Materials and methods

Liposome preparation

In total, 6000 nmol of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) (Avanti Polar Lipid Inc., Alabaster, AL) and 6000 nmol of cholesterol (Avanti Polar Lipid Inc, Alabaster, AL) were prepared at 20 mg/ml in chloroform. The lipids were mixed and dried for 1 hour under nitrogen. The resulting lipid films were hydrated in 2 ml of 5% USP dextrose (Abbott Laboratories, North Chicago, IL) to achieve a final lipid concentration of 6 M. The multilamellar vesicles (MLVs) generated were sonicated for 3–5 minutes using a sonicating bath (Laboratory Supplies Co. Inc., Hicksville, NY). For the pegylated liposomes, 6000 nmol DOTAP, 4800 nmol cholesterol, and 1200 nmol of 1,2-disteraoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5K] (DSPE-PEG_5K) (Avanti Polar Lipid Inc, Alabaster, AL) or DSPE-PEG_5K-Folate (Northern Lipids Inc., Vancouver, BC) were prepared as described above.

Precompacted DNA–protamine complex preparation

The pCMVinLUC plasmid containing the fire fly luciferase gene under the control of the CMV promoter was constructed by placing the luciferase gene from the pGL3-basic vector (Promega, Madison, WI, Genebank/EMBL accession # U47295), as a 1982 base pair SmaI to SalI restriction fragment, into the pUCCMVb (Clontech, Palo Alto, CA) vector backbone. The plasmid coding for the HSV-1-TK gene under the control of the CMV promoter pK2CMV-TK1 was constructed by excising the 1919 bp CMV-TK expression cassette from the plasmid construct tgCMV/TK²⁴ using EcoR1 and Xho1 and placing it into the K2 backbone derived from the pE1A-K2 plasmid.²⁵ The p(E1A)-K2-null plasmid was made by removing the 2.5 kb EcoRI to SacI fragment comprising the entire Adenovirus E1A gene sequence from pE1A-K2. All plasmids were isolated by standard molecular techniques (Althea Technologies Iin., San Diego, CA). Briefly, DNA and protamine sulfate USP (Elkins-Sinn, Cherry Hill, NJ) were prepared as equal volumes of 2 solutions and mixed into a glass reservoir via T-fitting using an Orion syringe pump (VWR, West Chester, PA) at a flow rate of 25 ml/minute resulting in a final ratio of 1:1 (weight/weight).

LPD preparation

LPD formulations with final DNA concentrations of 150 g/ml were prepared as described by Li et al.⁹ LPD particle size was measured with an N4Plus Coulter sizer using unimodal mode with all formulations having mean diameters in the range of 150–200 nm. The surface zeta potential was determined using a Malvern zeta sizer (Malvern Instrument Inc., Sacramento, CA). LPD formulations both with and without folate-conjugate-lipid had zeta-potentials of 17.3 or 5.5 mV, respectively, in 5% dextrose USP.

Cell line

The 410.4 murine breast adenocarcinoma cell line, syngeneic with Balb/c mice, was generously provided by Dr Fred Miller (Karmanos Cancer Center, Detroit, MI). Characterization of this cell line^26,27,28 demonstrated aggressive local growth when implanted into the mammary fat pad of syngeneic BALB/c female mice, and the propensity to metastasize to the lungs and other distant organs.³⁰

Di-I-labeled LPD binding to cells

LPD formulations were labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Di-I) (Molecular Probes, Eugene, OR). Di-I is a nonexchangeable, nonmetabolized fluorescent lipid tracer.³¹ In all, 100 l of 5% Di-I-labeled LPD formulations were incubated with 1 10⁶ 410.4 cells at 37°C for 1 hour, followed by three washes in PBS and resuspended in 1.0 ml of 2% paraformaldehyde solution. A total of 10,000 cells per sample were analyzed on a BD FACScan cytometer.

In vitro transfection

At 16 hours prior to transfection, 5 10⁴ 410.4 cells per well were seeded in 48-well plates (Costar, Corning, NY) and incubated overnight at 37°C in 10% CO₂. Transfections were performed using 0.1 g DNA/well and the cells were incubated for 4 hours at 37°C in 10% CO₂ in 500 l of serum-free media. Post incubation, 500 l fresh media containing 10% FBS was added per well and the cells were incubated for another 48 hours at 37°C in 10% CO₂. Cells were then washed with 1 ml PBS, pH 7.4 and 200 l of 1 luciferase reporter buffer (RLB) (Promega, Madison, WI) was added. The cells were then subjected to three freeze–thaw cycles. In all, 20 l of cleared lysate from each of six replicates per LPD formulation were assayed for luciferase activity using a Berthold Autolumat B953 luminometer (Oak Ridge, TN). The total protein concentration per sample was determined using a Coomassie Plus dye kit (Pierce, Rockford, IL).

TK/GCV cure study in 410.4 syngeneic breast cancer model

Female BALB/c mice (6-week old) (Animal Technologies Limited, Kent, WA) were injected with 1 10⁴ 410.4 cells into the right upper mammary fat pad. At 1 day post implantation, mice were randomized into groups for treatment. Mice were injected intravenously (i.v.) via the lateral tail vein with 333 l of LPD formulations. Once the same day and twice a day for 2 consecutive days following LPD administration, animals received GCV (Cytovene^®-IV, Roche, Nutley, NJ) intraperitoneally at a 100 mg/kg dose. Treatment groups were: (1) untreated (n=10), (2) vehicle (DOTAP:CHOL:PEG liposomes and protamine sulfate) (n=10), (3) LPD-PEG-TK (n=9), (4) LPD-PEG-Folate-Null (n=9), and (5) LPD-PEG-Folate-TK (n=8) (16 pmol DNA/mouse). The treatment schedule was well tolerated by the mice and concluded after 4 consecutive weeks of treatment, as described above. The tumor volume (mm³) was determined by caliper, twice weekly, using the formula [(length width depth of tumor)/2]. The weekly body weights and survival were monitored during the course of the study. Animal studies were in compliance with the "Guide for the Care and Use of Laboratory Animals" (NIH, Bethesda, MD) and the internal IACUC (Institutional Animal Care User Committee).

Statistical analysis

All values reported in this study are reported as the meanstandard deviation (SD) or standard error of the mean (SEM) as noted in the figure legends. Statistical evaluation of transfections was performed using the paired Student's t-test. Differences in tumor volumes between groups were assessed using the Mann–Whitney nonparameteric analyses. Additionally, differences in survival between groups were determined using Kaplan–Meier analyses and the Mantel–Cox test. A P-value of <.05 was considered statistically significant. Statistical tests were performed in MicroSoft Excel or STATView software for MacIntosh version 5.01 (Cary, NC).

Results

In vitro binding and transfection of LPD formulations

To determine if the LPD-PEG formulations could bind to the 410.4 murine breast adenocarcinoma cell line, cells were incubated with LPD formulations labeled with the fluorescent tracer, Di-I, and assessed by FACS analysis. Results demonstrated that cell-associated fluorescence, which detected both binding and potential internalization, occurred with the LPD formulation (MFI=833.32) and was partially suppressed with the addition of 2% PEG (MFI=741.22) (Table 1). The addition of 2% DSPE-PEG_5K-Folate to the LPD formulation not only restored binding to the level of the LPD (MFI=1162.79) effect but actually superceded it, demonstrating a 1.4-fold increase (Table 1). More importantly, when assessing the binding ratio of LPD-PEG-Folate to LPD-PEG, a 1.6-fold increase in binding is observed (Table 1). These results clearly demonstrate that the LPD formulations bind to the 410.4 cells. Competition experiments have demonstrated that the LPD-PEG-Folate formulations bind to cells in a folate-dependent manner.²³ Moreover, these results suggest that folate receptor mediated binding in the 410.4 cell line and provide support for the presence of the folate receptor on these cells, as antibodies to the murine folate receptor are not available commercially.

Table 1 - Cell-associated mean fluorescence intensity of Di-I-labeled formulations.

Full table

To evaluate whether the binding characteristics of the LPD formulations were reflected in transfection potency, the 410.4 cells were transfected with the LPD formulations containing the luciferase transgene and harvested 48 h after transfection. As demonstrated in Figure 1, transfections with the LPD formulations resulted in expression of luciferase activity which was significantly suppressed (22-fold) with the addition of 2% PEG (P=.043, Fig 1). Similar to the binding and internalization data, addition of 2% PEG-Folate restored transfection potency to the levels of unmodified LPD (P=.764, Fig 1). More significantly, the increased transfection capabilities of the 2% LPD-PEG-Folate demonstrated a 26-fold enhancement over the 2% LPD-PEG formulation (P=.0235, Fig 1). Again, these results suggest specific folate-mediated binding, internalization, and transfection over the base LPD-PEG formulation alone.

Figure 1.

Luciferase activity in 410.4 cells following transfection with the LPD formulations. 410.4 cells were transfected and processed for luciferase expression as described in Materials and methods. Formulations were prepared using 2 mol% DSPE-PEG or DSPE-PEG-Folate. Data are represented as luciferase activity normalized to mg protein in each samples (RLU/mg protein). Error bars represent meanSEM. *P=.043, **P=.0235.

Full figure and legend (30K)

LPD-PEG-Folate-TK mediated antitumor efficacy in a syngeneic murine tumor model

The antitumor efficacy of the various LPD-PEG formulations containing the HSV-TK gene was assessed using the 410.4 syngeneic breast carcinoma model. Similar to previous studies performed by our group, in vivo experiments were conducted using LPD formulations containing 10 mol% DSPE-PEG5K (LPD-PEG) or DSPE-PEG_5K-Folate (LPD-PEG-Folate) as it was shown that in contrast to non-PEG-bearing formulations these formulations possessed the greatest serum stability, likely due to reduced levels of complement activation, 23 minimal effect on inflammation responses as measured by TNF- levels, serum chemistries or white blood cell counts.²⁹ BALB/c mice were inoculated with 410.4 cells into the mammary fat pad and the tumor size monitored twice weekly. LPD treatment (16 pmol DNA/mouse), in combination with GCV, was initiated 1 day following cell implantation and continued weekly for 4 consecutive weeks as described in Materials and methods. As demonstrated in Figure 2a, tumor growth occurred rapidly in the untreated, vehicle, LPD-PEG-TK, and LPD-PEG-null groups. However, in the LPD-PEG-Folate-TK-treated group, tumor growth was significantly suppressed through day 25 following cell inoculation. For instance, at day 25, the LPD-PEG-TK group demonstrated a significant reduction in mean tumor volume (260.1 mm³, range=25–711.1 mm³) compared to the untreated (825.3 mm³, range=260.7–1968.2 mm³), vehicle (749.7 mm³, range=25–1398.5 mm³), and the untargeted LPD-PEG-TK (914.1 mm³, range=250.9–1874.8 mm³) groups (P<.019) (Fig 2a). While the control group containing the null gene within the formulation, LPD-PEG-Folate-Null (474.1 mm³, range=0–1242.8 mm³), did not demonstrate a significant difference with respect to mean tumor volumes compared to the LPD-PEG-Folate-TK group (P=.211), the trend suggests the partial antitumor effect that could have resulted from nonspecific immune/inflammatory responses elicited by CpG motifs expressed in the null DNA.⁹ Lastly, in Figure 2b when comparing the tumor growth rates at day 25, the LPD-PEG-Folate-TK group demonstrated a significant decrease compared to the untreated (68.4%), vehicle (65.22%), and LPD-PEG-TK (71.47%) groups (P<.018). Again, however, while no statistical difference was observed between the LPD-PEG-Folate-TK and LPD-PEG-Folate-Null groups, a 44.99% decrease in the tumor growth rate was shown (P=.184). Overall, these results suggest that LPD-PEG-Folate-TK gene therapy in combination with GCV is capable of reducing tumor growth over the base LPD-PEG-TK formulations (3.5-fold) and indicates the potential of Folate-targeted formulations.

Figure 2.

In vivo antitumor effects of systemically administered LPD-PEG-Folate-TK. (a) Effect on tumor volume. Data represents the mean tumor volume up to day 28 post cell inoculation. Cells were implanted and treatments administered as described in Materials and methods. Treatment groups were as follows: untreated (squares, n=10), vehicle (stars, n=10), LPD-PEG-TK (diamonds, n=9), LPD-PEG-Folate-Null (circles, n=9), and LPD-PEG-Folate-TK (triangles, n=8). Tumor volume was calculated by multiplying the length, width, and depth of each tumor and dividing by 2 [(L W D)/2]. Error bars represent meanSEM. *P-value <.019. (b) Suppression of tumor growth rates. At day 25 post 410.4 cell inoculation, tumor growth rates were determined in BALB/c mice by dividing the tumor volume by day (mm³/day). LPD-PEG-Folate-TK treatment resulted in a statistical significant decrease in tumor growth rate compared to the untreated, vehicle, and PEG-TK groups (*P<.018). Data represents mean tumor growth ratesSEM.

Full figure and legend (137K)

Treatment with LPD-PEG-Folate-TK demonstrates enhanced survival

In addition to suppression of tumor growth, LPD-PEG-Folate-TK treatment also resulted in a significant increase in survival time. As shown in Figure 3, median survival was increased from day 25 to day 31 comparing the LPD-PEG-TK to LPD-PEG-Folate-TK group (P=.0011). More specifically, at day 25, 100% of the animals in the LPD-PEG-Folate-TK remained, whereas 70% in the untreated (P=.0048), 50% in the vehicle (P=.092), 55.6% in the LPD-PEG-TK (P=.0011), and 77.8% in the LPD-PEG-Folate-Null (P=.1526) groups remained. While statistical significance was not achieved when comparing the vehicle and LPD-PEG-Folate-Null groups to the LPD-PEG-Folate-TK groups, the trend does suggest that LPD-PEG-Folate-TK enhanced animal survival. Lastly, in an additional survival study, preliminary data suggest LPD-PEG-Folate-TK treatment demonstrates a statistically significant increase in survival compared to LPD-PEG-Folate-Null group (P<.042), suggesting that statistical difference to the null control can be achieved (data not shown). Together, the data establish that LPD-PEG-Folate-TK treatment has the potential to not only decrease tumor growth but to also enhance overall survival.

Figure 3.

LPD-PEG-Folate-TK systemic treatment enhances median survival. The 410.4 tumor- bearing mice treated as described in Figure 2 were monitored for survival. Data represents percent survival over time as a Kaplan–Meier plot. At day 25 post cell inoculation, 100% of the animals remained in the LPD-PEG-Folate-TK groups, whereas only 70% in the untreated, 50% in the vehicle, 55.6% in the PEG-LPD-TK, and 77.8% in the LPD-PEG-Folate-Null groups remained. *P-value <.049 comparing the LPD-PEG-Folate-TK groups to the untreated and LPD-PEG-TK groups independently.

Full figure and legend (23K)

Discussion

Most gene therapy studies assess in vivo efficacy using xenograft models where a subcutaneous human cell line/tumor is used in an immunodeficient mouse. While this practice is useful in initial screening processes, the benefits or complications of an intact immune system cannot be addressed nor overlooked in importance. In the current study, the efficacy of LPD-PEG-Folate-TK gene therapy was evaluated in a syngeneic model for breast cancer and provides insight into the possible benefits, for instance enhanced antitumor efficacy of an intact immune system. Initial in vitro studies demonstrated that the 410.4 breast cancer cell line could be preferentially transfected with the LPD-PEG-Folate formulations and suggest the presence of the murine folate receptor on this cell line (Fig 1). While it would be ideal to assess direct expression of the murine folate receptor, there are no commercially available antibodies to the murine folate receptor. Additionally, as an attempt to assess specific folate–folate receptor interactions, previous studies in our laboratory have shown that competition of LPD-PEG-Folate binding and internalization with monovalent folate is not capable of competing with the multivalent folate ligands present on our formulations (data not shown). Furthermore, our results based on the transfection data, as well as the binding/internalization data, provide firm evidence that the 410.4 cell line does possess the murine folate receptor (Fig 1 and Table 1).

The therapeutic efficacy of the LPD-PEG-TK formulations, in conjunction with GCV treatment, was next examined using the 410.4 model in syngeneic BALB/c mice. Of note, in evaluating the antitumor activity of the LPD-PEG-Folate formulation, a formulation containing 10 mol% of DSPE-PEG_5K-Folate ligand was used compared to the in vitro Di-I binding and transfection experiments where 2 mol% of DSPE-PEG_5K-Folate was used. Previous studies demonstrated that 2 mol% was the optimal concentration for a maximum enhancement of LPD-PEG-Folate-mediated transfection enhancement in vitro.²³ Additionally, previous in vivo studies established that higher levels of DSPE-PEG_5K-Folate in the formulation offered a superior LPD surface protection, reduced serum protein interactions, and enhanced circulatory half-life allowing for more effective passive and targeted tumor deposition. Those in vivo effects are not readily amenable to in vitro analysis; therefore, ligand effects were modeled using the 2% PEG formulations. Using LPD-PEG-Folate-TK, a significant decrease in tumor volume and tumor growth rates was observed over the untargeted LPD-PEG-TK formulation, as well as vehicle and untreated control groups (Fig 2). In this aggressive tumor model, the significant differences were observed at day 25 following cell inoculation, but could not be achieved at later time points. Interestingly, this correlated to the termination of treatment. Therefore, alterations in treatment regimen, such as continued treatment or more frequent administration of the LPD-PEG formulations, have the potential to enhance and extend the observed antitumor effects. Additionally, statistical significance was not achieved comparing the LPD-PEG-Folate-TK group to the LPD-PEG-Folate-Null control group; however, the trend suggests that LPD-PEG-Folate-TK treatment is more efficacious (Figs 2, 3). The moderate antitumor effects observed following LPD-PEG-Folate-Null treatment potentially result from nonspecific immune and inflammatory responses to the CpG motif in the bacterially derived plasmid DNA. Recent work has demonstrated that these effects can be overcome when oligonucleotides with an NF-B consensus binding sequence are coadministered with the LPD formulations.¹⁰ While this holds promise for delineating the specific vs nonspecific effects of the therapeutic transgene, it may also detract from the potential antitumor benefits of the nonspecific components and add substantial unwanted complexity to a therapeutic approach.

Lastly, in very aggressive in vivo models of cancer, such as the 410.4 model, often the therapeutic modality is incapable of suppressing tumor growth and survival parameters are key to the evaluation of drug. Therefore, the significantly enhanced survival observed in the LPD-PEG-Folate-TK group compared to the untargeted LPD-PEG-TK group provides strong support for LPD-PEG-Folate-TK-based gene therapy (Fig 3). This observation is also extended through day 30 following cell inoculation and, as mentioned above, additional enhancements in survival rates may be achieved by altering the treatment regimen. Therefore, the reductions in tumor volumes and growth rates, as well as the enhanced survival following LPD-PEG-Folate-TK treatment provide a strong basis for the continued preclinical development of systemically delivered, tumor-targeted liposomal cancer gene therapy products for the treatment of primary and metastatic tumors at inaccessible or distant sites.

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Acknowledgements

We greatly acknowledge Drs Ziv Sandalon and Edward Kelly for their constructive comments and review of the manuscript.