Purpose: We have developed a PEGylated transferrin-conjugated liposomes (PTf-Ls) system for the combined tumor imaging and targeted delivery of the IFN-γ–inducible protein-10 (IP-10) gene in a single macromolecular construct. Here, we characterize and analyze the use of this system in a mouse model of breast cancer.

Experimental Design: The biophysical and cell transfection properties of PTf-Ls were determined through a series of in vitro experiments. A nude mouse/breast cancer cell line xenograft model (mouse xenograft model) was used to image the tumor internalization of fluorescently labeled PTf-Ls. The clinical use of the system was tested by treating tumor-bearing mice with PTf-Ls loaded with IP-10 plasmid DNA or fluorescent lipoplexes.

Results: The resulting 165-nm liposomes (zeta potential = −10.6 mV) displayed serum resistance, low cytotoxicity (<5%), and high transfection efficiency (≤82.8%) in cultured cells. Systemic intravenous administration of fluorescent PTf-Ls in the mouse xenograft model resulted in nanoparticle circulation for 72 hours, as well as selective and efficient internalization in tumor cells, according to in vivo fluorescence and bioluminescence analyses. Tumor fluorescence increased gradually up to 26 hours, whereas background fluorescence decreased to near-baseline levels. Treatment of mice with PTf-Ls entrapped pcDNA3.1-IP-10 suppressed tumor growth in mice by 79% on day 50 and increased the mean survival time of mice. Fluorescent pcDNA-IP-10–entrapped PTf-Ls showed good properties for simultaneous tumor-targeted imaging and gene-specific delivery in an animal tumor model.

Conclusions: Our developed transferrin-conjugated liposome system possesses promising characteristics for tumor-targeting, imaging, and gene therapy applications. Clin Cancer Res; 19(15); 4206–17. ©2013 AACR.

This article is featured in Highlights of This Issue, p. 4019

Translational Relevance

We describe a highly efficient PEGylated transferrin-conjugated liposomes (PTf-Ls) system for combined tumor imaging and targeted IFN-γ–inducible protein-10 (IP-10) gene therapy in a single macromolecular construct. The system displays multiple clinically relevant advantages including low cytotoxicity, serum resistance, high transfection efficiency, long blood circulation time, and targeted tumor delivery. The PTf-Ls were found to exhibit efficient targeted tumor imaging and gene delivery in a xenograft mouse model of breast cancer. Use of PTf-Ls loaded with IP-10 plasmid DNA significantly suppressed tumor growth and increased the mean survival time of mice. We conclude that this new bifunctional PTf-Ls system has important clinical implications for tumor imaging and gene therapy applications.

Nanomedicines, such as sterically stabilized liposome-encapsulated therapeutic agents, have been used to increase the selective toxicity of chemotherapeutics. Tumor-targeting treatments can improve cancer therapeutic outcomes and minimize damage to normal tissues (1, 2). PEGylated liposomes, in which polyethylene glycol (PEG)-containing lipids are incorporated into the lipid bilayer, have long half-lives in the blood circulation and are stable in solution for more than 1 week, thus meeting the requirements of an effective therapeutic delivery system (3, 4). Many ligands have been evaluated for liposome targeting including monoclonal antibodies, single-chain antibody variable region fragments, peptides, growth factors, glycoproteins, folate, and oligonucleotide aptamers (3–7). Transferrin has been extensively studied as a ligand for tumor-targeting and synthetic targeting systems because transferrin receptor (TfR) levels are elevated in various cancer cells types as compared with normal cells (8). Transferrin-coupling PEGylated liposomes exhibit interesting properties, including prolonged circulation time, low uptake by the reticuloendothelial system (RES; ref. presumably because transferrin is an abundant serum glycoprotein), and in vivo accumulation and internalization in tumors (9, 10). Targeting liposomes provide advantages over nontargeting liposomes, showing increased localization to tumor sites and increased interaction with the target cell population (11).

For targeted tumor cell internalization and imaging in a single macromolecular construct, fluorescent proteins, small organic dyes, heavy metals, and quantum dots have been used as fluorescent contrast agents in liposome vesicles (12–15). These vesicles offer great potential for tumor imaging applications, due to their rapid accumulation and prolonged retention within the tumor. Targeted delivery systems, by incorporating both cancer therapy agents and fluorescent dyes in different compartments of a single vesicle, offer many opportunities for the development and application of nanomedicines in vivo.

IFN-γ–inducible protein-10 (IP-10) plays important roles in the regression of tumor angiogenesis and the chemotaxis of activated T cells via its interactions with CXCR3 (16). We recently showed that IP-10 enhances the antitumor efficacy of glioma lysate-pulsed dendritic cells and an flk1-based DNA vaccine (17, 18). In this study, we present an efficient system of PEGylated liposomes (P-Ls) coupled with transferrin (PTf-Ls) for gene transfer and cancer imaging in vivo. We conducted a series of in vitro experiments to characterize the biophysical and cell transfection properties of the PTf-Ls. A nude mouse/breast cancer cell line xenograft model (mouse xenograft model) was used to image the tumor internalization of fluorescently labeled PTf-Ls. Finally, we tested the clinical use of our system by treating tumor-bearing mice with PTf-Ls loaded with IP-10 plasmid DNA.

Materials and animals

The plasmid DNA, pEGFP-N1, was purchased from Invitrogen. The IP-10 plasmid containing the EGFP reporter gene, pcDNA3.1-IP-10, was constructed in our laboratory by the method of Lu and colleagues (17). High-quality plasmid DNA was prepared with an Endo-free Giga kit (Qiagen), according to the manufacturer's instructions. The fluorescent molecule, X-SIGHT 670 Large Stokes Shift Dye (LSS670), was purchased from Kodak (Carestream Health, Inc.). The HeLa cell line was purchased from the Type Culture Collection of the Chinese Academy of Sciences. The MCF-7 and MDA-MB-231 cell lines were purchased from American Type Culture Collection. Cells were cultured in Dulbecco's modified Eagle medium (DMEM) or Leibovitz's L-15 medium with FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL).

Ten-week-old female BALB/c nude mice and nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice purchased from the Model Animal Research Center of Medical College of Xiamen University were housed in a laminar flow hood under sterilized conditions. Animal handling was conducted in accordance with the guidelines of the Animal Care and Use Committee of Xiamen University.

Liposome preparation

Chloroform solutions of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC,14.12 mg); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (PEG)-2000] ammonium salt (DSPE-PEG2000, 1.67 mg); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide (PEG)-2000] ammonium salt (DSPE-PEG2000-maleimide, 0.59 mg); and dimethyldioctadecyl ammonium bromide salt (DDAB, 0.38 mg) were mixed in a glass tube. Lipids used for liposome preparation were purchased from Avanti Polar Lipids, Inc. To prepare the fluorescent liposomes, 0.8 mg of DSPE-PEG2000 was replaced with DSPE-rhodamine (Avanti Polar Lipids, Inc.). The lipids were dried, rehydrated with 0.2 mL of Tris-HCl buffer (50 mmol/L, pH 7.0), and sonicated in a water bath for 5 cycles.

Plasmid DNA (400 μg of pEGFP-N1 or pcDNA3.1-IP-10) or calcein solution (2 μL, 0.01 mol/L) was added to the lipid solution with different volumes of ethanol or calcium solution (see Supplementary Data). The vials were closed firmly, shaken gently, and subjected to 5 freeze thaw cycles. Using a hand-held extruder (Avestin), the liposomes were extruded 20 times through 100-nm pore size filters, followed by 21 times through 50-nm filters. The liposomes were dialyzed against HEPES buffer (25 mmol/L HEPES, 140 mmol/L NaCl, pH 7.0) for 2 hours, with fresh buffer added after 1 hour.

Protein coupling was conducted at a protein:lipid molar ratio of 1:1,000. Human transferrin (>98% pure, Sigma-Aldrich Corp.) was chemically activated by reacting the calculated amount with 2-iminothiolane (200 μL, 7 mmol/L; Sigma) in sodium borate buffer (50 mmol/L, pH 9.4) in the dark for 2 hours at room temperature. Transferrin was washed twice with HEPES buffer and incubated overnight at room temperature with liposomes in sealed vials in a nitrogen atmosphere under gentle shaking. The vesicles were subjected to size exclusion chromatography with a Sepharose CL-4B column (GE Healthcare) equilibrated with HEPES buffer to separate the PTf-Ls from unconjugated proteins and nonencapsulated plasmid DNA. The fractions were collected, sterilized, and stored at 4°C. Transferrin was quantified by immunonephelometry on the BN* II system with the N AS TRF test (Siemens).

Biophysical characterization of nanoparticles

Particle sizes were measured 3 times in 150 mmol/L NaCl by dynamic light scattering (DLS; N4 PLUS Submicron Particle Size Analyzer, Beckman-Coulter). Zeta potentials were measured in 150 mmol/L (pH 7.0) with a laser electrophoresis zeta potential analyzer (LEZA-700, Otsuka Electronics). Transmission electron microscopy (TEM) was used to analyze the morphology of the liposomes (JEM2100HC, JEOL). The calcein fluorescence quenching method described by MacDonald and colleagues (19) was used to determine the encapsulation efficiency of liposomes. The liposome concentration was determined by the phosphorus assay (20). Nonreducing SDS-PAGE, matrix-assisted laser desorption-ionization time-of-flight/mass spectrometry (MALDI-TOF/MS), and X-ray photoelectron spectroscopy (XPS) analyses were applied to confirm the conjugation of transferrin to the liposome surface (see Supplementary Data).

Cell uptake analysis

The cell uptake of rhodamine-labeled nontargeting or targeting liposomes was determined in HeLa, MCF-7, and MDA-MB-231 cells by confocal fluorescence microscopy and flow cytometry. TfR expression in these cells types has been proved by Calzolari and colleagues (21) and Kawamoto and colleagues (22). Cells cultured in Millicell EZ SLIDE 8-well plates (Millipore) were treated with rhodamine-labeled liposomes. After incubation for 2 hours at 37°C, the cells were washed 4 times and fixed with 4% formaldehyde for 30 minutes. Cell nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI; Invitrogen). Cells were imaged with an MRC 1024 confocal spectral microscope (Bio-Rad). Images were analyzed with Leica confocal software.

Cells were incubated with rhodamine-labeled liposomes containing 100 μg of lipid diluted in 1 mL of medium with or without FBS for 2 hours at 37 °C. After incubation, liposomes were removed. The cells were washed several times with PBS or citric buffer (pH 3.0) for 3 minutes (acid wash) and then washed again with PBS buffer twice to remove all of the liposome outside the cell (bound and unbound). The cells were analyzed by flow cytometry.

In vitro gene transfection

The in vitro gene transfection efficiency of the PTf-Ls in tumor cell lines was assessed with the plasmid pEGFP-N1 or pcDNA3.1-IP-10 in HeLa and MDA-MB-231 cells. Cells were incubated with the lipoplexes for 5 hours in 1 mL of medium containing 10% FBS. For the competition experiment, cells were preincubated with transferrin (4 or 40 μg of protein in 1 mL of medium) for 1 hour, the same amount of lipoplexes was added, and the cells were incubated again for 5 hours. EGFP-expressing cells were directly visualized and photographed by fluorescence microscopy and analyzed by flow cytometry.

In vivo optical imaging

The MDA-MB-231-Luc breast cancer cell line, which had been transfected with a lentiviral construct containing the luciferase gene (Luc), was used to generate solid tumors in the mouse xenograft model. Briefly, 4 × 106 MDA-MB-231-Luc cells were implanted under the front right axilla of immunocompromised Balb/c nude mice. Tumor growth was monitored daily until the longitudinal diameter of the tumor was 0.8 to 1.0 cm, as measured with calipers. Xenograft-bearing mice were randomly divided into 3 groups (n = 3 mice per group) and injected with LSS670, LSS670-labeled P-Ls, or LSS670-labeled PTf-Ls (dose equivalent to ∼0.01 μmol of LSS670, ∼0.81 μmol of lipids, and ∼0.035 mg transferrin), respectively. Labeling reactions were conducted according to the manufacturer's protocol (see Supplementary Data). Animals were anesthetized by 3.5% isoflurane and maintained unconscious with 2.0% to 2.5% isoflurane during injection and imaging.

Optical imaging was conducted with the Kodak in vivo imaging system FXPro (Carestream Health, Inc.), which combines advanced multispectral fluorescence, luminescence, digital X-ray, and radioisotopic imaging in a single system. The imaging system consisted of a dark chamber with a heated stage and gas anesthesia inlet and outlet ports. The excitation and emission filters were set at 650 and 700 nm, respectively. Images were acquired with the following optimized parameters: 60,000 seconds exposure time, 2 × 2 binning, 60 seconds acquisition time, 120 × 120 mm2 field of view, and f 2.25 aperture stop.

The animals were carefully wiped with alcohol to remove any fluorescing contaminants, and baseline imaging was conducted. Nanoparticles (100 μL) were injected through the tail vein, with the animals still anesthetized. The animals were imaged again at several time points. For bioluminescent imaging, the light-sensitive substrate d-luciferin was given by intraperitoneal injection of about 2.5 mg luciferin/kg body weight for each mouse. The exposure time was 3 minutes, with 4 × 4 binning.

In vivo tumor-targeted therapy with PTf-Ls entrapped pcDNA3.1-IP–10

To establish subcutaneous tumors, 5 × 106 MDA-MB-231 cells in 0.5 mL of PBS were injected into the mammary fat pad of NOD/SCID mice, which were randomly allocated to 4 groups (n = 10 each) and intravenously treated with 200 μL of either PTf-Ls entrapped pcDNA3.1-IP-10, P-Ls entrapped pcDNA3.1-IP-10, naked pcDNA3.1-IP-10, or PBS. The quantity of pcDNA3.1-IP-10 used corresponded to 50 μg DNA. Animals were treated three times each on days 5, 15, and 25 after tumor establishment.

Tumors were measured with Vernier calipers at 5-day intervals after the tumor cells were inoculated. The tumor volume was calculated according to the formula: d1 × (d2)2 × 0.5, where d1 is the largest diameter and d2 is the perpendicular diameter. Mice were monitored daily for survival. They were sacrificed when any single or combined tumor linear measurement exceeded 20 mm. Three independent experiments were carried out.

For simultaneous in vivo imaging and gene therapy, LSS670-labeled pcDNA3.1-IP-10–entrapped PTf-Ls were prepared according to the protocol of LSS670-labeled PTf-Ls. Mice at 9 days after tumor cell injection were randomly allocated to 2 groups (n = 5 each) and treated with LSS670-labeled PTf-Ls or LSS670-labeled pcDNA3.1-IP-10–entrapped PTf-Ls every 5 days. Images were acquired at 32 hours after intravenous injection, according to the method above. Tumor size was determined region of interest (ROI) analysis in the Carestream Molecular Imaging (Carestream) software in the Kodak in vivo imaging system FXPro.

Statistical analysis

Data were analyzed with the SPSS 10.0 software package (SPSS). A 2-tailed Student t test was used to determine the significance of differences between experimental groups and controls. Survival rates of animals were calculated by the Kaplan–Meier method. Differences between survival curves were examined by the log-rank test. All tests were 2-sided. P < 0.05 was considered statistically significant.

Physical properties of liposomes

Use of 20% ethanol and 4 mmol/L Ca2+ (method IV) produced vesicles that were smaller in diameter than those created with no ethanol or calcium (method I) but with almost no change in trapping efficiency. However, the percent entrapment was sensitive to small changes in diameter when 35% ethanol without Ca2+ (method II) was used. Method III, with 35% ethanol and 4 mmol/L Ca2+, maximized calcein entrapment (up to 66.4%) inside stable liposomes with an average diameter of 142 nm. Therefore, this method was chosen for subsequent experiments (Supplementary Table S1). The phosphorus assay results revealed that 65% ± 3% of the polymer was incorporated into the liposomal formulation, and the binding level of transferrin was 40.5 μg/μmol lipids.

Before protein binding and DNA encapsulation, the PEGylated liposomes were homogenously distributed as individual nanoparticles, with a well-defined spherical shape and a diameter of about 100 nm. The liposomes showed both hydrophilic and hydrophobic dissections. Protein binding and plasmid DNA encapsulation increased the liposome size to about 160 nm by TEM (Fig. 1A–C and Supplementary Fig. S1). Similarly, the DLS results showed an increase in the particle size and size distribution from 106 ± 14 nm to 162 ± 24 nm for P-Ls and P-Ls/plasmid DNA, respectively. However, the DLS studies indicated that there was no significant size difference between targeting (165 ± 14 nm) and nontargeting (162 ± 24 nm) particles. Using the detection methods described in the Supplementary Data, we found that the encapsulation efficiency of PTf-Ls entrapped pcDNA3.1-IP-10 was 70.5% ± 2.8%.

Figure 1.

TEM images and dynamic light scattering particle size distributions for P-Ls (A), P-Ls entrapped pcDNA3.1-IP-10 (B), and PTf-Ls entrapped pcDNA3.1-IP-10 (C). Scale bar, 0.2 μm. D, analysis of transferrin conjugation to P-Ls by nonreducing SDS-PAGE and matrix-assisted laser desorption/ionization–time-of-flight/mass spectrometry (MALDI-TOF/MS). Nonreducing SDS-PAGE (left to right): molecular marker; thiolated transferrin; PTf-Ls mixture before Sepharose CL-4B treatment, with bands shown for thiolated transferrin before (i) and after (ii) conjugation; and PTf-Ls mixture after Sepharose CL-4B treatment, with band shown for transferrin after conjugation. MALDI-TOF/MS results show the molecular weights of transferrin before thiolation, after thiolation, and after conjugation.

Figure 1.

TEM images and dynamic light scattering particle size distributions for P-Ls (A), P-Ls entrapped pcDNA3.1-IP-10 (B), and PTf-Ls entrapped pcDNA3.1-IP-10 (C). Scale bar, 0.2 μm. D, analysis of transferrin conjugation to P-Ls by nonreducing SDS-PAGE and matrix-assisted laser desorption/ionization–time-of-flight/mass spectrometry (MALDI-TOF/MS). Nonreducing SDS-PAGE (left to right): molecular marker; thiolated transferrin; PTf-Ls mixture before Sepharose CL-4B treatment, with bands shown for thiolated transferrin before (i) and after (ii) conjugation; and PTf-Ls mixture after Sepharose CL-4B treatment, with band shown for transferrin after conjugation. MALDI-TOF/MS results show the molecular weights of transferrin before thiolation, after thiolation, and after conjugation.

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The zeta potential of the P-Ls was 4.4 mV. When the encapsulated plasmid DNA molecules were electrostatically adsorbed to the inner leaflet of the cationic liposome membrane, the zeta potential value (−1.7 mV) indicated a nearly neutral surface with a slight negative charge. This value became −10.6 mV after transferrin was conjugated to the outside of the membrane (Supplementary Table S1).

Coupling of transferrin to PEGylated liposomes

Using nonreducing SDS-PAGE, we identified 2 bands in the mixture before chromatographic separation (Fig. 1D), which corresponded to (a) thiolated transferrin and (b) DSPE-PEG2000-maleimide–conjugated transferrin. A single band of transferrin was detected after thiolation or chromatographic separation. The results of MALDI-TOF/MS indicated that thiolation and conjugation increased the molecular weight of transferrin by 2,378 and 7,912 Da, respectively. This finding indicated that every molecule of transferrin had 17 thiolated sites and 2 DSPE-PEG2000-maleimide chains.

The XPS scan summarizes the chemical compositions on the polymerized liposome surfaces (Supplementary Table S2). In transferrin-conjugated liposomes, about 2.06% sulfur was detected in addition to carbon, oxygen, nitrogen, and phosphorous. For the lipid mixture and P-Ls, high-resolution scans on nitrogen revealed a single peak, with a binding energy of 402.2 eV. The PTf-Ls exhibited an extra peak, with a slightly lower peak binding energy of 398.4 eV for (=N—),which arose from the thiolated transferrin. In the sulfur scan, PTf-Ls showed peaks with a binding energy of 167.8 eV. The presence of sulfur indicated the existence of transferrin on these liposome surfaces (Supplementary Fig. S2).

In vitro cellular association of PTf-Ls

The uptake of PTf-Ls was much higher than that of the nontargeting P-Ls in MCF-7 and MDA-MB-231 cells (Fig. 2A). The uptake efficiency of PTf-Ls by HeLa cells was increased in cell culture medium with 10% FBS as compared with culture in medium without 10% FBS (Fig. 2B), even after the cells were washed with citric buffer (pH 3.0) to remove all of the liposomes outside of the cells (bound or unbound). Cells incubated with rhodamine-labeled PTf-Ls showed high levels of fluorescence, and some of this fluorescence was located near the nucleus and showed a granular pattern, indicating active transport into the cell (Fig. 2C and Supplementary Fig. S3). All of the cells seemed to take up the PTf-Ls.

Figure 2.

Flow cytometric histograms of cellular fluorescence uptake by (A) MCF-7 and MDA-MB-231 cells; (B) HeLa cells in medium with 10% FBS, after washing with PBS (a) or citric buffer (b); and HeLa cells in medium without 10% FBS, after washing with PBS (c) or citric buffer (d). Thin line, control; gray line, P-Ls; black line, PTf-Ls. C, confocal fluorescence images showing uptake of free rhodamine (control), rhodamine-labeled P-Ls, or rhodamine-labeled PTf-Ls by MCF-7 cells. DAPI/nuclei (blue), rhodamine (red).

Figure 2.

Flow cytometric histograms of cellular fluorescence uptake by (A) MCF-7 and MDA-MB-231 cells; (B) HeLa cells in medium with 10% FBS, after washing with PBS (a) or citric buffer (b); and HeLa cells in medium without 10% FBS, after washing with PBS (c) or citric buffer (d). Thin line, control; gray line, P-Ls; black line, PTf-Ls. C, confocal fluorescence images showing uptake of free rhodamine (control), rhodamine-labeled P-Ls, or rhodamine-labeled PTf-Ls by MCF-7 cells. DAPI/nuclei (blue), rhodamine (red).

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In vitro gene expression assay

In HeLa cells, EGFP-containing P-Ls or PTf-Ls facilitated endocytosis and showed higher transfection efficiencies than the naked plasmid DNA (Fig. 3A). The results of flow cytometric analysis showed that the transfection efficiency of PTf-L–entrapped pEGFP-N1 (82.8% for HeLa cells, 79.7% for MDA-MB-231 cells) or pcDNA-3.1-IP-10 (76.5% for HeLa cells, 74.2% for MDA-MB-231 cells) was better than that of nontargeting nanoparticles (Fig. 3B and C). Low concentrations of transferrin significantly decreased the transfection efficiency in the competition experiment, and almost no positive cells were observed in the high concentration group (Fig. 3C and Supplementary Fig. S4). The transfection efficiency of PTf-Ls was higher in the presence of serum (Supplementary Figs. S5 and S6).

Figure 3.

A, fluorescence micrographs and differential interference contrast (DIC) of HeLa cells transfected with P-Ls or PTf-Ls entrapped pEGFP-N1. B, flow cytometric histograms of HeLa cells transfected with liposomes entrapped pEGFP-N1 or liposomes entrapped pcDNA3.1-IP-10 (IP-10 plasmid containing EGFP reporter gene). C, flow cytometric histograms of MDA-MB-231 cells transfected with liposomes entrapped pEGFP-N1 or liposomes entrapped pcDNA3.1-IP-10 for 5 hours or pretreated with 4 μg of transferrin in 1 mL of medium for 1 hour for competition experiment.

Figure 3.

A, fluorescence micrographs and differential interference contrast (DIC) of HeLa cells transfected with P-Ls or PTf-Ls entrapped pEGFP-N1. B, flow cytometric histograms of HeLa cells transfected with liposomes entrapped pEGFP-N1 or liposomes entrapped pcDNA3.1-IP-10 (IP-10 plasmid containing EGFP reporter gene). C, flow cytometric histograms of MDA-MB-231 cells transfected with liposomes entrapped pEGFP-N1 or liposomes entrapped pcDNA3.1-IP-10 for 5 hours or pretreated with 4 μg of transferrin in 1 mL of medium for 1 hour for competition experiment.

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In vivo tumor-targeting imaging

In athymic mice, liposome conjugation significantly prolonged the circulation of dye compared with free LSS670, which showed extremely rapid clearance and was only detectable in the bladder after 2 hours (Fig. 4). Both P-Ls and PTf-Ls accumulated in the tumor and showed more prominent fluorescence than free LSS670. However, the accumulation of targeting nanoparticles was extremely high than that of nontargeting nanoparticles. At 8 hours after injection, the tumor-targeting activity of PTf-Ls was confirmed by bioluminescent imaging of the tumor sites at 10 minutes after luciferin injection. Bioluminescence was readily detected at the tumor sites in all groups, but only in the PTf-Ls group did the bioluminescence images match perfectly to the fluorescence images at the tumor sites.

Figure 4.

In vivo fluorescence images using LSS670-labeled liposomes and bioluminescence images of nude mice bearing MDA-MB-231-Luc breast cancer xenografts implanted under the right front axilla. Left, in vivo fluorescence images acquired at 0.5 to 8 hours after intravenous injection of free LSS670, LSS670-labeled P-Ls, or LSS670-labeled PTf-Ls. Right, in vivo fluorescence (LSS670, red) and bioluminescence (BLI, green) of nude mice at 8 hours after injection of free LSS670, LSS670-labeled P-Ls, or LSS670-labeled PTf-Ls and 5 minutes after an intravenous injection of luciferin.

Figure 4.

In vivo fluorescence images using LSS670-labeled liposomes and bioluminescence images of nude mice bearing MDA-MB-231-Luc breast cancer xenografts implanted under the right front axilla. Left, in vivo fluorescence images acquired at 0.5 to 8 hours after intravenous injection of free LSS670, LSS670-labeled P-Ls, or LSS670-labeled PTf-Ls. Right, in vivo fluorescence (LSS670, red) and bioluminescence (BLI, green) of nude mice at 8 hours after injection of free LSS670, LSS670-labeled P-Ls, or LSS670-labeled PTf-Ls and 5 minutes after an intravenous injection of luciferin.

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In separate experiments, the tumor, liver, and bladder showed the most prominent fluorescence signals in all animals (Fig. 5). At 1 hour after injection, fluorescence signals were readily detected at the tumor site and in the mononuclear phagocytic system (MPS) organs, which mediate liposome clearance. At 4 hours, more prominent fluorescence was detected. The fluorescence in the tumor gradually increased over time up to 26 hours, whereas the fluorescence signal decreased to near-baseline levels in the MPS organs. Thereafter, the fluorescence signal decreased in the tumor. Tumor-specific fluorescence imaging was conducted at 32 hours and continued for 40 hours. The LSS670-labeled PTf-Ls showed extensive accumulation within the tumor tissue (Fig. 5C).

Figure 5.

A, in a separate experiment from Fig. 5, fluorescent images were acquired at 2 to 72 hours after i.v. injection of LSS670-labeled PTf-Ls into the mouse xenograft model. B, normalized fluorescence of LSS670-labeled PTf-Ls in the mouse xenograft model (n = 4) as a function of time. Normalized fluorescence signals (i.e., tumor-to-background signal ratios) were calculated by dividing the total fluorescence intensity in the tumor by the background intensity. C, two-color confocal microscopy images of 5-μm sections of frozen tumor tissues harvested at 8 hours after injection of free LSS670, LSS670-labeled P-Ls, or LSS670-labeled PTf-Ls. DAPI (blue), LSS670-labeled PTf-Ls (red).

Figure 5.

A, in a separate experiment from Fig. 5, fluorescent images were acquired at 2 to 72 hours after i.v. injection of LSS670-labeled PTf-Ls into the mouse xenograft model. B, normalized fluorescence of LSS670-labeled PTf-Ls in the mouse xenograft model (n = 4) as a function of time. Normalized fluorescence signals (i.e., tumor-to-background signal ratios) were calculated by dividing the total fluorescence intensity in the tumor by the background intensity. C, two-color confocal microscopy images of 5-μm sections of frozen tumor tissues harvested at 8 hours after injection of free LSS670, LSS670-labeled P-Ls, or LSS670-labeled PTf-Ls. DAPI (blue), LSS670-labeled PTf-Ls (red).

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Treatment of mice with pcDNA3.1-IP-10–entrapped P-Ls or PTf-Ls

The treatment of mice with pcDNA3.1-IP-10–entrapped PTf-Ls reduced tumor growth more effectively than treatment with pcDNA3.1-IP-10–entrapped P-Ls or naked plasmid (both P < 0.05; Fig. 6A). Treatment with pcDNA3.1-IP-10–entrapped PTf-Ls suppressed tumor growth in mice by 79% on day 50. The survival rate of tumor-bearing mice treated with pcDNA3.1-IP-10–entrapped PTf-Ls was higher than the rates of the other groups (both P < 0.05; Fig. 6B). All of the mice treated with PBS or pcDNA3.1-IP-10 died within 90 days, but 40% of mice treated with pcDNA3.1-IP-10–entrapped P-Ls and 90% of mice treated with pcDNA3.1-IP-10–entrapped PTf-Ls were alive at 140 days after implantation. Similar results were obtained in 3 replicate trials of this experiment.

Figure 6.

PTf-Ls entrapped pcDNA3.1-IP-10 can mediate effective tumor-targeted therapy in vivo. A, human breast cancer MDA-MB-231 cells (5 × 106) were injected s.c. into the mammary fat pad of NOD/SCID mice. Mice were intravenously injected with PBS (•), pcDNA3.1-IP-10 (▴), P-Ls–entrapped pcDNA3.1-IP-10 (▪), or PTf-Ls–entrapped pcDNA3.1-IP-10 (♦). n = 10. Tumor size was measured every 5 days after tumor implantation. B, long-term survival of mice bearing the MDA-MB-231 breast cancer cell line after treatment with PBS (♦), pcDNA3.1-IP-10 (▪), P-Ls–entrapped pcDNA3.1-IP-10 (▴), or PTf-Ls–entrapped pcDNA3.1-IP-10 (•). n = 10. C and D, LSS670-labeled PTf-Ls–entrapped pcDNA3.1-IP-10 showed effective simultaneous in vivo imaging and gene therapy. Xenograft mouse models at 9 days after tumor cell injection were treated with LSS670-labeled PTf-Ls (•) or LSS670-labeled PTf-Ls–entrapped pcDNA3.1-IP-10 every 5 days (♦). n = 5 each. D, images were acquired 32 hours after intravenous injection. C, tumor size was determined through ROI analysis.

Figure 6.

PTf-Ls entrapped pcDNA3.1-IP-10 can mediate effective tumor-targeted therapy in vivo. A, human breast cancer MDA-MB-231 cells (5 × 106) were injected s.c. into the mammary fat pad of NOD/SCID mice. Mice were intravenously injected with PBS (•), pcDNA3.1-IP-10 (▴), P-Ls–entrapped pcDNA3.1-IP-10 (▪), or PTf-Ls–entrapped pcDNA3.1-IP-10 (♦). n = 10. Tumor size was measured every 5 days after tumor implantation. B, long-term survival of mice bearing the MDA-MB-231 breast cancer cell line after treatment with PBS (♦), pcDNA3.1-IP-10 (▪), P-Ls–entrapped pcDNA3.1-IP-10 (▴), or PTf-Ls–entrapped pcDNA3.1-IP-10 (•). n = 10. C and D, LSS670-labeled PTf-Ls–entrapped pcDNA3.1-IP-10 showed effective simultaneous in vivo imaging and gene therapy. Xenograft mouse models at 9 days after tumor cell injection were treated with LSS670-labeled PTf-Ls (•) or LSS670-labeled PTf-Ls–entrapped pcDNA3.1-IP-10 every 5 days (♦). n = 5 each. D, images were acquired 32 hours after intravenous injection. C, tumor size was determined through ROI analysis.

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Compared with the animals treated with LSS670-labeled PTf-Ls, mice treated with LSS670-labeled pcDNA3.1-IP-10–entrapped PTf-Ls showed a tumor growth suppression rate of 66% on day 30 (Fig. 6C and D), when the tumor growth was calculated from the tumor volume in the images. When the growth was calculated according to the tumor-to-background ratio, the suppression was about 72%.

Biophysical characteristics of PTf-Ls

Different approaches have been used to improve DNA loading into targeting liposomes, including changing the concentration of the spacer unit between the cationic head group and hydrophobic lipid moieties on the liposomal membrane (23) and using different proportions of ethanol and calcium (24–26). Previous studies have shown average encapsulation efficiencies of 26% for liposomes of the size used here (27, 28). In contrast, we observed a plasmid DNA entrapment of up to 70.5% within stable 165 ± 14 nm liposomes. We achieved this result by taking advantage of the physical properties of liposomes and their interactions with ethanol and calcium ions (35% ethanol and 4 mmol/L Ca2+).

Liposomes of smaller sizes (< 200 nm) are less likely to be taken up by the RES. Use of smaller particles improves the utilization ratio, side effect profile, biodistribution, and circulation time of the liposomes (26, 29). Obata and colleagues (23) synthesized a series of pDNA-encapsulating cationic liposomes, reporting an approximate diameter of 200 nm, encapsulation efficiency of about 30% to 49%, and zeta potential of about −0.7 to 5.5 mV. In our article, because the P-Ls showed a positive zeta potential (4.4 mV), the DNA molecules were electrostatically adsorbed. After DNA encapsulation and protein conjugation, the lipoplexes showed an average zeta potential of −10.6 mV. The use of net negative charge carriers should result in comparatively less nonspecific tissue uptake and better overall biocompatibility (30).

The P-Ls and PTf-Ls formulations were physically stable. We did not observe any change in the size distribution after storage for over 1.5 years at 4°C under sterile conditions (Supplementary Fig. S7). The stability of liposomes in the presence of serum can provide insight into the behavior of liposomes in biological fluids (31). We found that the liposomal membranes were stable in the presence of serum, showing leakage as high as 20% when incubated in human serum or cell culture medium for 8 hours (Supplementary Table S3).

PTf-Ls show low cytotoxicity against cultured tumor cell lines

Liposomes containing different lipid compounds or spacer units display varying levels of cytotoxicity (23, 32). For example, poly (d,l-lactide-co-glycolide) (PLGA)-based nanoparticles and commercial Lipo2000 show about 90% and 86% cell viability, respectively (32). We observed that the cytotoxicity of P-Ls or PTf-Ls against HeLa and MCF-7 cells was remarkably low (<5%). Transferrin loading slightly increased the cytotoxicity compared with nontargeting P-Ls (Supplementary Fig. S8). Transferrin is one of the most widely used ligands for synthetic targeting systems (33, 34), and transferrin-coupled liposomes enhance cellular uptake via receptor-mediated endocytosis (35). Exposure of HeLa cells to 100 μg/mL P-Ls or PTf-Ls did not trigger cell-cycle alterations after 24 hours of culture (Supplementary Fig. S9).

Cell uptake and transfection efficiency of PTf-Ls

The cellular uptake of PTf-Ls was much higher than that of the nontargeting P-Ls into MCF-7, HeLa and MDA-MB-231 cells. Our confocal imaging studies indicated that the P-Ls were actively transported into the cells (Fig. 2) for the conjugation of transferrin to the liposomes. The uptake efficiency increased in the presence of FBS, consistent with the results of serum stability and transfection analyses, presumably because transferrin is a blood glycoprotein (34, 36, 37). PTf-Ls facilitated endocytosis and showed a higher transfection efficiency (∼80%) than that of the P-Ls or naked plasmid in the presence of 10% FBS. During the in vitro assessment of lipoplex-mediated gene transfection, it is important to emulate in vivo conditions by using serum, rather than serum-free medium (30, 38, 39).

Tumor imaging and treatment with PTf-Ls

In the in vivo imaging results, P-Ls and PTf-Ls showed brighter fluorescence, prolonged circulation time, and effectively no nonspecific binding when compared with free dye. Both nanoparticle types accumulated within the tumors, consistent with previous reports (3, 13). Suzuki and colleagues (40) previously found prolonged in vivo circulation and increased tumor accumulation when using transferrin–PEG–liposomes and PEG–liposomes with an average diameter of 100 to 200 nm. Later, Choi and colleagues (41) determined that nanoparticle localization within the tumor, but not nanoparticle accumulation, was influenced by the transferrin content. The tumor targeting of PTf-Ls was confirmed through the bioluminescent imaging of the tumor sites, an approach that has emerged as a powerful strategy for the validation of cell culture findings in animal models of breast cancer using MDA-MB-231-Luc cells (42).

IP-10 is a CXC chemokine that plays an important role in antitumor activity (16). We previously showed that IP-10 can enhance the antitumor efficacy of glioma lysate-pulsed dendritic cells and an flk1-based DNA vaccine (17, 18). The local production of IP-10 in tumor tissues contributes to the antitumoral effects through the recruitment of activated T cells, natural killer cells, monocytes, and macrophages, as well as through its inhibitory effects on the tumor vasculature (43). However, the enhancement of IP-10 gene delivery and expression in tumor cells is essential. In this study, we showed that the use of pcDNA3.1-IP-10–loaded PTf-Ls could potentially be used to enhance antitumor activity during the treatment of breast cancer.

The LSS670-labeled pcDNA-IP-10–entrapped PTf-Ls showed good properties for simultaneous tumor-targeted imaging and gene-specific delivery in an animal tumor models. This system may open up new avenues for future imaging-guided gene delivery and therapy applications.

To our knowledge, this study offers the first description of a highly efficient transferrin-conjugated liposome system possessing tumor-targeting, imaging, and gene therapy capabilities. Future work should focus on investigating the nanoparticle biodistribution, immunogenicity, and gene expression after targeted delivery, as well as the accuracy and sensitivity of in vivo tumor imaging. With these improvements, this transferrin-conjugated liposome system may provide a promising strategy for early tumor detection and therapy in vivo.

No potential conflicts of interest were disclosed.

Conception and design: H. Zhuo, Y. Peng, X. Lu, Y. Zhao

Development of methodology: H. Zhuo, Y. Peng, Q. Yao, N. Zhou, S. Zhou, J. He, Y. Fang, X. Li

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S. Zhou, J. He, Y. Fang, X. Li, H. Jin

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Y. Peng, Q. Yao

Writing, review, and/or revision of the manuscript: H. Zhuo, X. Lu

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): N. Zhou, H. Jin

Study supervision: X. Lu, Y. Zhao

The authors thank Medjaden Bioscience Limited and Jiaqi Shi for assisting in the preparation of the manuscript.

This work was supported, in part, by grants from the Program for New Century Excellent Talents in University (NECT-10-0098), the National Natural Scientific Foundation of China (Nos. 81072161, 81000769, 81172139, and 81060183), Programs for Changjiang Scholars and Innovative Research Team in University(No. IRT1119), and the Innovative Research Team in Guangxi Natural Science Foundation (No. 2011-18-5).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Supplementary data