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intratracheal gene therapy of early lung cancer. Yiyu Zou,1 Gang Zong,1 Yi-He Ling,2 and Roman Perez-Soler2. 1Department of Cancer Biology, University of ...
© 2000 Nature America, Inc. 0929-1903/00/$15.00/⫹0 www.nature.com/cgt

Development of cationic liposome formulations for intratracheal gene therapy of early lung cancer Yiyu Zou,1 Gang Zong,1 Yi-He Ling,2 and Roman Perez-Soler2 1

Department of Cancer Biology, University of Texas MD Anderson Cancer Center, Houston, Texas 77030; and 2New York University, Rita J. & Stanley Kaplan Comprehensive Cancer Center, New York, New York 10016. Regional (intratracheal or aerosol) delivery of cationic liposome-DNA complexes for gene therapy of lung disease offers distinct advantages over systemic (intravenous) administration. However, optimal formulations for early lung cancer treatment have not been established. Therefore, we investigated ⬎50 different liposome and micelle formulations for factors that may affect their transcription efficiency and tested the ideal formulations in an in vivo mouse model. Our data showed that cationic liposomes were generally more effective at transfecting genes than were micelles of the same lipid composition, thus suggesting a role for the bilayer structure in facilitating transfection. In addition, the transfection efficiency of liposome-delivered genes was highly dependent upon the lipid composition, lipid/DNA ratio, particle size of the liposome-DNA complex, and cell lines used. By optimizing these factors in vitro and in vivo, we developed a novel liposome formulation (DP3) suitable for intratracheal administration. Using G67 liposome as control, we found that DP3 was more effective than G67 in vitro and as effective as G67 at both preventing lung tumor growth and prolonging survival in our lung cancer mouse model. We observed a positive correlation between the in vitro p53 function and the in vivo antitumoral activities of liposome-p53 formulations, which had not been reported previously in studies of an intravenous liposome gene delivery system. This correlation may facilitate the development and optimization of a liposome-p53 formulation for aerosol use in lung cancer patients. Cancer Gene Therapy (2000) 7, 683– 696

Key words: Lung cancer; intratracheal administration; liposome-p53.

L

ung cancer, with a mortality of ⬃90%, is the leading cause of cancer-related deaths in the United States.1 Survival in the past 20 years has improved by only 2%, and to this day it remains dismal.2– 4 One reason for this poor survival is that the three traditional modalities of treatment have failed to treat lung cancer in its early stages or to adequately target lung cancer cells. Systemic chemotherapy has been used with little success because most drugs delivered in this way are immediately destroyed or inactivated by liver and blood components; as a result, not enough drug reaches the lung tumor. Those drug molecules that do reach tumor cells then must confront the intracellular obstacle of multiple drug resistance. Drugs delivered systemically may also cause life-threatening systemic toxicity. As for radiation therapy, it does not distinguish between tumor cells and normal cells and is not sensitive to lung cancer.5 Surgery, meanwhile, is useless for removing the whole pulmonary tree, most metastases, and most invisible tumors. Worse yet, none of these modalities can correct the genetic alterations and de-

Received April 29, 1999; accepted September 6, 1999. Address correspondence and reprint requests to Dr. Roman Perez-Soler, Rita J. Stanley Kaplan Comprehensive Cancer Center, New York University School of Medicine, 550 First Avenue, New York, NY 10016.

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fects in diffusely damaged bronchial epithelium that ultimately underlie all lung cancers,6 a failure that underscores the real need for gene therapy against lung cancers. Consequently, others and we have devoted much effort to devising effective approaches to the gene therapy of early lung cancers. To our advantage, recent advances in understanding the molecular genetics of lung cancer have made it possible to diagnose lung cancers even earlier than before7,8 Because the genetic lesion originates on the bronchoalveolar epithelium, the lesion, even though it can be diffuse, is usually superficially localized on the surface of the pulmonary airway. Thus, the best chance for gene therapy of early lung cancers seems to us to be airway administration, because this would most easily allow therapeutic agents to reach and penetrate most malignant cells. In general, researchers in the field have been considering two types of therapeutic gene delivery: viral and nonviral. Use of an adenoviral vector carrying a p53 gene has already been approved for clinical trial,9 –15 but the potential immunogenicity, high toxicity (including permanent damage to the genome), and limited methods of administration could limit its application.16 –20 In contrast, most of the nonviral gene delivery systems that are being considered have no immunogenicity.21 In our own work, we have focused on nonviral systems and have 683

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determined that an effective nonviral gene therapy system would include a gene delivery vector, administration method, and therapeutic gene. Among the nonviral gene delivery systems so far proposed by others and by us, the cationic liposome is the best candidate because of its low toxicity.22,23 The feasibility of its use in the airways has already been demonstrated in different animals,23–32 and studies of liposome-gene complexes administered by intranasal33 and aerosol34 routes in cystic fibrosis patients have revealed preliminary evidence of effective transfection of the bronchial epithelium. We ourselves have hypothesized that the intratracheal or aerosol administration of liposome-tumor suppressor gene complexes for early lung cancer gene therapy would be more effective and practical than intravenous chemotherapy and intratumoral viral gene therapy, especially for liposomes, because attacks from macrophages, high-density lipoproteins, and phospholipases would be presumably less fierce in the bronchial airway than in the liver and bloodstream.35 In previous studies, we developed an orthotopic animal model to mimic human early lung cancer and then used it to prove our hypothesis by showing that intratracheally injected liposome-p53 gene complexes could inhibit orthotopic human lung tumor growth and prolong the life span of the tumor-bearing mice.36 We used p53 as our prototype gene because of its proven tumor suppressor function37–39 and frequent (50 –70%) mutation in lung cancer.7,40,41 In this light, we therefore focused our present work on optimizing our liposome-p53 gene formulation and improving its therapeutic potential by pharmaceutical means. Because nonviral gene delivery systems are mainly hampered by their low delivery efficiency, our immediate goal was first to identify those factors that most affected our system’s ability to transfect cells and produce therapeutic results and then to modify them so as to optimize our liposome formulation for the delivery of therapeutic genes via the airway as a treatment for early lung cancers.

MATERIALS AND METHODS

Cell lines and cell culture H358 (human non-small cell lung carcinoma cells (NSCLC) with a p53 deletion mutation, developed from human bronchial epithelium), H322 (human NSCLC cells with a p53 mutation at codon 248, developed from human bronchial epithelium), Saos2 (human osteogenic sarcoma cells with a p53 deletion), HeLa (human cervical carcinoma cells with wild-type (wt) p53), A549 (human lung adenocarcinoma cells with mutant p53), and NIH/3T3 (murine embryo cells with wt p53) were purchased from the American Type Culture Collection (Manassas, Va). Cells were cultured in a cell culture incubator at a constant temperature (37°C) and humidity in filtered air containing 5% CO2. Only the first five passages of each cell line were used.

Expression plasmids Several expression plasmids were used. pC53SN is an 8.3-kb plasmid containing wt human p53 cDNA under the control of a cytomegalovirus (CMV) promoter. Empty vector plasmid was constructed by deleting p53 cDNA from the pC53SN plasmid. wwp-luc is a 7.9-kb plasmid containing the luciferase gene under the control of a p21 promoter, which is activated specifically by wt p53. pC53SN and wwp-luc plasmids were gifts of Dr. Bert Vogelstein at John Hopkins University (Baltimore, Md). ␤-galactosidase (␤-gal) is an 8.5-kb plasmid containing the lacZ gene (Escherichia coli lacZ gene encoding ␤-gal, a commonly used reporter gene) under the control of a CMV promoter. The pC53SN vector (p53 gene removed) was used as a nonexpression plasmid control (Vp).

Preparation of liposomes All lipids were obtained from Avanti Polar Lipids (Alabaster, Ala). Large liposomes (400 – 660 nm and 800-1200 nm) were prepared by hydration and centrifugation (1000 ⫻ g) methods. Medium liposomes (170 –240 nm) were prepared by detergent removal plus extrusion methods as described below. Small liposomes (20 – 40 nm and 60 –110 nm) were prepared by sonicating large or medium liposomes. Hydration was performed with a rotary evaporator for 2 hours at 5°C above the phase transition temperature of the cationic lipids at 50 rpm followed by five freeze-thaw cycles. A set of extrusion membranes with different pore sizes ranging from 0.1 ␮m to 1.0 ␮m (Gelman Sciences, Ann Arbor, Mich) was used to regulate the liposome particle size based on the requirements of the experiments. Each formulation was passed through at least two extrusion membranes with three filtration circles on each, first a larger pore size and then a smaller pore size at the same temperature as that used for the hydration phase. Liposomes containing stearylamine (ST) were prepared using the same method. The G67 liposomes (provided by Genzyme, Cambridge, Mass) were also treated by the extrusion method if required by the experimental design. The liposome particle size was measured by a multiple-angle dynamic light-scattering method using a Nicomp Submicron Particle Sizer 370 (Nicomp Particle Sizing System, Santa Barbara, Calif).

Preparation of cationic lipid micelles A dry lipid film containing cationic phospholipids was formed by drying the lipid chloroform-methanol solution on a rotor evaporator. The lipid film was then hydrated with a distilled water solution containing nonionic surfactant polyoxyethylene20 sorbitan monolaurate (Tween 20; Sigma, St. Louis, Mo) at room temperature. The phospholipid/surfactant ratios are listed in Table 1. The hydrated micelle suspensions were spun at maximum power in a vortex two times for 2 minutes each time. The particle size was measured as described above.

Liposome-DNA or micelle-DNA complex formation The liposome-DNA or micelle-DNA complexes were formed by mixing the preformed cationic liposomes or micelles with plasmids in Opti-Mem (Life Technologies, Gaithersburg, Md) at a weight ratio specified for each experiment and then incubating the mixture at 37°C for 20 minutes. All complexes were used immediately after the incubation. Opti-Mem is a cell culture medium that contains the basic nutrients needed for cell growth and a minimal amount of the serum proteins that can inhibit liposome-mediated gene transfection. The particle size of the complexes was measured immediately after incuba-

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Table 1. Effects of Micelle Formulation on Transfection Efficiency In Vitro* Name

Composition

Lipid ratio (mol)

Size (mean ⫾ SD, nm)

% Transfection in 3T3 cells

Micelle D1 Micelle D2 Micelle D3

DODAP/De DODAP/De DODAP/De

7:1 10:1 20:1

6.5 ⫾ 3.2 8.2 ⫾ 4.5 8.9 ⫾ 3.3

0.1 ⫾ 0.2 0.8 ⫾ 0.3 0.2 ⫾ 0.1

Micelle T1 Micelle T2 Micelle T3

DOTAP/De DOTAP/De DOTAP/De

7:1 10:1 20:1

6.6 ⫾ 3.7 7.6 ⫾ 3.5 7.8 ⫾ 3.9

0.1 ⫾ 0.3 1.4 ⫾ 0.7 0.2 ⫾ 0.3

Micelle L1 Micelle L2 Micelle L3

DLEP/De DLEP/De DLEP/DOPE

7:1 10:1 20:1

5.7 ⫾ 3.1 6.8 ⫾ 3.4 7.2 ⫾ 4.1

0.2 ⫾ 0.2 1.6 ⫾ 0.5 0.3 ⫾ 0.2

Micelle M1 Micelle M2 Micelle M3

DMEP/DOPE DMEP/DOPE DMEP/DOPE

7:1 10:1 20:1

7.1 ⫾ 3.0 7.5 ⫾ 3.6 8.3 ⫾ 4.5

0.5 ⫾ 0.3 3.1 ⫾ 1.4 2.2 ⫾ 0.9

Micelle P1 Micelle P2 Micelle P3

DPEP/DOPE DPEP/DOPE DPEP/DOPE

7:1 2:1 3:1

7.4 ⫾ 3.8 7.9 ⫾ 4.0 8.1 ⫾ 5.4

0.3 ⫾ 0.2 3.3 ⫾ 0.6 1.7 ⫾ 0.9

*The micelles were made by hydrating dry cationic lipid film with a distilled water solution containing the nonionic surfactant Tween 20. The phospholipid/surfactant ratio is given as the molar ratio. The hydrated micelle suspensions were spun at maximum power in a vortexer two times for 2 minutes each time. The particle size was measured by a light-scattering method using a Nicomp Submicron Particle Sizer. The micelle-␤-gal DNA complexes were used to transfect NIH/3T3 cells. The transfection efficiency was calculated by determining the percentage of total cells that turned blue after X-Gal staining. The DNA dose was 2 ␮g of DNA/106 cells. Data for each formulation represent the mean ⫾ SD of three independent experiments carried out with duplicate samples.

tion by the dynamic light-scattering method using the Nicomp Sizer.

number of viable nontreated cells, and Td equals number of viable liposome-p53-treated cells.

In vitro cell transfection

p53 function assay

Cells were plated in 6-well tissue culture plates overnight at 0.5–1 ⫻ 106 cells/well and then exposed to lipid-DNA complexes (DNA dose 2 ␮g/106 cells) in 1 mL of Opti-Mem for 6 hours. Next, an equal amount of RPMI 1640 medium containing 20% fetal bovine sera (Life Technologies) was added, and the cells were cultured for another 24 hours. Different lipid/ DNA ratios and DNA doses were tested. Transfection was terminated by replacing the medium with fresh RPMI 1640 medium containing 10% fetal bovine serum. All transfections were performed at 37°C in an atmosphere of 5% CO2 and 95% air.

p53 function was determined by cotransfecting the cells with the p53 gene and wwp-luc. In this system, measured luciferase activity is proportional to the cellular p53 function. A total of 2 ⫻ 106 cells were plated in 90-mm culture dishes. After 24 hours, the cells were transfected with liposome-DNA complexes containing equal amounts of three plasmids: wwp-luc, ␤-gal, and pC53SN (2 ␮g DNA/106 cells). Controls included cells transfected with DP3-DNA containing equal amounts of wwp-luc, ␤-gal, and empty vector plasmid (Vp, without p53), cells transfected with pC53SN alone, and cells treated with either liposomes or luminometry reaction buffer (100 mM K2PO4, 5 mM adenosine triphosphate, 15 mM MgSO4, and 1 mM dithiothreitol (pH 7.8)). Cells were incubated at 37°C for 36 hours in fresh medium after transfection, washed with 4°C phosphate-buffered saline (PBS), and harvested with a cell scraper in 1.0 mL of luminometry reaction buffer at 4°C. Cells were then carefully resuspended, sonicated for 2 minutes on ice, and centrifuged at 1000 ⫻ g for 5 minutes. A total of 25 ␮L of the supernatant was mixed with 325 ␮L of the reaction buffer and 100 ␮L of luciferin solution (0.3 mg/mL) on ice, and the mixture was measured immediately in a luminometer (TD20/20; Promega, Madison, Wis) at a wavelength of 550 – 570 nm. The ␤-gal activity in 25 ␮L of the supernatant was measured as described previously.42,43 Briefly, a 25-mL sample was diluted to 990 mL with a tris(hydroxymethyl)aminomethane-HCl buffer (pH 7.5) and then mixed with 10 mL of dimethylsulfoxide solution containing resorufin (7.5 mg/mL). After the cells had been incubated for 30 minutes at 30°C, the optical density (OD) of the treated samples was measured with a spectrometer at 558 nm. Transfection efficiency was mea-

Determination of transfection efficiency and killing efficiency The ␤-gal staining method was used to determine transfection efficiency after 48 hours of transfection as described above. In brief, cells were fixed and stained with 5-bromo-4-chloro-3indolyl ␤-D-galactoside (X-Gal) for 5 hours. For each kind of treatment, the total cell numbers from two parallel samples (two wells) were counted using a hemocytometer just before all cells were fixed. Transfection efficiency was determined by counting the blue cells in six randomly selected fields for at least 200 cells each under a microscope and was subsequently expressed as the percentage of blue cells per total cells. p53 transfection caused significant lung cancer cell death in tissue culture. To account for this, the percentage of killed cells, or killing efficiency, was calculated as follows: % Killing ⫽ (Tl/C ⫺ Td/C) ⫻ 100%, where Tl equals the number of viable cells treated with liposome alone, C equals the total

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sured by ␤-gal activity. p53 function was expressed as “corrected luminometry units,” which were obtained by dividing the luminometry units by the OD values of ␤-gal activity to factor out uneven transfection efficiency.

displayed multiple visible bilateral lung tumor nodules. By week 10 –16, the mice died of multiple bilateral lung tumors without distant metastases.

Antitumoral activity of liposome-p53 in mice bearing early endobronchial H358 and H322 tumors

Apoptosis assays The percentage of apoptotic cells was determined by a terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick end-labeling (TUNEL) reaction assay according to the manufacturer’s recommendation (Boehringer Mannheim, Indianapolis, Ind). Cells (106 cells/well) in 1.0 mL of Opti-Mem were plated in 6-well plates, grown for 24 hours at 37°C in a 5% CO2 atmosphere, and transfected with DP3-p53 as described above. Untreated cells and cells exposed to DP3 alone were used as controls. After transfection, cells were washed with PBS containing 1% bovine serum albumin, fixed with 4% paraformaldehyde in PBS at room temperature for 30 minutes, and permeabilized with 0.1% Triton X-100 (Sigma) in 0.1% sodium citrate at 4°C for 2 minutes. Cells were then washed once with PBS and incubated in 50 ␮L of TUNEL reaction mixture containing 2 U of terminal transferase and fluorescein-dUTP at 37°C for 60 minutes. After being washed three times with PBS, the fluorescently labeled cells were assessed by fluorescence microscopy and flow cytometry (Epics Profile Analyzer; Coulter, Hialeah, Fla).

Endobronchial H358 and H322 lung tumor models Male nu/nu mice (7– 8 weeks of age, 18 –22 g each; Harlan Sprague-Dawley, Indianapolis, Ind) were inoculated intratracheally with H358 or H322 cells (1–2 ⫻ 106 cells/mouse). Inoculated cells initially attached to the surface of the bronchial epithelium. By week 2– 4, multiple microscopic tumors arising in the bronchial epithelium and in connection with the bronchial lumen were well-established. By week 7–9, the mice

In all therapeutic experiments, treatment was started early (at day 4 postinoculation) because our main objective was to investigate the efficacy of liposome-p53 in the treatment of endobronchial lesions before they had invaded the lung parenchyma. A multiple dose schedule was used because under optimal conditions a single transfection with DP3-p53 causes apoptosis in only ⬃40% of cells in vitro (see Results). A dose was given every 4 days; however, the number of doses was limited to five because of the technical difficulties involved in repeatedly cannulating the trachea through the mouse’s mouth.

Tumor growth inhibition (TGI) experiments Male nu/nu mice were each inoculated intratracheally with 106 H358 cells and divided into eight groups with five mice in each group. One group was left untreated. The other seven groups were treated intratracheally with pC53SN plasmids alone, ST liposomes alone, G67 liposomes alone, DP3 liposomes alone, or ST-p53, G67-p53, or DP3-p53 complexes on days 4, 8, 12, 16, and 20 after H358 cell inoculation, respectively. The dose was 2 ␮g of DNA per administration. The liposome size for all formulations was 60 –110 nm. The lipid/DNA ratio was 6:1 for DP3-p53 and ST-p53 and 2:1 for G67-p53. All mice were killed on the day the first untreated tumor-bearing mouse became moribund (day 74 –77 in this study). The lungs were resected and weighed. The percentage of tumor growth inhibition (%TGI) was calculated as follows: %TGI ⫽ (1 ⫺ [lung weight difference between each p53-treated group and healthy group]/

Table 2. Effects of Liposome Formulation on Transfection Efficiency In Vitro* Composition

Lipid ratio (mol)

Size (mean ⫾ SD, nm)

% Transfection in 3T3 cells

Liposome D1 Liposome D2 Liposome D3

DODAP/DOPE DODAP/DOPE DODAP/DOPE

1:3 1:1 3:1

83.6 ⫾ 37.8 88.6 ⫾ 42.1 78.3 ⫾ 45.2

0.6 ⫾ 0.3 0.3 ⫾ 0.2 0.2 ⫾ 0.3

Liposome T1 Liposome T2 Liposome T3

DOTAP/DOPE DOTAP/DOPE DOTAP/DOPE

1:3 1:1 3:1

70.9 ⫾ 38.1 66.9 ⫾ 34.9 59.2 ⫾ 33.9

5.6 ⫾ 1.0 5.4 ⫾ 1.1 1.7 ⫾ 0.4

Liposome L1 Liposome L2 Liposome L3

DLEP/DOPE DLEP/DOPE DLEP/DOPE

1:1 2:1 3:1

457 ⫾ 273 230 ⫾ 175 146 ⫾ 85

3.7 ⫾ 1.9 3.8 ⫾ 1.5 4.7 ⫾ 1.4

Liposome M1 Liposome M2 Liposome M3

DMEP/DOPE DMEP/DOPE DMEP/DOPE

1:1 2:1 3:1

1573 ⫾ 446 1000 ⫾ 410 1250 ⫾ 317

4.1 ⫾ 1.8 7.4 ⫾ 1.4 11.4 ⫾ 5.2

Liposome DP1 Liposome DP2 Liposome DP3

DMEP/DOPE DPEP/DOPE DPEP/DOPE

1:1 2:1 3:1

1354 ⫾ 772 1250 ⫾ 782 80.0 ⫾ 25.2

3.7 ⫾ 1.1 3.1 ⫾ 0.9 11.2 ⫾ 6.6

G67

L#67/DOPE

1:2

380 ⫾ 280

8.5 ⫾ 2.1

Name

*The liposomes were prepared by thin-lipid film hydration as described in Materials and Methods, followed by five freeze-thaw cycles. G67 liposomes were provided by Genzyme. Particle size and transfection efficiency were determined as described in Table 1. The data for each formulation represent the mean ⫾ SD of three independent experiments carried out with duplicate samples.

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Table 3. Effect of Lipid/DNA Ratio on Transfection Efficiency* % Transfection in NIH/3T3 cells lipid/DNA ratio Formulation Liposome Liposome Liposome Liposome G67

ST T1 M3 DP3

2:1

3:1

6:1

12:1

24:1

0.4 ⫾ 0.1 ND† ND 2.0 ⫾ 0.4 6.8 ⫾ 1.7

0.6 ⫾ 0.2 0.3 ⫾ 0.1 4.8 ⫾ 1.1 5.4 ⫾ 1.0 8.5 ⫾ 2.1

2.9 ⫾ 0.1 3.2 ⫾ 0.4 9.2 ⫾ 2.1 10.1 ⫾ 3.5 4.3 ⫾ 1.4

6.2 ⫾ 0.2 5.6 ⫾ 1.0 11.4 ⫾ 5.2 11.2 ⫾ 6.6 0⫾0

0⫾0 ND 1.4 ⫾ 0.3 9.5 ⫾ 3.1 0⫾0

*The selected micelle and liposome formulations (which had higher transfection efficiencies than those listed in Tables 1 and 2) were incubated with ␤-gal plasmid together at different lipid/DNA ratios at 37°C for 20 minutes. The complexes were then used to transfect NIH/3T3 cells. The data for each formulation represent the mean ⫾ SD of three independent experiments carried out with duplicate samples. † ND, transfection efficiency not detected.

[lung weight difference between the untreated tumor-bearing group and healthy group]) ⫻ 100%.

Survival experiments Both p53-null (H358, p53 deletion) and p53-mutant (H322, p53 248 codon point mutation) tumor-bearing mice were used in the survival experiments. Animals were inoculated intratracheally with 1–2 ⫻ 106 H358 or H322 cells/mouse. Treatment consisted of five intratracheal doses given on days 4, 8, 12, 16, and 20. The dose was 2– 8 ␮g of DNA per administration. Increased life span (%ILS) was calculated as follows: %ILS ⫽ ([average survival days of the treatment group/average survival days of the control group] ⫺ 1) ⫻ 100%. Differences in survival among groups were analyzed for statistical significance by the two-sided log-rank test. All statistical tests used in this study were two-sided statistical tests.

RESULTS

Liposomes were generally more effective than micelles in vitro and in vivo Many different cationic liposome formulations have been tested for their ability to mediate transfection. In contrast, the ability of micelles to mediate transfection has not been investigated extensively. It is not clear whether the liposome bilayer is necessary for transfection. To answer this question, we compared the transfection efficiency of ⬎50 different micelle and liposome

formulations made of various lipid components in various ratios. A wide variety of lipids were used to prepare micelles and liposomes. Tables 1 and 2 show the lipid composition, lipid ratio, particle size, and transfection efficiency for each micelle and liposome formulation used in NIH/3T3 cells. For formulations of the same lipid composition, the three with the highest transfection efficiencies were listed. Transfection efficiency was measured by ␤-gal transfection and X-Gal staining. The data in these tables show that although micelles successfully mediated transfection in vitro, they were in general much less effective than the liposomes made of the same phospholipids. The reason for this was not clear, but two of the main factors were particle size and membrane structure. The relationship between transfection efficiency and the size of liposome-DNA complexes was investigated later. A similar result was also confirmed in vivo. The micelle formulation (micelle P2) with the highest transfection efficiency (3.3.%) among the micelle formulations was tested for TGI (see Fig 5A). The result was parallel with the in vitro transfection: micelle-p53 had much less of an effect on tumor inhibition than the liposome-p53 formulations tested. It seems that the micelle may not be able to transfect enough of the tumor cells and inhibit the tumor growth in vivo.

Table 4. Effect of the Complex Size on Transfection Efficiency* % Transfection in NIH/3T3 cells Size (nm) Formulation Liposome Liposome Liposome Liposome G67

ST T1 M3 DP3

1250-900

710-500

430-320

280-190

150-110

2.3 ⫾ 1.2 0.3 ⫾ 0.1 0.8 ⫾ 0.4 0.9 ⫾ 0.5 1.9 ⫾ 1.1

2.1 ⫾ 0.8 1.8 ⫾ 0.2 4.2 ⫾ 1.0 2.1 ⫾ 0.4 3.2 ⫾ 0.5

5.2 ⫾ 1.5 2.3 ⫾ 0.2 5.9 ⫾ 1.2 4.1 ⫾ 0.7 6.0 ⫾ 1.3

4.8 ⫾ 1.1 6.2 ⫾ 0.4 11.2 ⫾ 1.7 10.6 ⫾ 1.2 7.2 ⫾ 1.2

0.9 ⫾ 0.5 6.1 ⫾ 0.4 7.6 ⫾ 1.1 8.5 ⫾ 1.1 0.8 ⫾ 0.7

*Five liposome formulations with different size ranges (from 20 nm to 1200 nm) were separately incubated with ␤-gal plasmid at 37°C for 20 minutes. The lipid/DNA ratio was 12:1. The particle size of the liposome-DNA complex was measured by a light-scattering method using a Nicomp Submicron Particle Sizer immediately after incubation. The size range encompassed ⬎99.9% of particles (by number) in each formulation. Data represent transfection efficiency for each complex, which represents the mean ⫾ SD of three independent experiments carried out with duplicate samples.

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Figure 1. Effects of liposome-DNA size and DNA dose on transfection efficiency in vitro. The liposome-DNA complexes DP3-␤-gal and G67-␤-gal (three different sizes of each: large, 900-1250 nm (L); medium, 190 –280 nm (M); and small, 110 –150 nm (S)) were used to transfect H358 and H322 cells in 6-well plates using three different DNA doses (2, 6, and 10 ␮g of DNA/106 cells). The transfection efficiency was measured by determining the percentage of blue cells. Data represent the mean ⫾ 95% confidence intervals from three independent experiments carried out with duplicate samples. Here, mean transfection efficiency is shown as a function of DNA dose and liposome-DNA complex size.

Transfection efficiency was highly dependent upon lipid composition and lipid/DNA ratio As Tables 1 and 2 also show, transfection efficiencies varied greatly according to the lipid compositions of different micelles or liposomes, suggesting that transfection efficiency was highly dependent upon lipid composition. Of all of the micelle formulations tested, micelle P2 had the highest transfection efficiency (3.3%) (Table 1); liposomes M3 and DP3 had the highest transfection efficiencies (11.4% and 11.2%, respectively) of all of the liposome formulations tested (Table 2). Liposomes M3 and DP3 both performed better than the control liposome G67 under these experimental conditions.

Different lipid/DNA weight ratios were also tested for the micelle and liposome formulations whose compositions corresponded to the highest transfection efficiency. The DNA amount was fixed at 2 ␮g of DNA/well. The experimental conditions and the determination of transfection efficiency were the same as described above. As Table 3 shows, transfection efficiency could be optimized by altering the lipid/DNA ratios, and different liposomes had a different optimal lipid/DNA ratio. Other than micelle P2 and G67, which have optimal lipid/DNA ratios of 2:1 or 3:1, respectively, the optimal lipid-DNA ratio for the rest of the liposome formulations was 12:1 under these experimental conditions. The 6:1 ratio also

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Table 5. Effect of Cell Type on Transfection Efficiency* % Transfection Cell type Formulation

NIH/3T3 mouse embryo

Bronchial epithelial

Saos2 osteosarcoma

A549 lung carcinoma

H358 lung carcinoma

H322 lung carcinoma

Liposome ST Liposome T1 Liposome DP3 G67

5.2 ⫾ 1.5 6.1 ⫾ 0.4 13.8 ⫾ 3.4 9.2 ⫾ 2.4

1.7 ⫾ 0.8 5.1 ⫾ 1.7 9.4 ⫾ 2.3 7.4 ⫾ 1.7

3.2 ⫾ 1.4 5.5 ⫾ 2.1 4.7 ⫾ 1.4 13.4 ⫾ 3.5

1.1 ⫾ 0.6 2.0 ⫾ 0.8 3.9 ⫾ 1.5 4.5 ⫾ 1.2

1.2 ⫾ 0.8 1.7 ⫾ 1.1 10.7 ⫾ 3.6 7.2 ⫾ 2.8

0.8 ⫾ 0.3 0.5 ⫾ 0.2 5.8 ⫾ 2.1 4.1 ⫾ 1.7

*Five different cell lines and human normal bronchial cells were used to test the transfection efficiency of three different liposome-DNA (␤-gal) complexes. G67-␤-gal complexes were used as a benchmark. The transfection procedure was the same as that described in Tables 1 and 2. The data for each formulation represent the mean ⫾ SD of three independent experiments carried out with duplicate samples.

led to a very good transfection efficiency that was only slightly lower than that given by the 12:1 ratio. Because of its relatively low toxicity, the 6:1 ratio seemed more suitable for in vivo experiments.

Transfection efficiency was highly dependent upon the size of liposome-DNA complexes As shown in Tables 1 and 2, the liposome formulations had a wide particle size distribution and different average sizes before the size selection. Therefore, the particle sizes of the liposome-DNA complexes formed by those liposomes with DNA were also different. Suspecting that transfection efficiency may also be affected by the size of the liposome-DNA complexes, we subsequently tested the effect of size on transfection efficiency (Table 4). The size ranges shown in Table 4 encompassed ⬎99% of the liposome-DNA complexes in a sample measured with a Nicomp Submicron Particle Sizer 370. Transfection efficiency data are the mean ⫾ SD of three different experiments. The DNA (␤-gal) dose was 2 ␮g of DNA/ well. The results showed that transfection efficiency was also highly dependent upon the particle size of the liposome-DNA complexes, consistent with the idea that the right size range of the liposome-DNA complexes is crucial for achieving high transfection efficiency. Interestingly, for all liposomes, the highest transfection efficiency in NIH/3T3 cells was obtained with liposomesDNA in the same size range, 190 –280 nm. The only exception was liposome ST-DNA (LST-␤-gal), which had two almost equally effective size ranges of 320 – 430 nm and 190 –280 nm. Thus, the optimal size of liposomeDNA complexes was not lipid composition-dependent. Whether it was cell line-dependent was our next question.

Different cell lines could have different optimal sizes of liposome-DNA complexes It has been shown that the transfection efficiency of the same liposome formulation can differ in different cell lines (i.e., can be cell line-dependent).44 Except for varying characteristics of different cell lines, the formulation factors will also significantly affect the transfection efficiency. However, almost all of the results from previous studies were obtained by using a mixture of

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heterogeneously sized liposomes. Therefore, it is not clear whether such differences were due to the special liposome-DNA size preferences of different cell lines or to the lipid compositions of the liposomes. To determine whether different cell lines need different optimal sizes of liposome-DNA complexes to achieve the highest transfection efficiency, three groups of DP3-␤-gal complexes with size ranges of 110 –150 nm (small), 190 –280 nm (medium), and 900-1250 nm (large) were used to transfect two human NSCLC cell lines (H358 and H322) as described above. The lipid/DNA ratio was 12:1, and the DNA dosage was 2, 6, or 10 ␮g of DNA/well. Our results, as shown in Figure 1, led us to two conclusions: First, the transfection efficiency was not significantly changed by increasing the DNA dose from 2 to 10 ␮g of DNA/106 cells. Second, even when using the same liposome-DNA, the optimal complex size for H358 cells was 190 –280 nm, whereas the optimal size for H322 cells was 900-1250 nm. Therefore, for different cell lines, the optimal sizes of liposome-DNA complexes differed even for liposome-DNA with the same lipid composition and the same DNA construction.

Different cell lines could have different optimal lipid compositions To determine whether different cell lines needed different optimal lipid compositions to achieve the highest transfection efficiency, we constructed different liposome-DNA complexes with a fixed size range of 190 –280 Table 6. Effect of Adding ST to Liposome on Transfection Efficiency In Vitro* Name Liposome ST1 Liposome ST2 Liposome ST3

Lipid ratio (mol) Size % Transfection (DPPC†/CHOL‡/ST) (mean ⫾ SD, nm) (in 3T3 cells) 2:2:1 3:3:2 7:4:5

216 ⫾ 145 204 ⫾ 136 164 ⫾ 88

1.3 ⫾ 0.8 0.7 ⫾ 0.6 8.2 ⫾ 2.5

*Liposomes containing ST of optimal size were prepared by hydration followed by extrusion through a 0.2-␮m membrane. The lipid/DNA ratio was 12:1. The data for each formulation represent the mean ⫾ SD of three independent experiments carried out with duplicate samples. †DPPC, dipalmitoyl phosphatidylcholine. ‡CHOL, cholesterol.

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Figure 2. p53 function in p53-null H358 (A) and p53 mutant H322 (B) cells transfected with liposome-p53 complexes. Buffer, p53, liposome-Vp, or liposome-p53 represent cells treated with luminometry reaction buffer (100 mM K2PO4, 5 mM adenosine triphosphate, 15 mM MgSO4, and 1 mM dithiothreitol (pH 7.8)) alone; pC53SN alone; a combination of vector plasmid (without the p53 gene), wwp-luc, and ␤-gal complexed to different liposomes; or a combination of pC53SN, wwp-luc, and ␤-gal complexed to the liposomes. p53 function was measured by luciferase activity. Transfection efficiency was measured by ␤-gal activity. Luminometry units were divided by the OD values of ␤-gal activity to factor out uneven transfection efficiency, which gave the corrected luminometry units. Data represent the mean ⫾ 95% confidence intervals of three independent experiments carried out with duplicate samples. Vp, vector plasmid; LST, liposome ST; G67, liposome G67.

nm and then transfected them into five different cell lines. The results are shown in Table 5. The DNA dosage was 2 ␮g of DNA/well. The experimental conditions and methods were the same as those described previously. Under these experimental conditions, liposome DP3 was the best formulation for transfecting NIH/3T3 cells, normal bronchial epithelial cells, H358 cells, and H322 cells; liposome G67 was the best formulation for transfecting Saos2 cells and A549 cells. Therefore, when the liposomes size was fixed, transfection efficiency was dependent upon both cell line and lipid composition.

ST liposome-mediated transfection efficiency ST is a small cationic molecule with a single-carbon frame. It is not a phospholipid, but its positive charge and surfactant properties are similar to those of cationic phospholipids used in liposomes. ST is easy to insert into lipid bilayers to make different liposomes; its positivecharge portion will remain on the liposome surface. Therefore, liposomes made of neutral phospholipids and ST will have a positive charge from the amino group of ST but not from cationic phospholipids. Furthermore, because the relative amount of phospholipid will be reduced by inserting ST into the liposomes, the ST liposomes will theoretically have less lipid exchange with cells than the liposomes made of cationic phospholipids. ST has not been investigated extensively for mediating transfection.45 To understand whether phospholipid structure affects transfection and whether reducing lipid exchange affects transfection efficiency, we tested several liposome formulations made with two neutral lipids and

supplemented with ST. We selected dipalmitoyl phosphatidylcholine and cholesterol because neither has a charge and because the combination easily forms a relatively stable liposome in vivo. Table 6 shows the results. When the lipid ratio was appropriate (e.g., 7:4:5), the ST liposome was capable of initiating and carrying out transfection with a high degree of efficiency. These results demonstrated that the phospholipid chemical structure may not be critical for the transfection, and that lipid exchange may not be the main pathway for the liposome-mediated transfection. Because of its relatively high toxicity (our unpublished data), we did not use ST in the survival experiments.

H358 cells were effectively transfected via liposomep53 complexes in vitro We subsequently tested the efficacy of several liposome formulations at delivering p53 into H358 cells in vitro. To ascertain whether p53 plasmid could be successfully delivered into the cells and functional p53 protein could be expressed, liposomes ST, G67, and DP3 were used to form liposome-p53 complexes with three plasmids, pC53SN (containing a CMV promoter-driven human wt p53 gene), wwp-luc (containing a p21 promoter-driven luciferase gene), and ␤-gal (containing the CMV promoter driven lacZ gene), to transfect H358 cells. Theoretically, if p53 plasmid is delivered into the cells and is expressed, the functional p53 protein will activate the p21 promoter and induce the expression of luciferase. Luciferase and ␤-gal activities were measured at 48 hours after the completion of transfection. Luciferase

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Figure 3. Effects of liposome-DNA size and DNA dose on the killing efficiency of liposome-p53 complexes in vitro. H358 and H322 cells were transfected with the liposome-p53 complexes described in the legend to Figure 1. The numbers of viable cells were counted in a hemocytometer under a microscope after trypsinization and trypan blue staining. Untreated cells were used as controls. Data represent the mean ⫾ 95% confidence intervals of three independent experiments carried out with duplicate samples.

activities, which were corrected for transfection efficiency by measuring the ␤-gal activity, showed a 60-, 120-, or 130-fold induction in cells transfected with liposome ST-p53, G67-p53, or DP3-p53 complexes, respectively, compared with the normal saline controls, but no induction in cells transfected with pC53SN plasmid alone, liposome ST-vector plasmid, G67-vector plasmid, or DP3-vector plasmid (Fig 2). Thus, cells transfected with any of the three liposome-p53 complexes expressed functional p53 protein, and DP3 was slightly better than G67 at delivering p53 plasmids in vitro.

the p53 killing efficiency was 30 – 40%. One possible reason we considered was that the ␤-gal assay was not sensitive enough, so the transfection efficiency was actually much higher than that measured by ␤-gal staining. This possibility was later confirmed using a more sensitive green fluorescent protein (GFP) plasmid as the reporter gene (see Discussion). Another possibility was that a “bystander effect” might exist. To demonstrate that the cell death seen after DP3-p53 transfection was due to apoptosis, TUNEL assays were performed. DP3-p53-transfected cells showed a strong

Killing efficiency of liposome-p53 in H358 and H322 cells was much higher than transfection efficiency as measured by ␤-gal staining H358 and H322 cells can be killed through the apoptosis induced by transfected wt p53. Therefore, we compared the killing efficiency with the transfection efficiency of DP3-p53 and G67-p53 in both cell lines. Three groups of DP3-␤-gal (Fig 1) or pC53SN plasmid (Fig 3) complexes with size ranges of 110 –150 nm (small), 190 –280 nm (medium), and 900-1250 nm (large) were used to transfect H358 and H322 cells. The lipid/DNA ratio was 12:1 and the DNA dose was 2, 6, or 10 ␮g of DNA/well. Untreated cells and cells treated with liposome alone were used as controls. Transfection efficiency and p53 killing efficiency were determined as described above. By comparing Figures 1 and 3, it can be seen that DP3-p53 effectively killed both p53-null and p53-mutant lung cancer cells. However, the p53 killing efficiency was much higher than the transfection efficiency under optimal transfection conditions for both the H358 and H322 cell lines. The transfection efficiency measured by ␤-gal staining was ⬃10% for H358 and H322 cells, whereas

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Figure 4. Lung biopsies of mice bearing orthotopic H358 human NSCLC. Male nude mice were inoculated with 1–2 ⫻ 106 H358 cells/mouse intratracheally. After 3 weeks, the lungs were resected, fixed with 10% formalin, and embedded in paraffin. The embedded lung tissue was sectioned and stained with hematoxylin and eosin. Bronchial lumina and lung tumors are labeled 1 and 2, respectively. As shown in this figure, H358 tumors grew orthotopically in the lungs of nude mice in a multinodular pattern without evidence of distant metastatic spread.

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Figure 5. In vivo lung TGI by intratracheal administration. A: Male nude mice were inoculated intratracheally with H358 cells. The inoculated mice were then divided into eight groups of five mice each. One inoculated group was used as a control and received no treatment; the other seven groups were treated intratracheally with liposomes (LST, DP3, or G67) alone, with pC53SN plasmid alone, or with micelle P2-p53 complexes or liposome-p53 complexes (LST-p53, DP3-p53, and G67-p53) on days 4, 8, 12, 16, and 20 after tumor inoculation. The DNA dose was 2 ␮g of DNA per mouse per injection. The lipid/DNA ratio was 6:1. A ninth group of mice that were left uninoculated and untreated was used as a normal control. All mice were killed on day 77, and the lungs were resected and weighed. TGI was calculated as described in Materials and Methods. Differences in %TGI between groups were analyzed for statistical significance using the two-sided log-rank test. Liposome-p53 versus liposome or plasmid alone, P ⫽ .009; liposome-p53 versus no treatment, P ⫽ .004. B: The human NSCLC model H322 with mutant p53 was use to confirm the antitumoral activity of the best liposome-p53. Experiments similar to those described in (A) were performed by inoculating mice with H322 cells intratracheally. DP3-Vp (Vp: nonexpression plasmid) was used as a control. The DNA dose was 8 ␮g of DNA per mouse per injection. Liposome-p53 versus liposome, plasmid alone, or DP3-Vp, P ⫽ .008; liposome-p53 versus no treatment, P ⫽ .004.

fluorescent signal, indicating the presence of DNA fragmentation in the apoptotic cells (data not shown). Apoptotic cells were examined by flow cytometry, and the results showed that at 30 hours after DP3-p53 transfection, 85% of the detached cells and 15% of the attached cells were apoptotic, indicating that DP3-p53 transfection had induced apoptosis in H358 cells. Apoptosis in the transfected cells was also confirmed by agarose gel electrophoresis (data not shown).

DP3-p53 and G67-p53 administered intratracheally were both effective at inhibiting lung tumor growth in vivo To determine the antitumoral efficacy of liposome-p53 treatment in vivo, we developed a human lung cancer model in nude mice that mimics human bronchial malignancy and early lung cancer. Repeated experiments showed that human lung cancer cells implanted intratracheally in mouse bronchi attached to the bronchial epithelium and gave rise to multiple tumors, and that the life spans of these mice correlated with the number of cancer cells inoculated. Figure 4 shows a mouse lung biopsy at 3 weeks after intratracheal H358 tumor inoculation. The results indicate that H358 cells grew orthotopically in the lungs of nude mice in a multinodular pattern similar to that seen for human bronchioalveolar

carcinoma and caused death by local growth without evidence of metastatic spread. To determine whether our liposome-p53 formulations would inhibit tumor growth in vivo, lung TGI experiments were performed. As Figure 5A shows, both DP3p53 and G67-p53 treatment significantly inhibited tumor growth by day 77; the %TGI was 97% for DP3-p53 and 94% for G67-p53 (P ⬎ .05). There were no visible tumors in the lung tissues of G67-p53-and DP3-p53treated mice, and all five mice in these two groups were alive until the day they were killed. The best micelle formulation (micelle P2-p53) had little antitumoral activity compared with the liposome formulations; perhaps the micelles were not able to transfect enough tumor cells. Groups treated with either p53 DNA alone or empty liposome alone showed limited tumor inhibition in both tumor models (%TGI of ⬍27%). Most of the lung parenchymas in these animals were replaced by tumors by day 77. The average lung/body weight ratios of the five control groups (untreated and those treated with pC53SN, LST, G67, or DP3 liposome) were 6- to 13-fold higher than those of the three liposome-p53-treated groups. There was no significant difference between the average lung/body weight ratios of G67-p53- or DP3-p53-treated mice and normal mice not inoculated with tumor (8.0 vs. 7.3 mg/g, n ⫽ 13, P ⬎ .1). The tumor inhibition experiment was

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Figure 6. Effect of DP3-p53 on the survival of human lung tumor-bearing mice. p53 null (H358, p53 deletion) (A) and p53 mutant (H322, p53 mutation) (B) tumor-bearing mice identical with those used in the TGI tests (Fig 5) were used in the survival experiments. Treatment consisted of five intratracheal doses given on days 4, 8, 12, 16, and 20. The dose was 2 ␮g or 8 ␮g of DNA per mouse per administration for H358- or H322-inoculated mice, respectively. Increased life span (%ILS) was calculated. Differences in survival among groups were analyzed for statistical significance by the two-sided log-rank test. All statistical tests used in this study were two-sided statistical tests.

repeated in a p53 mutant human NSCLC model in mice, and similar results were obtained (Fig 5B).

Dp3-p53 and G67-p53 administered intratracheally significantly prolonged the life of lung tumor-bearing mice The optimal liposome-p53 complex sizes (190 –280 nm for H358 treatment and 900-1250 nm for H322 treatment) and lipid/DNA ratios (6:1 for DP3-p53 and 2:1 for G67-p53) were used. The DNA dose was 2 ␮g and 8 ␮g of p53 DNA per mouse per injection for H358 and H322 treatment, respectively. In the H358 survival experiments, the mean median survival of the three control groups (no treatment, p53 alone, DP3 alone) was 107 days compared with 179 and 167 days of mean survival for the DP3-p53- and G67-p53-treated groups (Fig 6A). In the H322 survival experiments, the mean median survival of the three control groups was 93 days compared with 219 and 199 days for the DP3-p53- and G67-p53-treated groups (Fig 6B). Both liposome-p53 formulations significantly prolonged the lives of the tumor-bearing mice by ⬃1.6- to 2.4-fold (P ⬍ .009 by log-rank test) after only five doses. DP3-p53 was slightly more or at least equally as effective as G67-p53. DISCUSSION A cationic liposome delivery system has distinct advantages over a viral delivery system. The main advantages are minimal toxicity, no immunogenicity, and repeat-

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able, or chronic, administration. Repeatable administration is necessary for treating early lung cancers in the bronchial epithelium for three reasons. First, just one administration of either liposome-gene or virus-gene particles may not penetrate deeply enough into the bronchial epithelium to reach all malignant cells, even though in most cases bronchial premalignant and early malignant lesions are superficial and limited in thickness to a few layers of cells. With repeated administrations, however, surface malignant cells can be killed first and then deeper layers of malignant cells can be exposed and subsequently killed. Second, the cancer cells remaining alive after the first administration will continue to grow and develop tumors. Third, some patients most likely will need repeated administrations if they continue to be exposed to airborne carcinogens such as cigarette smoke,46 asbestos, ground radon, and certain chemicals. Compared with a viral delivery system, a cationic liposome delivery system has the major disadvantage of its low transfection efficiency. Indeed, in our experiments, the in vitro transfection efficiencies for cationic liposome-DNA complexes were generally ⬃10% as measured by the ␤-gal staining method. However, the p53 killing efficiency (i.e., the number of cells undergoing apoptosis after a single transfection with DP3-p53) was ⬃40 –50%. Possible reasons for this discrepancy include the low sensitivity of ␤-gal staining in determining transfection efficiency and/or the existence of a bystander effect, as described previously for adenoviralp53 transfection.47– 49 To assess transfection efficiency more accurately, we therefore used the GFP plasmid as a reporter plasmid, because GFP expression is much

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easier to detect and much more sensitive in measuring transfection efficiency. As a result, we found that the transfection efficiency of DP3-GFP in H358 or H322 cells in vitro is constantly ⬃40% (data not shown). Therefore, we now think the discrepancy between transfection efficiency and p53 killing efficiency was most likely due to the insensitivity of the ␤-gal staining method rather to a bystander effect. In fact, in the case of DP3-p53, there was little difference between its p53 killing efficiency and its transfection efficiency as measured by GFP, which raises the question of whether the bystander effect of p53 truly exists. For future development of an effective nonviral gene therapy for treating early lung cancer, optimization of a liposome-gene delivery system will be crucial. This idea drove our present study and led us to explore two important ways to improve cationic lipid gene delivery systems: selection of the best cationic material and optimization of the formulations. We identified several important factors that significantly affect gene transfection. First, we noted that liposomes made of cationic phospholipids were more effective than liposomes made of ST, and that liposomes were more effective than micelles made of the same cationic phospholipids. In micelle structures, the hydrophobic portion of the lipids associates to form regions from which water is excluded, whereas the hydrophilic head groups remain on the outer surface to maximize their interaction with the water and oppositely charged ions. Unlike liposomes, micelle particles made of phospholipids usually are smaller and have no lipid bilayer structures. However, it was not clear before our study whether the liposome bilayer was necessary for transfection. Nevertheless, our study partially answered the question: cationic phospholipid micelles do have the potential to mediate transfection, and a bilayer structure is not necessary. Our results also hinted that the differences between liposome-mediated transfection and micelle-mediated transfection may reveal certain aspects of the mechanism of the transfection process. Second, our results implied that the liposome-DNA complex was a suitable form for delivering DNA into cells and that, during transfection, bilayer structure may play an important role. However, the nonphospholipid ST was not effective as cationic phospholipid. This can be explained by the fact that cationic phospholipid and cell phospholipid exchange is also important for transfection. Cell lines differ in their cell size and possibly in their cell membrane lipid compositions and surface protein distribution. Thus, we hypothesized that the optimal size of liposome-DNA complexes may be cell line-dependent. In other words, liposome-DNA complexes with the optimal size might attach more easily and tightly to certain types of cell surfaces than to others, so as to induce endocytosis or create the right surface tension needed for optimal fusion and lipid exchange with the cell membrane. Our results confirmed this hypothesis and showed that the optimal size of liposome-DNA complexes for different cell lines may differ even when the same liposomes, the same DNA, and the same lipid

composition are used. Because the optimal complex size needed to transfect a particular cell line was the same for different liposome-DNA formulations, we also concluded that it is best to find the optimal liposome-DNA size first and then optimize its lipid composition and lipid/DNA ratio using homogeneous liposomes within the optimal size range. We also hypothesized that, in the case of treating early lung cancer in bronchi, the functions of p53 in liposomep53 formulations in vitro correlate positively with their TGI effects in vivo. Our rationale was that, compared with physical environment in the circulatory system, the physical environment in the bronchi is relatively simple36 and similar to that in the culture dish. The bronchial environment also contains fewer substances, such as high-density lipoprotein and phospholipase, that can destroy liposomes immediately and contains too little liquid to significantly dilute the liposome-DNA concentration or change the physical properties of the complex. Therefore, we reasoned that liposome-DNA complexes delivered intratracheally to the bronchi would more likely retain their physical properties and their transducing ability. The opposite assumption was that liposomeDNA complexes administered intravenously or intratumorally would be less effective because blood and tissue fluids might change their physical properties and therefore their ability to induce transfection. Furthermore, we assumed that certain enzymes and proteins in the blood and in tissue fluids would destroy most liposome-DNA complexes, further decreasing the in vivo transfection efficiency. In those situations, the transfection efficiency of liposome-DNA complexes in vitro most likely will not correlate with their transfection efficiency in vivo. In short, our data supported our hypothesis and showed that those formulations with higher in vitro p53 function (Fig 2) also had higher in vivo antitumoral effects (Figs 5 and 6). We used p53 as prototype gene because its tumor suppressor function has been solidly confirmed,37–39 and because its mutation is frequent in lung cancer cases (50 –70%).7,40,41 Theoretically, the technique we developed in this study also can be used to deliver any other therapeutic genes by intratracheal or aerosol administration after minor modification. G67 has been shown previously to be highly effective in delivering plasmid into mouse lungs at levels comparable with those attained with recombinant adenovirus vectors at multiplicities of infection ranging from 1 to 20.29,50 Through this study, we have developed and tested a novel cationic liposome formulation, DP3, that is at least as effective as G67 for the treatment of early lung cancer both in vitro and in vivo and is a promising gene delivery system for lung cancer gene therapy. ACKNOWLEDGMENTS We thank Dr. Bert Vogelstein for providing the pC53SN and wwp-luc plasmid. This work was supported by a research grant

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(to Y.Z.) from the Physicians Referral Service of the University of Texas MD Anderson Cancer Center.

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Cancer Gene Therapy, Vol 7, No 5, 2000