Interleukin-4 receptor-targeted liposomal doxorubicin as a model for enhancing cellular uptake and antitumor efficacy in murine colorectal cancer

Abstract

Our previous studies showed that colorectal tumor has high interleukin-4 receptor α (IL-4Rα) expression, whereas adjacent normal tissue has low or no IL-4Rα expression. We also observed that human atherosclerotic plaque-specific peptide-1 (AP1) can specifically target to IL-4Rα. In this study, we investigated the therapeutic efficacy and systemic toxicity of AP1-conjuagted liposomal doxorubicin. AP1 bound more strongly to and was more efficiently internalized into IL-4Rα-overexpressing CT26 cells than CT26 control cells. Selective cytotoxicity experiment revealed that AP1-conjugated liposomal doxorubicin preferentially killed IL-4Rα-overexpressing CT26 cells. AP1-conjugated liposomal doxorubicin administered intravenously into mice produced significant inhibition of tumor growth and showed decreased cardiotoxicity of doxorubicin. These results indicated that AP1-conjugated liposomal doxorubicin has a potent and selective anticancer potential against IL-4Rα-overexpressing colorectal cancer cells, thus providing a model for targeted anticancer therapy.

Introduction

Colorectal cancer (CRC) is one of the most common cancers in the world and is a significant cause of morbidity and mortality.¹ The cause of CRC mortality is mainly due to resistance to treatment and occurrence of distant metastasis. Approximately 45% CRC patients have regional or distant metastasis at the time of presentation.^2,3 Prognosis is not satisfactory in this group of patients under modern treatment strategy and effective therapy may depend on development of a more specific agent targeting the primary and metastatic tumor cells.

The IL-4Rα is the receptor for IL-4 and IL-13 and couples to the JAK1/2/3-STAT6 pathway.⁴ The IL4/IL13 responses are involved in regulating IgE production as well as chemokine and mucus production at sites of allergic inflammation. In certain cell types, IL4/IL13 responses are triggered through activation of insulin receptor substrates, IRS1/IRS2.^4,5 Over the past few years, several in vitro and in vivo studies showed that IL-4 can be produced as autocrine by various types of cancer such as colon, thyroid, breast and lung, and plays crucial role in anti-apoptosis contributing to tumor growth, chemotherapy-induced cell death and resistance to death receptors.^6,7 In addition, recent study shows myeloid IL-4R plays a role in tumor metastasis in mouse model of breast cancer.⁸ Exogenous IL-4Rα expression can promote colon tumor proliferation and growth in tumor cells and IL-4Rα -null mice experiments.^9,10

Increased interleukin-4 receptor α (IL-4Rα) expression has been demonstrated in many human cancers including AIDS-related Kaposi's sarcoma,¹¹ breast carcinoma,^12,13 melanoma,¹² renal cell carcinoma,¹⁴ ovarian carcinoma,^12,15 glioblastoma,¹⁶ and head and neck squamous cell carcinoma.¹⁷ and CRC.^9,18 IL-4Rα is associated with adenoma-carcinoma progression and lymph node metastases in CRC.^19,20 In addition, exogenous IL-4Rα expression can promote colon cancer cell proliferation and growth.^9,10 On the other hand, clinical trial on anti-IL-4 in respiratory diseases has already been carried out and no significant side effects were observed.^21,22 Also, several studies revealed IL-4Rα as an effective target and has being applied in clinic for different cancer therapy.^18,23-27

Liposomal drug delivery systems have been instrumental in elevating antitumor drug levels with limited systemic drug exposure and toxicity.²⁸ These are already used in standard clinical therapies that provide considerable benefits to patients, one example is Doxil®, a liposomal form of doxorubicin (DOX) for the treatment of ovarian and some other types of cancer.^29-32 In the liposomal chemistry studies, liposomes conjugated to antibodies or targeting ligands was found to optimize and enhance local drug delivery and exhibit better cell internalization than free drug.^33,34 With this in mind, we designed a novel peptide, ligand from atherosclerotic plaque-specific peptide-1 (AP1) consisting of 9 amino acids sequence (CRKRLDRNC) which was selected from phage display libraries that can locate atherosclerotic plaque tissue and bind to IL- 4R, since it has the same binding motif to the IL-4 protein.³⁵ AP1-conjugated nanoparticles has been used for targeted drug delivery to tumors.^36-40

In this study, we established a stable IL-4Rα expressing colorectal CT-26 cells and evaluated the specific cytotoxicity and therapeutic efficacy of AP1-conjugated liposomal DOX (Lipo-DOX-AP1) in CT26 cells and tumor-bearing animal model. The information obtained from this study may help in developing a model for optimizing drug delivery through nanoparticle-conjugated liposomal anti-cancer drugs.

Results

Expression of IL-4Rα in human colorectal tissues and mouse CT26 cells

Based on the literature that several epithelial cancers including breast, brain and colon had up-regulated IL-4Rα expression, we examined 13 pairs of human CRC and adjacent normal tissues for IL-4Rα expression by immunohistochemical (IHC) staining. Each IHC staining was scored by 2 experienced pathologists according to the staining intensity and distribution. A higher expression for IL-4Rα was observed in CRC tissue compared with adjacent normal tissue (Fig. 1A–B, P < 0.0001) .

Figure 1.

Immunohistochemistry analysis of IL-4Rα distribution and expression in paired CRC and adjacent normal tissue. (A) Tumors exhibited high expression of IL-4Rα in IHC staining. Scale bar = 50 μm. (B) The level of IL-4Rα IHC was quantified by calculating DxI, where D is the percentage of IL-4Rα-positive cells, and I is the IHC staining intensity. Data points represent individual adjacent normal or CRC tissue and the bars indicate the median value for each group. ***, P < 0.0001 (Mann–Whitney U-test) for the difference between tissues.

The murine CT26 cells represent carcinogen-induced CRC in Balb/c mouse. To establish in vitro and in vivo experiments in CT26 cells, we started with evaluating the cytotoxicity effect of various antitumor drugs on CT26 cells in vitro. CT26 cell is most sensitive to doxorubicin (DOX) compared to oxaliplatin and irinotecan, 2 agents that are now standard treatment for CRC (Fig. S1). The IC50 concentration of DOX is about 1 μM at 24 hour treatment (Fig. S1A). To evaluate the targeting efficiency of AP1 peptide, we established CT26 cells stably expressing IL-4Rα (CT26-IL4R) and determined the expression of IL-4Rα in CT26-IL4R cells compared to CT26 control cells (CT26-ctrl) as shown in Figure 2A. IL-4Rα was not detected in CT26-ctrl cells by Western blot analysis (Fig. 2A left panel) and barely detectable by flow cytometry analysis (Fig. 2C). Low IL-4Rα expression was detected in CT-26 control cells only after prolonged exposure of the membrane to the X-ray film (data not shown). IL-4Rα overexpression and membrane localization was observed in CT26-IL4R cells but not in CT26-ctrl cells by cell fractionation and immunofluorescence analysis, respectively (Fig. 2A-B right panel). MTT assay revealed that the proliferation ability of CT26-IL4R was similar to both CT26-ctrl and wild type CT26 (Fig 2D) .

Figure 2.

IL-4Rα expression and distribution, and proliferation of CT26-IL-4R and CT26-ctrl cells. (A) Western blot analysis of whole cell extract (left) and cellular fractionation (right) in CT26-IL4R and CT26-ctrl cells. Immunofluorescence (B) and flow cytometry analysis (C) of CT26-IL-4R and CT26-ctrl cells. Scar bar=10 μm (D) Proliferation assay of wild type CT26 (CT26 WT), CT26-IL-4R and CT26-ctrl cells. β-actin were used as cytosolic fraction makers; and Na, K ATPase α1, as a membrane marker.

In vitro binding and internalization of the AP1 peptide into CT26 cells

To investigate the cellular binding and internalization of the AP1 peptide, AP1 peptide was synthesized and conjugated with a green-fluorescent dye (FITC) at the N-terminus. In vitro binding and internalization experiments were performed at 4°C and 37°C, respectively. Immunofluorescence imaging and relative fluorescence intensity quantification demonstrated that the FITC-conjugated AP1 peptide bound more strongly to the surface of CT26-IL4R cells than CT26-control cells, while little binding was observed with the FITC-control peptide at 4°C (Fig. 3A). Binding of the AP1 peptide to CT26-IL4R cells versus CT26-control cells at 4°C was measured by flow cytometry analysis (Fig. 3C-D). The percentage of the AP1 peptide bound CT26-IL4R cells and CT26-control cells was 60 ± 5 % and 38 ± 7 %, respectively, while a very small percentage was observed in the control peptide binding. More AP1 peptide was internalized into CT26-IL4R cells than CT26-control cells at 37°C (Fig. 3B). These results indicated that the binding and internalizing of the AP1 peptide to CT26-IL4R cells was more intense compared to CT26-control cells.

Figure 3.

In vitro binding and internalization of AP1 peptide by CT26-IL4R and CT26-ctrl cells. FITC-conjugated AP1 or control peptide (20 μμ) was incubated with CT26-IL4R and CT26-ctrl cells at indicated temperature for 1 h. (A) In vitro binding to the cells at 4°C determined by confocal microscope analysis. (B) In vitro internalization of AP1 peptide by cells at 37°C determined by confocal microscope analysis. (C) Flow cytometry analysis of peptide binding at 4°C. (D) Statistic analysis of flow cytometry data. Bars represent the percentage of fluorescent cells bound with the peptide as mean ±SD of data obtained from 3 independent experiments. Scale bar, 20 μm.

Targeted killing of CT26-IL4R cells by Lipo-DOX-AP1

Based on our finding that CT26 cell death was DOX concentration-dependent (Fig. 1S), we next evaluated the efficiency of Lipo-DOX-AP1 induced cell apoptosis. We used the FAM-FLICA Poly Caspase Assay Kit and flow cytometry to monitor cell apoptosis (Fig. 4A). We found the percentage of poly-caspase positive cells with Lipo-DOX-AP1 treatment in CT26-ILR4R cells was more than CT26 ctrl cells. In addition, the percentage of poly-caspase positive cells with Lipo-DOX-AP1 treatment was more than Lipo-DOX or DOX treatment, respectively. Interestingly, the DOX and untreated cells showed no significant difference in the percentage of poly-caspase positive cells. It may due to short treatment time (10 hours). To investigate the specific targeting cytotoxic activity of Lipo-DOX-AP1 drug, time-lapse microscopy was used to monitor the progression of cell death under different drug formulation treatments. CT26-IL4R cells labeled with Green CMFDA cell tracker dye were co-cultured with an equal amount of unlabeled CT26-control cells. The mixed cells were then treated with the same concentration (10 μM) of free DOX (free doxorubicin), Lipo-DOX (liposomal doxorubicin) and Lipo-DOX-AP1, respectively, and monitored for dead cell morphology by 15 hours time-lapse microscopy (Fig. 4B and Supplementary Video). Our results show that Lipo-DOX-AP1 induced significantly more cell death (54.4%) compared to Lipo-DOX (29.5%) or free DOX (15.6%) in labeled CT26-IL4R; whereas the free DOX or Lipo-DOX or Lipo-DOX-AP1 did not induce significant cell killing of unlabeled CT26-control cells during 15 hours of observation (Fig. 4C). These results suggest that Lipo-Dox-AP1 has more specific cytotoxic activity against IL4Rα overexpressing CT26 cells compared to CT26-control cells.

Figure 4.

Apoptosis and selective cytotoxicity in CT26-IL4R and CT26-ctrl cells. (A) Poly-caspase activity in CT26-IL4R and CT26-ctrl cells. Cells were untreated or treated with different drug formulation for 10 hours. A FAM-FLICA detection probe was used to assess poly-caspase activity by flow cytometry. A representative of 3 separate experiments is shown. (B) Sequential images of cytotoxicity assay were cropped from Supplementary Video. CT26-IL4R cells labeled with Green CMFDA were mixed with unlabeled CT26-ctrl cells, treated with different drug formulation and continuously observed for 15 hours. The green arrow indicates the CT26-IL4R cell and white arrowhead indicates CT26-ctrl cell. Cell undergoing death progression is marked with an asterisk. (B) Quantitative analysis of cell death in CT26-IL4R or CT26-ctrl cells. *, P < 0.05 Lipo-DOX-AP1 compared to Lipo-DOX treatment in CT26-IL4R cells; ***, P < 0.001 CT26-IL4R cells compared to CT26-control cells in Lipo-DOX-AP1 treatment. Bar represent the percentage of cell death as mean ±SD of data obtained from 3 separate experiments. Bar scale, 20 μm.

Enhanced therapeutic efficacy and reduced systemic toxicity of Lipo-DOX-AP1 in vivo

We evaluated the antitumor efficacy of Lipo-DOX-AP1 in the CT26-IL4R (Fig. 5A, left panel) and CT26-ctrl (Fig. 5A, right panel) cells in tumor-bearing mouse model. Once the tumor reached the average size of approximately 100 mm³ (about 14 d after transplantation), we randomly distributed the mice into 6 groups (N = 6) according to the tumor size in order to minimize variations in weight and tumor size among the groups. Mice bearing subcutaneous tumors were intravenously (i.v.) administered once a week for 4 weeks with the following regimens: (i) saline buffer (Control); (ii) free DOX (DOX) (10 mg/kg); (iii) liposomal DOX (Lipo-DOX) (10 mg/kg); (iv) empty Liposome (Lipo); (v) AP1-conjugated liposomal DOX (Lipo-DOX-AP1) (10 mg/kg); (vi) empty AP1-conjugated liposome (Lipo-AP1). No significant differences in body weight were observed between these treatment groups. Mice treated with saline control (Control) or empty carrier (Lipo or Lipo-AP1) showed significantly bigger tumor volume compared to mice treated with free DOX or doxorubicin-containing drug carrier (Lipo-DOX or Lipo-DOX-AP1). Treatment with Lipo-DOX-AP1 significantly inhibited tumor growth compared to saline control or Lipo or Lipo-AP1 or free DOX treatment (Fig. 5). In addition, the tumor suppression efficacy of Lipo-DOX-AP1 was significantly improved compared to Lipo-DOX treatment in CT26-IL-4R tumor-bearing mouse (Fig. 5A, right panel), whereas no tumor suppression was not observed in CT26-ctrl tumor-bearing mouse (Fig. 5A, left panel). Meanwhile, we evaluated the systemic toxicity of different treatments in mice by collecting blood to measure the serum levels of indicated proteins that provided a sensitive assessment of cardio-toxicity (LDH and CKMB), hepatotoxicity (GOT and GPT) and renal toxicity (BUN-P and CRE-P) at the end point of the experiments (Fig. S2). As shown in Figure 5B, the hematologic toxicity analysis showed that free DOX treatment had a 2.fold4- higher CKMB and 1.fold6- higher LDH serum level compared to Lipo-DOX-AP1 treatment, confirming the known cardio-toxicity of free DOX. Thus, our data suggest that the targeted Lipo-DOX-AP1 treatment shows the best therapeutic efficacy and minimal cardio-toxicity among the various drug treatments.

Figure 5.

The AP1-conjugated liposomal doxorubicin treatment had significantly reduced tumor volume and systemic toxicity. (A) Mice bearing CT26-ctrl cells (left) or CT26-IL4R cells (right) xenograft tumor (approximately 100 mm³) were intravenously injected with PBS buffer (Control), free doxorubicin (DOX), liposomal doxorubicin (Lipo-DOX), liposome (Lipo), AP1-conjuated liposome (Lipo-AP1) and the AP1-conjugated Lipo-DOX (Lipo-DOX-AP1), respectively (10 mg of doxorubicin/kg body weight at days were indicated). Data represent tumor volumes as mean ±SD (n = 6). *, P < 0.05. **, P < 0.01. ***, P < 0.001. (B) Analysis of systemic toxicity. Blood serum was collected immediately after the tumor-bearing mice were sacrificed upon reaching the end point. Data shown are means ± S .E. (n = 6). *, P < 0.05. **, P < 0.01.

Discussion

In this study, we established IL-4Rα overexpressing clones and demonstrated that these cells have high affinity for AP1 peptide and were highly sensitive to the cytotoxicity effect of Lipo-DOX-AP1 in vitro. We also evaluated the antitumor activity and toxicity profile of Lipo-DOX-AP1 in vivo. Lipo-DOX-AP1 displayed an enhanced therapeutic efficacy in tumor-bearing mice as well as markedly lower cardio-toxicity compared to free DOX.

Over the past few years, several in vitro and in vivo experimental studies showed autocrine IL-4 production by various types of cancer cells such as colon, thyroid, breast and lung, and it plays crucial role in anti-apoptosis contributing to tumor growth, chemotherapy-induced cell death and resistance to death receptors.^36,37 In addition, recent study shows myeloid IL-4R plays a role in tumor metastasis in a breast cancer mouse model.⁸ Exogenous IL-4Rα expression can promote colon tumor proliferation and growth in tumor cells and IL-4Rα-null mice.^12,15 Our study in mice also showed that CT26-IL4R tumors grew faster than CT26-ctrl tumors (Fig. 5). Recent studies show IL-4Rα acts as an autocrine survival signal and contributes to chemoresistance in colon tumor stem cells.^41-44 There have also been reports of clinical trials using IL-4Rα as a target to treat recurrent and metastatic kidney cancer, non-small cell lung cancer or breast cancer.⁴⁵ Therefore, IL-4Rα is a potential tumor therapeutic targeting molecule and in this study, we have contrived an animal model system to evaluate the anti-cancer efficacy of IL-4Rα−targeting therapy.

Several studies have adopted nanoparticle-containing or -fused cytotoxicity peptide- IL-4 receptor targeting drug to kill tumor cells in glioblastoma, Hodgkin lymphoma, pancreatic ductal adenocarcinoma and ovarian cancer.^{15,25,38,40,46-48} Encapsulating drugs in nanoparticles reduces drug concentration in normal tissues and increases the concentration of active drug within the tumor. Liposomal DOX has a half-life of several days, therefore increasing exposure of the tumor to drug. Another advantage of PEGylated liposomal DOX in cancer treatment is reduction of cardiotoxicity, which is the main side effect of DOX.^49-51 Significant reduction of cardiotoxicity was observed by monitoring CKMB and LDH in the blood of tumor-bearing mice (Fig. 5B). To further improve the specificity, we used AP1-conjuagted therapeutic liposomes coated with maleimide-functionalized polyethylene glycol chains.^38,40,46 Targeting nano-drugs using antibodies or antagonists that recognize the tumor-associated antigens or receptors is a widely adopted approach. However, its application might be limited by altered or low expressions of the specific antigen or receptor on the tumor cells. The AP1-conjugated liposome we used had been successfully applied to treat glioblastoma multiforme in mouse model.^38,40,46 Several studies demonstrated that IL-4 peptide could be conjugated to different drug-containing nanoparticles to improve drug uptake in cancer cells and antitumor efficacy, and enhance retention time in tumor tissue.^23,39,52 These findings showed that therapeutic agents that bind to IL-4R allow selective drug delivery that help tumor treatment.

Previous report showed that poor retention of anticancer drug is caused by overly twisty and porous vessels in tumor mass.⁵³ The porosity causes anti-cancer drug to leak out of tumor cells and tumor mass. Irinotecan and oxaliplatin are 2 of the most commonly used chemotherapeutic drugs for colorectal cancer. Irinotecan is a P-glycoprotein (P-gp/MDR1/ABCB1) substrate, oxaliplatin is a multidrug resistance protein 1 (MRP1/ABCC1) substrate and DOX is an MDR1, MPR1 and breast cancer resistance protein (BCRP/ABCG2) substrate.^54,55,56 In this study, we found that among them, Dox is the most efficient in killing CT26 cells in vitro (Fig. S1). Therefore, CT26 cells may have other mechanism that contribute to the irinotecan and oxaliplatin resistance. Furthermore, we designed the AP1-conjugated liposome to actively target colorectal cancer cells overexpressing IL-4R and provided a potential targeting anticancer drug formulation to enhance the permeability and retention (EPR) effect. Herein, we observed IL-4R overexpression in clinical colorectal tumor tissue compared to adjacent normal tissue (Fig. 1). Our in vitro selective cytotoxicity study compared cell death of CT26-IL4R cells treated with Lipo-DOX-AP1 or Lipo-DOX or free DOX using time-lapsed microscope analysis and showed that Lipo-DOX-AP1 possessed more overt and specific cytotoxicity than Lipo-DOX and free DOX (Fig. 4). This work demonstrated that AP1-conjuagated liposome could specifically kill cells overexpressing IL-4R in vitro. Moreover, Lipo-DOX-AP1 significantly decreased tumor growth in tumor-bearing mouse model. The selective cytotoxicity result implies that Lipo-DOX-AP1 has stronger cytotoxic effect than Lipo-DOX and free DOX, it has then lower IC50 value than Lipo-DOX and free DOX. This is consistent with others reports on the in vitro cytotoxicity of the free and liposomal drug.^57,58 However, other studies found free DOX to have higher intracellular uptake and displayed higher cytotoxicity than liposomal DOX.⁵⁹ The discrepancy might be attributed to the different cell lines used, the nature of the liposomes and drug treatment duration. Liposomes may facilitate cellular internalization of drug, thereby providing higher drug concentration that may account for the higher efficacy of liposomal DOX than free DOX which is dependent on diffusion only.

Clinical trials have successfully used IL-4R as a target to treat recurrent and metastatic kidney cancer, non-small cell lung cancer and breast cancer.⁴⁵ CRC was shown to overexpress IL-4R, while liposome has been well developed as a safe nano-carrier with fewer side effects. Together, it is expected that the liposomal IL-4R targeting strategy could be applied to CRC treatment in the future.

Materials and Methods

Clinical specimens

A total of 13 pairs of CRCs and corresponding adjacent normal tissues were collected from Taipei Veterans General Hospital following the ethical regulations passed by Institutional Review Board (IRB). Written informed consent was obtained from all patients and the study was approved by IRB of the Hospital (Permit Number: TCHIRB-1000401-E).

Cell culture

The CT-26-IL4R mouse colon cell line was engineered to stably overexpress IL-4Rα by lentiviral infection. Cultured cells were grown in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution, and incubated in a humidified atmosphere containing 5% CO2.

Vector Constructions and Lentivial Infection

For lentiviral expression vector construction, the IL-4Rα gene was amplified by PCR from the cDNA of CT-26 cells and cloned into pLKO_AS3w.neo vector. Transfection of 3 plasmids (mouse IL-4Rα gene-transfer plasmid pLKO_AS3w-IL4R.neo or control plasmid pLKO_AS3w.neo, the packaging plasmid pCMVR8.91, and the vesicular stomatitis virus glycoprotein envelope-encoding plasmid pMD.G) into 293T cells was conducted using T-Pro NTR II (T-Pro Biotech, Taiwan) transfection reagent. The virus supernatants were collected at 48 hours post-transfection and filtered using a 0.45 μm filter. CT-26 cells were infected with the virus supernatant. Transduced cells were selected in G418-containing (10 mg/ml) culture medium for one week. Transgene expression in the transduced CT-26 cells was confirmed by Western blot analysis using anti-IL-4Rα antibody.(R&D, AF530)

Subcellular fractions extraction

To examine the subcellular distribution of stably expressed IL-4Rα, confluent cells were fractionated using the ProteoExtract Subcellular Proteome Extraction kit (Calbiochem, La Jolla, CA) according to the manufacturer's instructions.

IL-4Rα immunohistochemistry and quantification

For immunohistochemical (IHC) staining, 3-μm thick sections from the colorectal cancer and adjacent normal tissues were prepared and mounted on silane-coated microscope slides (Dako). Sections were deparaffinized and rehydrated through graded alcohols. For antigen retrieval, slides were immersed in Trilogy (Cell Marque) and boiled for 20 min in a pressure cooker. Endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxide (H₂O₂) for 5 min. Non-specific binding was blocked by incubation with 5% BSA in PBS for 1 hour at room temperature. The slides were incubated with the specific anti-human IL-4Rα antibody (Santa Cruz, sc-684) at 1:100 dilution for 24 h at 4°C. Bound antibodies were detected with biotinylated horse universal secondary antibodies and streptavidin–peroxidase complex. Diaminobenzidine tetrahydrochloride (DAB) was used as the substrate followed by counterstaining with hematoxylin. The stained slides were scanned using a Scanscope CS (Aperio). Sections were reviewed and the overall intensity and distribution of IHC staining were scored independently by 2 surgeons (CY Chen and JK Jiang) blinded to all clinical and pathological information at the time of evaluation. For IHC evaluation, protein expression in the tissues was quantified using a visual grading system based on the intensity and distribution of staining. Intensity (I) of staining was graded from 0 to 3 (graded as 0, 1, 2, and 3 for no signal, faint, week but definite, and strong signals, respectively) and distribution (D) of staining graded from 0 to 4 (graded as 0, 1, 2, and 3 for 0–25%, 26–50%, 51–75% and 76–100% positive cells, respectively). The value of D × I for each sample, ranging from 0 to 12, represents the level of its IL-4Rα content.

Flow cytometry analysis of peptide Binding

FITC-AP1 peptide (FITC-CRKRLDRNC) and FITC-control (FITC-NSSSVDK) peptide were synthesized by Taiwan Biotools company. 1×10⁵ cells in suspension were incubated with culture medium containing 1% BSA at 37°C for 30 min for blocking and then treated with 25 μM FITC-conjugated peptide in serum-free medium at 4°C for 1 h. After extensive washing with PBS containing 0.1% Tween-20, cells were subjected to flow cytometry.(BD Bioscience, USA).

Immunofluorescence assays of peptide binding and internalization

Cells (1 × 10⁶/well) were plated into a 6-well plate. After overnight culture, cells were blocked with culture medium containing 1% BSA at 37°C for 30 min and then incubated with 50 μM FITC-conjugated peptide in serum-free culture medium at 37°C (for peptide internalization )or 4°C (for peptide binding) for 1 h. After extensive washing, cells were fixed with 4% paraformaldehyde, incubated with 4′, 6′-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) for nuclear staining and then were visualized under a fluorescent microscope (Leica Microsystems). The fluorescent intensity of 80 cells was quantified using MetaMorph Premier software (Universal Imaging).

Preparation of various liposomal doxorubicin formulation

Lipo-DOX was manufactured using a solvent injection method plus remote loading procedures. Concisely, cholesterol (31.9 mg, Sigma-Aldrich), hydrogenated soybean L-α-phosphatidylcholine (95.8 mg, Avanti Polar Lipids) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPEPEG2000, 31.9 mg, Avanti Polar Lipids) were dissolved and well mixed in 1 ml of absolute ethanol at 60°C. The ethanol and lipid mixture was then injected into 9-ml solution of 250 mM ammonium sulfate and stirred for 1 h at 60°C. The mixture was then extruded 5 times through polycarbonate membranes (Isopore Membrane Filter, Millipore) with pore sizes of 0.4, 0.2, 0.1, and 0.05 μm, consecutively, at 60°C with high-pressure extrusion equipment (Lipex Biomembranes) to produce small liposomes. The liposome suspension was then dialyzed 5 times against large amounts of 10% sucrose containing 5 mM NaCl to remove the unentrapped (free) ammoniumsulfate and ethanol. After dialysis, the liposome suspension was placed in a 50-ml glass bottle in a 60°C water bath and mixed with DOX to a final DOX concentration of 2 mg/ml in 10% sucrose solution. The bottle was kept in a 60°C water bath for 1 h with intermittent shaking and then immediately cooled down to 4°C, culminating in the production of Lipo-DOX.

The presence of a thiol group on each cystine of the AP1 peptide (CRKRLDRNC) makes it possible to couple AP1 to liposomes via the thiol-maleimide reaction. Briefly, AP1 peptide was conjugated to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [maleimide(polyethylene glycol)-2000] (DSPE-PEG2000-MAL, Avanti Polar Lipids) by adding AP1 to the DSPE-PEG2000-MAL micelle solution at a 2:1 molar ratio while mixing at 4°C overnight. The free thiol groups were measured with 5,5′-dithiobis-(2-nitrobenzoic acid) (Ellman's reagent, Sigma-Aldrich) at 420 nm to confirm that most of the AP1 was conjugated with DSPE-PEG2000-MAL after the reaction. AP1-conjugated DSPE-PEG2000 was transferred into the preformed Lipo-Dox at 1.5% molar ratio of total lipid components and incubated at 60°C for 1 h to obtain AP1-labeled Lipo-DOX.

The resulting unconjugated Lipo-DOX and Lipo-DOX-AP1 were found to have particle diameters of 100–120 nm, as measured by a dynamic light-scattering apparatus (Coulter N4 plus, Beckman), as well as a surface zeta potential of between −20 and −30 mV, as measured by electrophoretic light scattering.(ZetaPlus, Brookhaven)

Apoptosis analysis

Cells were untreated or treated with 10 μM indicated drug formulation for 10 hours. Apoptotic cells were detected using the FAM-FLICA Poly Caspase Assay Kit (ImmunoChemistry Technologies) according to the manufacturer's instructions and as previously described.⁶⁰ Cells were counted and data acquisition was performed using FACS Canto flow cytometer (BD Biosciences), and data were analyzed using FACS Diva software (BD Biosciences).

Time lapse video microscopy and analysis of live cell imaging

Cells were cultured in 60-mm dishes, incubated at 37°C in 5% CO2 and monitored using a live cell instrument (Chamlide) fitted into a Leica DM IRB immunofluorescence microscope (Leica Microsystem Inc..). Image acquisition was achieved and processed using MetaMorph Premier software (Universal Imaging). To study cell death progression, we used live-cell imaging in combination with time lapse microscopy to monitor the co-culture cells containing Green CMFDA (Invitrogen molecular probe)-labeled CT26-IL4R cells and unlabeled CT26 control cells. Here, both fluorescent and phase contrast images of live cell cultures were taken at 6 minutes intervals for the duration of experiments lasting to 15 hours. In each experiment, more than 75 cells in the images were analyzed at every time point for the duration of the experiment. Analysis of sequential live images allows us to quantitate cell death number of labeled and unlabeled cells induced by the various drug treatments.

Animals and tumor model

All mice experiments were carried out with approval of the Institutional Animal Care and Use Committee (IACUC) of National Yang-Ming University. BALB/c mice (6-weeks-old males, National Laboratory Animal Center, Taiwan) were used to perform the tumor model for various drug formulations treatment. CT26-IL4R and CT26-ctrl cells (2.0 × 10⁵) were subcutaneously (s.c.) injected into the right and left flanks of mice, respectively. Tumor-bearing mice were used 2 weeks after tumor implantation when the tumor volume had grown to approximately 100 mm³. Mice were injected intravenously via the tail vein with different drug formulations once a week for 4 weeks. Animal weight and tumor volumes were measured every other day. The tumor length (L) and width (W) were calculated by equation: WR = 1/2 × L × W².

Analysis of systemic toxicity

Blood serum was collected when mice were sacrificed at the end point of the therapeutic experiment. Multi-organ toxicity was analyzed including lactate dehydrogenase (LDH), creatine phosphokinase (CKMB), aspartate aminotransferase (GOT), alanine aminotransferase (GPT), blood urea nitrogen (BUN-P) creatinine (CRE-P) by Taiwan Mouse Clinic (National Phenotyping and Drug Testing Center, Taiwan).

Statistical analysis

The difference of statistical analysis between treatment conditions was performed by Student's t-test for pairs of groups and one-way analysis of variance (ANOVA) for multiple groups. All statistical analyses were carried out using GraphPad Prism Software (Version 5.0, San Diego, CA) and SPSS software (v19.0, Chicago, IL, USA). The level of statistical significance was set at P ≤ 0 .05.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Dr. Li-Li Li for language revision of the article.

Funding

This work was supported by research grants NSC 102–2321-B-075−001, NSC 102–2325-B-010–013 and NSC102–2319-B-010–001, National Science Council and MOST 104-2320-B-532-001, Ministry of Science and Technology, Taiwan.

Supplemental Material

Supplemental data for this article can be accessed on the publisher's website