EGFR +ve A549 cells than that of non-targeted Nps confirming the in vitro therapeutic activity of CET conjugated Nps. 3.5.3. JC-1 assay Mitochondria are the major cellular component involved in the intrinsic pathway of apoptosis and also DTXL is reported to act via
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Fig. 6. Cytotoxicity assays. (A) Live dead staining performed on A549 cells treated with (a) Control A549 cells, (b) DTXL, (c) DTXL-␥-PGA Nps and (d) CET-DTXL-␥-PGA Nps followed by sequential staining with AO and EtBr. (B) Cell viability profiles of (a) A549 cells and (b) NIH3T3 cells obtained from 24 h MTT assay. (c) Cell viability profile of A549 cells obtained from 24 h LDH assay. Data shown are the mean values ± SD (n = 3). * Represented the statistical significance with p < 0.05.
Fig. 7. Cell cycle analysis. Flow cytometric analysis of cell cycle progression of A549 cells treated with DTXL, DTXL-␥-PGA Nps and CET-DTXL-␥-PGA Nps for 24 h with percentage of cells in each phase depicted in corresponding chart.
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Fig. 8. Apoptosis/necrotic assay (A) Percentage of early apoptotic, late apoptotic and necrotic A549 cells represented in scatter plots (B) Graphical representation of flow cytometric quantification of cancer cell death (C and D) Fluorescent microscopic images of A549 cells labeled with Annexin V/PI. Annexin V labels green and PI labels red. Data shown are the mean values ± SD (n = 3). * Represented the statistical significance with p < 0.05.
Fig. 9. JC-1 assay for mitochondrial membrane potential ( m) analysis (A) Scatter plot showing percentage of A549 cells based on their mitochondrial membrane potential (B) Graphical representation of cancer cells with reduced m (C and D) Fluorescent microscopic images of A549 cells fluorescing red (polarized m) due to aggregated JC-1 and fluorescing green (unpolarized m) due to monomeric form of JC-1. Data shown are the mean values ± SD (n = 3). * Represented the statistical significance with p < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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the intrinsic mitochondrial pathway [31]. Flow cytometric evaluation of mitochondrial membrane potential was performed since the process of apoptosis is marked by the depletion of mitochondrial membrane depolarization. JC-1, a cationic fluorescent dye exhibited potential-dependent accumulation in the mitochondria, indicated by a fluorescence emission shift from green (∼525 nm, monomeric form in apoptotic cells) to red (∼590 nm, aggregate form in healthy cells). Both targeted and non-targeted Nps induced a reduction in the m but in comparison, there was a significant difference in the percentage of cells with membrane potential loss due to the treatments with CET-DTXL-␥-PGA Nps and DTXL-␥-PGA Nps. Scatter plot from flow cytometer (Fig. 9A) and the graphical representation of percentage of cells quantified by flow cytometer (Fig. 9B) showed that 25% of A549 cells treated with DTXL-␥-PGA Nps showed disrupted m, where as m of 59% cell was disrupted due to CET-DTXL-␥-PGA Nps treatment. The fluorescence microscopic image (Fig. 9C) very well distinguishes live cells with red fluorescence and dead cells fluorescing green. Thus the result referred to the enhanced uptake of targeted Nps by EGFR +ve A549 cells, resulting in disrupted mitochondrial membrane potential in higher number of cells compared to that by non-targeted Nps, thereby causing enhanced cancer cell death [31]. 4. Conclusions We have developed a targeted biocompatible nanosystem based on ␥-PGA and chitosan which is well tolerated and non-toxic. To prepare this nanosystem, chitosan was used as a cross-linker which is more biocompatible compared to other chemical cross-linkers. The DTXL-␥-PGA Nps prepared by the chitosan crosslinking was further surface modified with CET antibody using EDC/NHS chemistry and potential of these CET conjugated DTXL-␥-PGA Nps to serve as a receptor specific and selective drug delivery system has been analyzed by various in vitro cell culture experiments. The flow cytometric analysis showed differential cellular binding and uptake of the targeted Nps by EGFR +ve A549 cells compared to that of NIH3T3 (EGFR −ve) cells. The competitive binding of CET-DTXL␥-PGA Nps with free CET and free AF-647-anti-EGFR antibody has been proved by flow cytometry and confocal microscopy, thereby revealing CET mediated active targeting to EGFR over-expressing NSCLC cells. EGFR mediated internalization of the targeted CETDTXL-␥-PGA Nps was confirmed by confocal microscopy. The cytotoxicity assays showed enhanced cancer cell death by targeted Nps due to the selective delivery of DTXL. The targeted Nps induced a mitotic arrest in G2/M phase in higher number of A549 cells. CETDTXL-␥-PGA Nps reduced the mitochondrial membrane potential and resulted in inducing apoptotic and necrotic cancer cell death. Comparing targeted and non-targeted Nps CET-DTXL-␥-PGA Nps significantly enhanced cancer cell death than DTXL-␥-PGA Nps and hence proved to enhance receptor specific therapeutic potential for improving the treatment strategy for EGFR +ve NSCLC. This targeted nanoplatform based on bioplymeric carriers could further facilitate a treatment protocol for EGFR overexpressing NSCLC in combination with therapeutic agents (chemodrugs like taxanes and targeting agents like mAb). They enhance the cumulative therapeutic payload specifically at the tumor and after proper preclinical validation may lead to possible clinical application. Acknowledgements The authors acknowledge the Department of Science & Technology (DST), India and Portuguese Science Foundation (FCT), Portugal for providing financial support under Indo-Portugal Joint
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Program (Ref. no. INT/PORTUGAL/P-05/2009). This work was also partially supported by the Indian Council of Medical Research (Ref. No.35/11/2013-BMS). Author S. Maya is thankful to Council of Scientific and Industrial Research (CSIR), India for providing Senior Research Fellowship (SRF Award No: 9/963 (0012) 2K11-EMR-I). References [1] A. Jemal, M.J. Thun, L.A. Ries, H.L. Howe, H.K. Weir, M.M. Center, E. Ward, X.C. Wu, C. Eheman, R. Anderson, U.A. Ajani, B. Kohler, B.K. Edwards, J. Natl. Cancer Inst. 100 (2008) 1672–1694. [2] Y. Yang, Y.X. Hu, Y.H. Wang, J. Li, F. Liu, L. Huang, Mol. Pharmaceutics 9 (2012) 2280–2289. [3] P.P. Lv, W. Wei, H. Yue, T.Y. Yang, L.Y. Wang, G.H. Ma, Biomacromolecules 12 (2011) 4230–4239. [4] Y. Haijun, Z. Yonglong, J. Lei, Y. Qi, H. Xinyu, C. Lingli, Z. Zhiwen, G. Wangwen, L. Yaping, Biomaterials 34 (2013) 2738–2747. [5] R. Rosell, T.G. Bivona, N. Karachaliou, Lancet 382 (2013) 720–731. [6] F. Grossi, M. Aita, C. Defferrari, F. Rosetti, O. Vinante, G. Pappangallo, Oncologist 14 (2009) 497–510. [7] M. Reck, D.F. Heigener, T. Mok, J.C. Soria, K.F. Rabe, Lancet 382 (2013) 709–719. [8] W.J. Curran Jr., R. Paulus, C.J. Langer, R. Komaki, J.S. Lee, S. Hauser, J. Natl. Cancer Inst. 103 (2011) 1452–1460. [9] S. Shobha, D. Sarah, Thorac. Surg. Clin. 23 (2013) 411–419. [10] M. Soda, Y.L. Choi, M. Enomoto, S. Takada, Y. Yamashita, S. Ishikawa, M. Soda, Y.L. Choi, M. Enomoto, S. Takada, Y. Yamashita, S. Ishikawa, Nature 448 (2007) 561–566. [11] K. In-Young, K. Young-Sook, L. Doo-Sung, P. Heon-Joo, C. Eun-Kyung, O. YuKyoung, S. Hye-Jung, K. Jin-Seok, J. Controlled Release 140 (2009) 55–60. [12] L.R. Chirieac, S. Dacic, Surg. Pathol. Clin. 3 (2010) 71–82. [13] E.M. Toloza, T.A. D’Amico, Semin. Thorac. Cardiovasc. Surg. 17 (2005) 199–204. [14] J. Baselga, Signal 1 (2000) 12–21. [15] J. Baselga, C.L. Arteaga, J. Clin. Oncol. 23 (2005) 2445–2459. [16] J. Cadranel, A. Marie Ruppert, M. Beau-Faller, M. Wislez, Crit. Rev. Oncol./Hematol. 88 (2013) 477–493. [17] G.M. Stella, M. Luisetti, S. Inghilleri, F. Cemmi, R. Scabini, M. Zorzetto, E. Pozz, Respir. Med. 106 (2012) 173–183. [18] J. Baselga, Eur. J. Cancer 37 (2001) 16–22. [19] J. Capdevila, E. Elez, T. Macarulla, F.J. Ramos, M. Ruiz-Echarri, J. Tabernero, Cancer Treat. Rev. 35 (2009) 354–363. [20] S.S. Ramalingama, A. Kotsakisb, A.A. Tarhinic, D.E. Herond, R. Smithc, D. Friedlande, D.P. Petroe, L.E. Raezf, J.R. Brahmerg, J.S. Greenbergerd, S. Dacich, P. Hershbergeri, R.J. Landreneauj, J.D. Luketichj, C.P. Belanik, A. Argiris, Lung Cancer 81 (2013) 416–421. [21] S. Azarmi, W.H. Roa, R. Löbenberg, Adv. Drug Delivery Rev. 60 (2008) 863–875. [22] C. Li, Adv. Drug Delivery Rev. 54 (2001) 695–713. [23] T. Gonzalo, G. Lollo, M. Garcia-Fuentes, D. Torres, J. Correa, R. Riguera, E. Fernandez-Megia, P. Calvo, P. Avilés, M.J. Guillén, M.J. Alonso, J. Controlled Release 169 (2013) 10–16. [24] F. Hellmers, P. Ferguson, J. Koropatnick, R. Krull, A. Margaritis, Biochem. Eng. J. 75 (2013) 72–78. [25] M. Li, W. Song, Z. Tang, S. Lv, L. Lin, H. Sun, Q. Li, Y. Yang, H. Hong, X. Chen, ACS Appl. Mater. Interfaces 5 (2013) 1781–1792. [26] Y. Luo, Q. Wang, Int. J. Biol. Macromol. 64 (2014) 353–367. [27] S. Maya, B. Sarmento, V.K. Lakshmanan, D. Menon, S.V. Nair, R. Jayakumar, J. Biomed. Nanotechnol. 10 (2014) 1416–1428. [28] M. Valodkar, R.N. Jadeja, M.C. Thounaojam, R.V. Devkar, S. Thakore, Mater. Sci. Eng. C 31 (2011) 1723–1728. [29] A. Anitha, K.P. Chennazhi, S.V. Nair, R. Jayakumar, J. Biomed. Nanotechnol. 8 (2012) 29–42. [30] D. Menon, R.T. Thomas, S. Narayanan, S. Maya, R. Jayakumar, F. Hussain, V.K. Lakshmanan, S.V. Nair, Carbohydr. Polym. 84 (2011) 887–893. [31] Y. Luo, Y. Ling, W. Guo, J. Pang, W. Liu, Y. Fang, X. Wen, K. Wei, X. Gao, J. Controlled Release 147 (2010) 278–286. [32] S. Maya, G.K. Lekshmi, B. Sarmento, R.N. Sanoj, D. Menon, S.V. Nair, R. Jayakumar, Carbohydr. Polym. 93 (2013) 661–669. [33] B. Sun, S.S. Feng, Nanomedicine (Lond.) 4 (2009) 431–445. [34] J. Li, L. Zheng, H. Cai, W. Sun, M. Shen, G. Zhang, X. Shi, Biomaterials 34 (2013) 8382–8392. [35] V.G. Deepagan, B. Sarmento, D. Menon, A. Nascimento, J. Aswathy, S. Maya, K. Manzoor, S.V. Nair, R. Jayakumar, Nanomedicine 7 (2012) 507–519. [36] R.V. Kutty, S. Feng, Biomaterials 34 (2013) 10160–10171. [37] F.A. Shepherd, F.V. Fossella, T. Lynch, J.P. Armand, J.R. Rigas, M.G. Kris, Semin. Oncol. 28 (Suppl 2) (2001) 4–9. [38] C. Bernard, A. Fellous, A. Di Leo, Eur. J. Cancer 37 (2001) 182–183. [39] D. Liu, Z. Liu, L. Wang, C. Zhang, N. Zhang, Colloids Surf., B: Biointerfaces 85 (2011) 262–269. [40] K.J. Pienta, Semin. Oncol. 28 (2001) 3–7. [41] D.L. Morse, H. Gray, C.M. Payne, R.J. Gillies, Mol. Cancer Ther. 4 (2005) 1495–1504.
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Chitosan cross-linked docetaxel loaded EGF receptor targeted nanoparticles for lung cancer cells S. Maya a , Bruno Sarmento b,c , Vinoth-Kumar Lakshmanan a , Deepthy Menon a , Vitor Seabra b , R. Jayakumar a,∗ a Amrita Centre for Nanosciences and Molecular Medicine, Amrita Institute of Medical Sciences and Research Centre, Amrita Vishwa Vidyapeetham University, Kochi 682041, India b IINFACTS, Department of Pharmaceutical Sciences, Instituto Superior de Ciências da Saúde, CESPU, Rua Central de Gandra, 1317, 4585-116 Gandra, Portugal c INEB, Institute of Biomedical Engineering, University of Porto, Rua do Campo Alegre, Porto, Portugal
a r t i c l e
i n f o
Article history: Received 24 March 2014 Received in revised form 16 May 2014 Accepted 5 June 2014 Available online 17 June 2014 Keywords: Targeted nanoparticles Poly(␥-glutamic acid) nanoparticles Chitosan Cetuximab Epidermal growth factor receptor
a b s t r a c t Lung cancer, associated with the up-regulated epidermal growth factor receptor (EGFR) led to the development of EGFR targeted anticancer therapeutics. The biopolymeric nanoparticles form an outstanding system for the targeted delivery of therapeutic agents. The present work evaluated the in vitro effects of chitosan cross-linked ␥-poly(glutamic acid) (␥-PGA) nanoparticles (Nps) loaded with docetaxel (DTXL) and decorated with Cetuximab (CET), targeted to EGFR over-expressing non-small-cell-lungcancer (NSCLC) cells (A549). CET-DTXL-␥-PGA Nps was prepared by ionic gelation and CET conjugation via EDC/NHS chemistry. EGFR specificity of targeted Nps was confirmed by the higher uptake rates of EGFR +ve A549 cells compared to that of EGFR −ve cells (NIH3T3). The cytotoxicity of Nps quantified using cell based (MTT/LDH) and flowcytometry (Cell-cycle analysis, Annexin V/PI and JC-1) assays showed superior antiproliferative activity of CET-DTXL-␥-PGA Nps over DTXL-␥-PGA Nps. The A549 cells treated with CET-DTXL-␥-PGA NPs underwent a G2/M phase cell cycle arrest followed by reduction in mitochondrial membrane potential of A549 cells, inducing apoptosis and necrosis resulting in enhanced cancer cell death. CET-DTXL-␥-PGA Nps exhibited enhanced cellular internalization and therapeutic activity, by actively targeting EGFR on NSCLC cells and hence could be an effective alternative to non-specific, conventional chemotherapy by increasing its efficiency by many folds. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Heavy toxicity burden and drug resistance from conventional chemotherapeutics and late stage diagnosis have made NSCLC a disease of high mortality with a median survival of less than a year [1–5]. The overall survival in lung cancer patients was improved with the introduction of third-generation cytotoxic drugs such as gemcitabine, vinorelbine, docetaxel and paclitaxel [6]. The disadvantage and serious adverse effects of conventional anticancer drug application lies in the fact that a fraction of the administered drug accumulates in normal tissue. Now the efficiency of the treatment modalities for NSCLC has reached a therapeutic plateau which urges for improvements in diagnostics and treatment of NSCLC [7–10].
∗ Corresponding author. Tel.: +91 484 2801234; fax: +91 484 2802020. E-mail addresses: [email protected], [email protected] (R. Jayakumar). http://dx.doi.org/10.1016/j.ijbiomac.2014.06.009 0141-8130/© 2014 Elsevier B.V. All rights reserved.
Currently targeted therapies inhibiting growth factor receptor systems, angiogenesis or both, are the focus of interest to improve the outcome of systemic therapy in patients with NSCLC [11–13]. The development of targeted therapies requires the identification of aberrant biochemical and molecular pathways which makes the cells metastatic. The genetic profiling showed that NSCLC is associated with 85% epidermal growth factor receptor (EGFR) expression and mutations in EGFR play a major role in the growth, invasion, metastasis and poor prognosis of the disease. Hence the identification of driver mutations as the primary oncogenic event led to the identification of EGFR as a target for therapeutic intervention in NSCLC treatment [14–16]. Anti-EGFR-targeted therapies based on monoclonal antibodies and tyrosine kinase inhibitors have improved the efficacy of conventional chemotherapy in both preclinical and clinical studies [17,18]. Cetuximab (Erbitux) is an immunoglobulin G1 mouse–human chimeric monoclonal antibody that targets human EGFR with high affinity and abrogates ligand-induced EGFR phosphorylation, thereby blocking EGFR signaling cascades. Cetuximab has been evaluated as a single agent
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or in combination for patients with advanced NSCLC in several phase II and two phase III trials [19,20]. Nanotechnology inculcates innovative strategies to overcome the limitations of conventional chemotherapeutics by entrapping the anticancer agents in biocompatible and biodegradable nanocarrier systems with varying architecture resulting in its controlled and specific release to the target cancer tissue [21]. ␥Poly(glutamic acid) (␥-PGA) are naturally occurring polyamides composed of amide linkages formed between the ␣-amino group and the ␥-carboxyl group in the polymer backbone [22,23]. The biodegradability and non-toxicity (as its breakdown product, glutamic acid, can enter normal cellular metabolism) of ␥-PGA made it a good candidate for various biological applications including sustained release material, drug carrier, curable biological adhesive, biodegradable fibers etc. It has carboxyl groups on the side chains that offer attachment points for the conjugation of chemotherapeutic agents, thereby rendering the drug more soluble and easier to administer [24,25]. Similarly chitosan in one among the very popular biopolymer which have been exploited as an efficient drug delivery carrier owing to its biocompatibility and biodegradability. This polycationic polysaccharide has been utilized in our study as a cross-linker wherein nanoparticles were formed by the electrostatic interaction between anionic ␥-PGA and cationic chitosan [26]. Our approach is to exploit nanotechnology in developing nanomedicines where ␥-PGA nanocarriers deliver the potent cytotoxic agent docetaxel (DTXL) in a targeted manner with the help of Cetuximab antibody conjugated to the Nps. The present work aims in studying the in vitro effects of targeted Nps (CET-DTXL-␥-PGA Nps) toward EGFR over expressing NSCLC cells compared to that of non-targeted Nps (DTXL-␥-PGA Nps). The potential of CET-DTXL-␥PGA Nps in actively targeting and inducing toxicity in A549 cells in receptor mediated fashion was evaluated and compared with EGFR −ve normal cells. Thus CET-DTXL-␥-PGA Nps has been proved to be advantageous as a targeted therapeutic nanomedicine for EGFR +ve NSCLC.
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FACS Aria II (Beckton and Dickinson, Sanjose, CA). Later the cells were analyzed under confocal microscopy for confirming the receptor expression. 2.3. In vitro targeting efficiency Uptake of targeted Nps (CET-␥-PGA Nps) by A549 (EGFR +ve) cells were quantified using flow cytometry and compared to that of NIH3T3 (EGFR −ve) cells by labeling the Nps with Rhodamine123 (Rho-123). 50,000 cells/well were seeded in a 24 well plate and treated with 0.1 mg/mL Rho-123-CET-␥-PGA Nps for 1 and 4 h. The flow cytometer measured the fluorescence intensity after excitation with a 488 nm argon laser using FACS Aria II (Beckton and Dickinson, Sanjose, CA) [27]. The efficiency of targeting EGFR by CET-␥-PGA Nps was further confirmed by analyzing the cellular uptake of these CET-␥-PGA Nps after pretreating with free cetuximab. After reaching confluence, cells were treated with 50 g of free CET for 2 h and then the media were removed followed by nontargeted and targeted Nps treatment for 1 and 4 h. The media were removed, washed well and the cells were trypsinized to measure the fluorescence intensity using flow cytometer. Similarly a qualitative analysis was performed to confirm the targeting efficiency of the CET conjugated Nps with confocal microscopy of A549 cells treated with AF-647-anti-EGFR antibody (633 laser excitation) and Rho-123-CET-␥-PGA Nps (488 laser excitation) simultaneously and also with individual pretreatments. After reaching confluence, one set of A549 cells were treated with a mixture of 20 g AF-647-anti-EGFR antibody and 100 g Rho-123CET-␥-PGA Nps for 4 h, second set of cells pretreated with 20 g AF-647-anti-EGFR antibody for 2 h followed by 100 g Rho-123CET-␥-PGA Nps for 4 h and third set of cells pretreated with 100 g Rho-123-CET-␥-PGA Nps for 2 h followed by 20 g AF-647-antiEGFR antibody for 4 h. Following sample treatments, the coverslips were taken out washed well with PBS to remove unbound particles and analyzed using confocal microscopy. 2.4. In vitro qualitative cellular uptake study
2. Materials and methods 2.1. Materials ␥-Poly glutamic acid (Mw —60 kDa) was purchased from Vedan (Taichung, Taiwan), Chitosan (Molecular Weight 100–150 kDa) from Koyo Chemical Co, Ltd., Japan and Docetaxel from AK Scientific, Inc, USA. Propidium iodide and RNAase for cell cycle analysis and Rhodamine-123 were purchased from Sigma Aldrich. Cetuximab (Erbitux) was kindly provided by Dr. Bruno Sarmento, University of Porto, Portugal. Alexa fluor-647 conjugated anti EGFR antibody (AF-647-anti EGFR antibody) was purchased from Santa Cruz, USA. The cell lines used for the study, viz A549 (NSCLC cells) and NIH3T3 (mouse fibroblast cells) were obtained from NCCS, Pune. Minimal Essential Medium (MEM) for culturing A549 and NIH3T3 cells were purchased from Sigma Aldrich. 2.2. EGFR expression by the cells Expression of EGFR by A549 and NIH3T3 cells were analyzed with AF-647-anti-EGFR antibody (Santa Cruz, USA) using flow cytometer and confocal microscopy. 105 cells were seeded in a 6well plate and after reaching confluence; cells were trypsinized, and re-suspended in 1% FBS (fetal bovine serum) containing phosphatebuffered saline (PBS; pH 7.4). Then 1 g of AF-647-anti EGFR antibody was added to the cell suspension and incubated at 37 ◦ C for 2 h. The cells were washed three times with PBS and analyzed using flow cytometer after excitation with a 633 nm laser using
Confocal microscopy qualitatively analyzed the internalization of Nps within EGFR +ve A549 cells. Cells were seeded onto coverslips and once confluent, cells were treated with 0.1 mg/mL Rho-123-CET-␥-PGA Nps for 6 h. Following sample incubation, cells were washed with PBS, fixed with PFA and mounted using DPX. Similarly Actin/DAPI staining of Rho-123-CET-␥-PGA Nps treated A549 cells were performed and imaged using confocal microscope. The cells were washed and permeabilized using 1% triton after treating with samples for 6 h. The fluorochrome for staining actin was added and incubated for 1 h followed by incubating the cells with DAPI. The cells were washed with PBS, dried and mounted using DPX. The slides were then viewed under the confocal microscope (Leica SP 5 II). 2.5. In vitro cytotoxicity assays 2.5.1. Live dead staining assay Live and dead A549 cells after treatment with Nps was qualitatively distinguished by sequential staining with acridine orange (AO) and ethidium bromide (EtBr). 25,000 A549 cells/coverslip were seeded onto the coverslips in 24 well plates followed by 24 h incubation with DTXL, DTXL-␥-PGA Nps and CET-DTXL-␥-PGA Nps. The cells were washed with PBS and stained with AO/EtBr (15 g/mL AO and 50 g/mL EtBr) for 20 min at room temperature and viewed under fluorescence microscope (Olympus). AO stains the live cells green whereas the EtBr labels the nuclei of dead cells red [28].
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2.5.2. MTT and LDH assays The cytotoxicity of CET-DTXL-␥-PGA Nps against A549 cells and NIH3T3 cells was evaluated by MTT assay and compared with that of DTXL-␥-PGA Nps. 104 cells/well were incubated with different concentrations of Nps (based on 20, 50, 100, 250 and 500 g/mL of DTXL) in media for 24 h and the cell viability was calculated according to standard MTT protocol [29]. The treated cells were incubated with MTT for 4 h, solubilized the formazan crystals and then absorbance were measured at a wavelength of 570 nm using a Beckmann Coulter Elisa plate reader (BioTek Power Wave XS). The toxicity induced by the targeted and non-targeted Nps was reconfirmed by LDH assay, where 104 A549 cells/well were incubated with Nps for 24 h followed by standard LDH protocol [30]. The 96 well plate was centrifuged at 300 × g for 10 min, 100 L supernatant was collected and placed on to another well plate containing 100 L of LDH reaction mixture for 30 min at 37 ◦ C. The plate was read at 490 nm using a micro plate reader. Triplicate samples were analyzed for each experiment. Cell viability was expressed as the percentage of the negative control calculated as Viability (%) =
N t
Nc
× 100;
Nt is the absorbance of cells treated with sample and Nc is the absorbance of the untreated cells.
glass slide and viewed under a fluorescent microscope for qualitative confirmation of cell death. 2.8. Mitochondrial membrane potential assay A549 cells were seeded at density of 105 per well in 6 well plates and treated with 0.25 mg/mL CET-DTXL-␥-PGA Nps and DTXL-␥-PGA Nps for 24 h after reaching 80% confluence. Mitochondrial membrane potential ( m) was determined based on JC-1 (5,5 ,6,6 -tetrachloro-1,1 ,3,3 tetraethylbenzimidazolylcarbocyanine iodide) assay according to the manufacturer’s instructions (BD Biosciences, San Diego, CA). The trypsinized cells after sample treatment were re-suspended in 0.5 mL of freshly prepared JC-1 working solution and incubated in a CO2 incubator at 37 ◦ C for 15 min followed by washing with assay buffer. Cells were resuspended in 0.5 mL assay buffer and analyzed by flow cytometer using green and red channel [33]. 2.9. Statistics analysis The experiments were carried out in triplicates and values were expressed as mean ± standard deviation (SD). A Student’s t-test was conducted to determine the significance. A probability level of p < 0.05 was considered to be statistically significant.
2.6. Cell cycle analysis 3. Results and discussions A549 cells (seeding density: 1 × 106 cells/well) were seeded in a 6 well plate until 80% confluence, followed by sample treatment (DTXL, DTXL-␥-PGA Nps and CET-DTXL-␥-PGA Nps) at a concentration of 0.25 mg/mL Nps for 12 h. After the required incubation time, cells were trypsinized and washed twice with cold PBS. The cells were fixed with 70% ice cold ethanol and stored at 4 ◦ C for 1 h. Centrifuge them at 1200 rpm for 5 min followed by 1 h incubation with 0.2 mg/mL of RNAase at 37 ◦ C. 10 g/mL of PI was added and the percentage of cells in each phase of the cell cycle were evaluated by a flow cytometer [31]. 2.7. Apoptosis assay The apoptotic or necrotic death induced by the Nps in A549 cells was measured using Annexin V-FITC/PI Vybrant apoptosis assay kit (Molecular probes, Eugene, OR). After reaching 80% confluence, the cells were treated with 0.25 mg/mL of CET-DTXL-␥-PGA Nps for 24 h. Cells were trypsinized and washed with ice-cold PBS and pelleted at 500 g for 5 min at 4 ◦ C. The samples were processed as described previously and the fluorescence intensity was analyzed by flow cytometry after excitation with a 488 nm argon laser [32]. Immediately after analysis, cell suspension was dropped on to a
Selectivity toward tumor tissue has become an important requirement for designing drug delivery systems wherein the ability of nanoparticles to passively (EPR effect) and actively (by specific ligands like folic acid, transferring, monoclonal antibodies etc.) target the tumor tissue are exploited to enhance the availability of anticancer drugs at the target site minimizing non-specific toxicity. The present work evaluated the in vitro targeting efficiency of DTXL loaded ␥-PGA Nps via CET conjugation toward EGFR over-expressing NSCLC cells (A549). The CET-DTXL-␥-PGA Nps was prepared by the simple technique of ionic gelation using chitosan as a cross-linker. Polycationic chitosan posses free NH2 group which is being utilized to cross-link with the carboxylic groups of ␥-PGA resulting in the formation of stable nanoparticles. The Nps was further surface conjugated with CET antibody exploiting the EDC/NHS chemistry. The synthesis and characterization of CET-DTXL-␥-PGA Nps has been recently reported by our group [27]. Non targeted DTXL-␥-PGA Nps (110 ± 40 nm) and targeted CET-DTXL-␥-PGA Nps (200 ± 20 nm) with −28 and −17 mV zeta potential, respectively, and with controlled release of DTXL was developed. CET was conjugated on to the DTXL-␥-PGA Nps with 42% efficiency [25]. Fig. 1 represents the SEM image of CET-DTXL-␥-PGA Nps.
Fig. 1. (A) SEM image of CET-DTXL-␥-PGA Nps showing 200–250 nm size Nps and (B) schematic representation of targeted CET-DTXL-␥-PGA Nps.
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Fig. 4. In vitro targeting (A) A549 cells treated with mixture of AF-647-anti-EGFR antibody and Rho123-CET-␥-PGA Nps (B) A549 cells pretreated with AF-647-anti-EGFR antibody followed by Rho123-CET-␥-PGA Nps treatment (C) A549 cells pretreated with Rho123-CET-␥-PGA Nps followed by AF-647-anti-EGFR antibody treatment.
the live and dead cells. Fig. 6A(a–d) shows the fluorescent microscopic images of control A549 cells, A549 cells treated with DTXL, non-targeted (DTXL-␥-PGA Nps) and targeted (CET-DTXL-␥-PGA Nps) Nps followed by AO/EtBr staining. The control cells appear green because at normal pH, AO fluoresce green, indicating live cells. Cells treated with DTXL appear red indicating dead cells where EtBr labels its nuclei red. Comparing the targeted and non-targeted Nps, cells treated with CET-DTXL-␥-PGA Nps showed enhanced cell death than with DTXL-␥-PGA Nps. Here AO shift its fluorescence from green at normal toward orange–red at acidic pH during apoptosis followed by EtBr labeling the nuclei of dead cells red [28].
Thus the cell viability assays supported the fact that enhanced cytotoxicity of CET-DTXL-␥-PGA Nps in EGFR +ve NSCLC cells was due to its targeted delivery through cellular binding and eventual uptake. Docetaxel is a well established potential antimicrotubule agent, which inhibit microtubule dynamics by stabilizing the tubulin polymers against depolymerization. Blocking microtubule dynamics impair mitotic progression, arresting the cell cycle, which prevents the cells to pass through the natural cell division process finally inhibiting cell proliferation. Here CETDTXL-␥-PGA Nps enhance the availability of DTXL to A549 cells more than that of non-targeted Nps, resulting in enhanced cell death [37,38].
3.4.2. MTT and LDH assays The cytotoxicity effect of free bare ␥-PGA Nps, DTXL-␥-PGA Nps and CET-␥-DTXL-␥-PGA Nps were evaluated using MTT assay (Fig. 6B(a and b)) for 24 h. The cell viability profiles of A549 (Fig. 6B(a)) and NIH3T3 (Fig. 6B(b)) exhibited dose-dependent cytotoxic effect by the targeted and non-targeted Nps. The doses for cytotoxic effect were determined based on IC50 values of these Nps. The CET-DTXL-␥-PGA Nps showed significant advantages in inhibiting cancer cell growth compared to DTXL-␥-PGA Nps. The IC50 value of CET-DTXL-␥-PGA Nps (50 g/mL) was 5 times less than that of DTXL-␥-PGA Nps (250 g/mL) for A549 cells (EGFR +ve). But there was no significant difference in the IC50 values of both targeted and non-targeted Nps toward normal NIH3T3 cells (250 g/mL). The carrier Nps (␥-PGA Nps) exhibited no toxicity in both cancer and normal cells. The cell viability based on membrane integrity can be primarily assessed by Lactate dehydrogenase (LDH) assay. The number of lysed cells is proportional to the amount of red formazan dye with absorption around 490 nm. The cytotoxicity based on the cell membrane integrity was further confirmed by the leakage of lactate dehydrogenase in the culture medium. The cell viability profile (Fig. 6B(c)) obtained from LDH assay also correlated MTT assay showing more cell death in cancer cells treated with the targeted Nps.
3.5. Flow cytomeric evaluation of cell death 3.5.1. Cell cycle analysis Flow cytometry determined the effect of targeted and nontargeted Nps on the cell cycle progression of A549 cells exposed to CET-DTXL-␥-PGA Nps and DTXL-␥-PGA Nps for 12 h. Fig. 7 represents the flow cytometry profile of cells in different phases of the cell cycle. Treatment with both CET-DTXL-␥-PGA Nps and DTXL␥-PGA Nps resulted in an arrest in G2/M phase followed by cancer cell death. The flow cytometry profile is divided into four quadrants four phases: P3 (G0/G1), P4 (S), P5 (G2/M) and P6 (dead cells). Compared to control cells, there was a reduction in the percentage of cells in the G0/G1 phase and an increase in the G2/M phase, when treated with Nps, indicating a mitotic arrest by the released DTXL which produces unstable microtubule, interfering with the mitotic spindle function, thereby arresting the cells in the G2/M phase of mitosis [39]. G2/M population (P5: 36%) and dead cells (P6: 23%) of A549 cells was higher when treated with CET-DTXL-␥-PGA Nps than that for cells treated with DTXL-␥-PGA Nps (P5: 29% and P6: 12%), indicating that targeted Nps exhibited higher arrest in the G2-M phase, followed by enhanced cancer cell death. The result indicated that EGFR +ve cells take up the targeted Nps (CET-DTXL␥-PGA Nps) more efficiently than the non-targeted (DTXL-␥-PGA)
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Fig. 5. (A) Qualitative analysis of cellular uptake by confocal microscopy (a) Control A549 cells alone (b) A549 cells treated with Rho123-CET-␥-PGA Nps (c) Representative Z-stacked images showing the section wise imaging of cellular localization of the Rho123-CET-␥-PGA Nps after 6 h treatment. Z stacking was performed for a depth of 16 m and 25 slices were obtained. (B) Confocal microscopic images of A549 cells stained with actin and DAPI. (a) Control A549 cells alone (b) A549 cells treated with Rho123-CET-␥-PGA Nps for 6 h.
Nps, causing G2/M phase arrest, inhibiting the cell division and further progressing into the death phase. 3.5.2. Annexin V-PI binding assay The ability of A549 cells to bind Annexin V/PI measured by flow cytometer correlated to the apoptotic or necrotic stage of A549 cells treated with CET-DTXL-␥-PGA Nps and DTXL-␥-PGA Nps. The scatter plot obtained provided four quadrants indicating different phenotypes: viable cells (Annexin −ve, PI −ve in the lower left quadrant Q3), early apoptotic cells (Annexin +ve, PI −ve in the lower right quadrant Q4), late apoptotic cells (Annexin +ve, PI +ve in the upper right quadrant Q2) and necrotic cells (PI +ve in the upper left quadrant Q1). Fig. 8A represents the scatter plot which showed 30% early apoptotic, 32% late apoptotic and 15% of necrotic cells when treated with CET-DTXL-␥-PGA Nps whereas only 22% early apoptotic, 3% late apoptotic and 5% of necrotic cells when treated with DTXL-␥-PGA Nps. The study showed that both CET-DTXL-␥PGA Nps and DTXL-␥-PGA Nps induced both necrotic and apoptotic cell death in A549 cells. But comparing targeted and non-targeted Nps, CET-DTXL-␥-PGA Nps induced significant enhancement in cancer cell death (Fig. 8B). From the fluorescent microscopic images (Fig. 8C), the early apoptotic cells clearly showed annexin V binding at the outer membrane which were stained green and late apoptotic or necrotic cells stained red due to PI.
Antimicrotubule agents are believed to phosphorylate Bcl-2, an important apoptosis regulator. The DTXL delivered by the targeted Nps to the EGFR +ve A549 cells causes taxane induced mitotic arrest in the G2/M cell cycle phase of the cells and also induces phosphorylation of genes regulating apoptosis (p21, p53, bcl-2) and these cascade of events resulted in apoptotic cell death. Our study via Annexin V/PI binding revealed co-existence of apoptotic and necrotic cell death of A549 cells after Nps treatment. Docetaxel is also reported to induce non-apoptotic cancer cell death (mitotic catastrophe, senescence and lytic necrosis) [40,41]. Taxanes are observed to induce dose- and cell line-specific mixtures of apoptotic and mitotic cell death [41]. At low concentrations, mitotic catastrophe and apoptosis are observed, whereas at high concentrations terminal mitotic arrest and necrosis are observed. So, here the Nps were taken up by A549 cells in EGFR specific (targeted Nps) pattern and the DTXL available to the cells might be in a concentration range inducing apoptotic as well as non apoptotic cell death. However, targeted Nps induce more cell death in EGFR +ve A549 cells than that of non-targeted Nps confirming the in vitro therapeutic activity of CET conjugated Nps. 3.5.3. JC-1 assay Mitochondria are the major cellular component involved in the intrinsic pathway of apoptosis and also DTXL is reported to act via
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Fig. 6. Cytotoxicity assays. (A) Live dead staining performed on A549 cells treated with (a) Control A549 cells, (b) DTXL, (c) DTXL-␥-PGA Nps and (d) CET-DTXL-␥-PGA Nps followed by sequential staining with AO and EtBr. (B) Cell viability profiles of (a) A549 cells and (b) NIH3T3 cells obtained from 24 h MTT assay. (c) Cell viability profile of A549 cells obtained from 24 h LDH assay. Data shown are the mean values ± SD (n = 3). * Represented the statistical significance with p < 0.05.
Fig. 7. Cell cycle analysis. Flow cytometric analysis of cell cycle progression of A549 cells treated with DTXL, DTXL-␥-PGA Nps and CET-DTXL-␥-PGA Nps for 24 h with percentage of cells in each phase depicted in corresponding chart.
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Fig. 8. Apoptosis/necrotic assay (A) Percentage of early apoptotic, late apoptotic and necrotic A549 cells represented in scatter plots (B) Graphical representation of flow cytometric quantification of cancer cell death (C and D) Fluorescent microscopic images of A549 cells labeled with Annexin V/PI. Annexin V labels green and PI labels red. Data shown are the mean values ± SD (n = 3). * Represented the statistical significance with p < 0.05.
Fig. 9. JC-1 assay for mitochondrial membrane potential ( m) analysis (A) Scatter plot showing percentage of A549 cells based on their mitochondrial membrane potential (B) Graphical representation of cancer cells with reduced m (C and D) Fluorescent microscopic images of A549 cells fluorescing red (polarized m) due to aggregated JC-1 and fluorescing green (unpolarized m) due to monomeric form of JC-1. Data shown are the mean values ± SD (n = 3). * Represented the statistical significance with p < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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the intrinsic mitochondrial pathway [31]. Flow cytometric evaluation of mitochondrial membrane potential was performed since the process of apoptosis is marked by the depletion of mitochondrial membrane depolarization. JC-1, a cationic fluorescent dye exhibited potential-dependent accumulation in the mitochondria, indicated by a fluorescence emission shift from green (∼525 nm, monomeric form in apoptotic cells) to red (∼590 nm, aggregate form in healthy cells). Both targeted and non-targeted Nps induced a reduction in the m but in comparison, there was a significant difference in the percentage of cells with membrane potential loss due to the treatments with CET-DTXL-␥-PGA Nps and DTXL-␥-PGA Nps. Scatter plot from flow cytometer (Fig. 9A) and the graphical representation of percentage of cells quantified by flow cytometer (Fig. 9B) showed that 25% of A549 cells treated with DTXL-␥-PGA Nps showed disrupted m, where as m of 59% cell was disrupted due to CET-DTXL-␥-PGA Nps treatment. The fluorescence microscopic image (Fig. 9C) very well distinguishes live cells with red fluorescence and dead cells fluorescing green. Thus the result referred to the enhanced uptake of targeted Nps by EGFR +ve A549 cells, resulting in disrupted mitochondrial membrane potential in higher number of cells compared to that by non-targeted Nps, thereby causing enhanced cancer cell death [31]. 4. Conclusions We have developed a targeted biocompatible nanosystem based on ␥-PGA and chitosan which is well tolerated and non-toxic. To prepare this nanosystem, chitosan was used as a cross-linker which is more biocompatible compared to other chemical cross-linkers. The DTXL-␥-PGA Nps prepared by the chitosan crosslinking was further surface modified with CET antibody using EDC/NHS chemistry and potential of these CET conjugated DTXL-␥-PGA Nps to serve as a receptor specific and selective drug delivery system has been analyzed by various in vitro cell culture experiments. The flow cytometric analysis showed differential cellular binding and uptake of the targeted Nps by EGFR +ve A549 cells compared to that of NIH3T3 (EGFR −ve) cells. The competitive binding of CET-DTXL␥-PGA Nps with free CET and free AF-647-anti-EGFR antibody has been proved by flow cytometry and confocal microscopy, thereby revealing CET mediated active targeting to EGFR over-expressing NSCLC cells. EGFR mediated internalization of the targeted CETDTXL-␥-PGA Nps was confirmed by confocal microscopy. The cytotoxicity assays showed enhanced cancer cell death by targeted Nps due to the selective delivery of DTXL. The targeted Nps induced a mitotic arrest in G2/M phase in higher number of A549 cells. CETDTXL-␥-PGA Nps reduced the mitochondrial membrane potential and resulted in inducing apoptotic and necrotic cancer cell death. Comparing targeted and non-targeted Nps CET-DTXL-␥-PGA Nps significantly enhanced cancer cell death than DTXL-␥-PGA Nps and hence proved to enhance receptor specific therapeutic potential for improving the treatment strategy for EGFR +ve NSCLC. This targeted nanoplatform based on bioplymeric carriers could further facilitate a treatment protocol for EGFR overexpressing NSCLC in combination with therapeutic agents (chemodrugs like taxanes and targeting agents like mAb). They enhance the cumulative therapeutic payload specifically at the tumor and after proper preclinical validation may lead to possible clinical application. Acknowledgements The authors acknowledge the Department of Science & Technology (DST), India and Portuguese Science Foundation (FCT), Portugal for providing financial support under Indo-Portugal Joint
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Program (Ref. no. INT/PORTUGAL/P-05/2009). This work was also partially supported by the Indian Council of Medical Research (Ref. No.35/11/2013-BMS). Author S. Maya is thankful to Council of Scientific and Industrial Research (CSIR), India for providing Senior Research Fellowship (SRF Award No: 9/963 (0012) 2K11-EMR-I). References [1] A. Jemal, M.J. Thun, L.A. Ries, H.L. Howe, H.K. Weir, M.M. Center, E. Ward, X.C. Wu, C. Eheman, R. Anderson, U.A. Ajani, B. Kohler, B.K. Edwards, J. Natl. Cancer Inst. 100 (2008) 1672–1694. [2] Y. Yang, Y.X. Hu, Y.H. Wang, J. Li, F. Liu, L. Huang, Mol. Pharmaceutics 9 (2012) 2280–2289. [3] P.P. Lv, W. Wei, H. Yue, T.Y. Yang, L.Y. Wang, G.H. Ma, Biomacromolecules 12 (2011) 4230–4239. [4] Y. Haijun, Z. Yonglong, J. Lei, Y. Qi, H. Xinyu, C. Lingli, Z. Zhiwen, G. Wangwen, L. Yaping, Biomaterials 34 (2013) 2738–2747. [5] R. Rosell, T.G. Bivona, N. Karachaliou, Lancet 382 (2013) 720–731. [6] F. Grossi, M. Aita, C. Defferrari, F. Rosetti, O. Vinante, G. Pappangallo, Oncologist 14 (2009) 497–510. [7] M. Reck, D.F. Heigener, T. Mok, J.C. Soria, K.F. Rabe, Lancet 382 (2013) 709–719. [8] W.J. Curran Jr., R. Paulus, C.J. Langer, R. Komaki, J.S. Lee, S. Hauser, J. Natl. Cancer Inst. 103 (2011) 1452–1460. [9] S. Shobha, D. Sarah, Thorac. Surg. Clin. 23 (2013) 411–419. [10] M. Soda, Y.L. Choi, M. Enomoto, S. Takada, Y. Yamashita, S. Ishikawa, M. Soda, Y.L. Choi, M. Enomoto, S. Takada, Y. Yamashita, S. Ishikawa, Nature 448 (2007) 561–566. [11] K. In-Young, K. Young-Sook, L. Doo-Sung, P. Heon-Joo, C. Eun-Kyung, O. YuKyoung, S. Hye-Jung, K. Jin-Seok, J. Controlled Release 140 (2009) 55–60. [12] L.R. Chirieac, S. Dacic, Surg. Pathol. Clin. 3 (2010) 71–82. [13] E.M. Toloza, T.A. D’Amico, Semin. Thorac. Cardiovasc. Surg. 17 (2005) 199–204. [14] J. Baselga, Signal 1 (2000) 12–21. [15] J. Baselga, C.L. Arteaga, J. Clin. Oncol. 23 (2005) 2445–2459. [16] J. Cadranel, A. Marie Ruppert, M. Beau-Faller, M. Wislez, Crit. Rev. Oncol./Hematol. 88 (2013) 477–493. [17] G.M. Stella, M. Luisetti, S. Inghilleri, F. Cemmi, R. Scabini, M. Zorzetto, E. Pozz, Respir. Med. 106 (2012) 173–183. [18] J. Baselga, Eur. J. Cancer 37 (2001) 16–22. [19] J. Capdevila, E. Elez, T. Macarulla, F.J. Ramos, M. Ruiz-Echarri, J. Tabernero, Cancer Treat. Rev. 35 (2009) 354–363. [20] S.S. Ramalingama, A. Kotsakisb, A.A. Tarhinic, D.E. Herond, R. Smithc, D. Friedlande, D.P. Petroe, L.E. Raezf, J.R. Brahmerg, J.S. Greenbergerd, S. Dacich, P. Hershbergeri, R.J. Landreneauj, J.D. Luketichj, C.P. Belanik, A. Argiris, Lung Cancer 81 (2013) 416–421. [21] S. Azarmi, W.H. Roa, R. Löbenberg, Adv. Drug Delivery Rev. 60 (2008) 863–875. [22] C. Li, Adv. Drug Delivery Rev. 54 (2001) 695–713. [23] T. Gonzalo, G. Lollo, M. Garcia-Fuentes, D. Torres, J. Correa, R. Riguera, E. Fernandez-Megia, P. Calvo, P. Avilés, M.J. Guillén, M.J. Alonso, J. Controlled Release 169 (2013) 10–16. [24] F. Hellmers, P. Ferguson, J. Koropatnick, R. Krull, A. Margaritis, Biochem. Eng. J. 75 (2013) 72–78. [25] M. Li, W. Song, Z. Tang, S. Lv, L. Lin, H. Sun, Q. Li, Y. Yang, H. Hong, X. Chen, ACS Appl. Mater. Interfaces 5 (2013) 1781–1792. [26] Y. Luo, Q. Wang, Int. J. Biol. Macromol. 64 (2014) 353–367. [27] S. Maya, B. Sarmento, V.K. Lakshmanan, D. Menon, S.V. Nair, R. Jayakumar, J. Biomed. Nanotechnol. 10 (2014) 1416–1428. [28] M. Valodkar, R.N. Jadeja, M.C. Thounaojam, R.V. Devkar, S. Thakore, Mater. Sci. Eng. C 31 (2011) 1723–1728. [29] A. Anitha, K.P. Chennazhi, S.V. Nair, R. Jayakumar, J. Biomed. Nanotechnol. 8 (2012) 29–42. [30] D. Menon, R.T. Thomas, S. Narayanan, S. Maya, R. Jayakumar, F. Hussain, V.K. Lakshmanan, S.V. Nair, Carbohydr. Polym. 84 (2011) 887–893. [31] Y. Luo, Y. Ling, W. Guo, J. Pang, W. Liu, Y. Fang, X. Wen, K. Wei, X. Gao, J. Controlled Release 147 (2010) 278–286. [32] S. Maya, G.K. Lekshmi, B. Sarmento, R.N. Sanoj, D. Menon, S.V. Nair, R. Jayakumar, Carbohydr. Polym. 93 (2013) 661–669. [33] B. Sun, S.S. Feng, Nanomedicine (Lond.) 4 (2009) 431–445. [34] J. Li, L. Zheng, H. Cai, W. Sun, M. Shen, G. Zhang, X. Shi, Biomaterials 34 (2013) 8382–8392. [35] V.G. Deepagan, B. Sarmento, D. Menon, A. Nascimento, J. Aswathy, S. Maya, K. Manzoor, S.V. Nair, R. Jayakumar, Nanomedicine 7 (2012) 507–519. [36] R.V. Kutty, S. Feng, Biomaterials 34 (2013) 10160–10171. [37] F.A. Shepherd, F.V. Fossella, T. Lynch, J.P. Armand, J.R. Rigas, M.G. Kris, Semin. Oncol. 28 (Suppl 2) (2001) 4–9. [38] C. Bernard, A. Fellous, A. Di Leo, Eur. J. Cancer 37 (2001) 182–183. [39] D. Liu, Z. Liu, L. Wang, C. Zhang, N. Zhang, Colloids Surf., B: Biointerfaces 85 (2011) 262–269. [40] K.J. Pienta, Semin. Oncol. 28 (2001) 3–7. [41] D.L. Morse, H. Gray, C.M. Payne, R.J. Gillies, Mol. Cancer Ther. 4 (2005) 1495–1504.
