Development of Cationic Liposome Formulations for Intratracheal Gene Therapy of Early Lung Cancer

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 1Department 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 Ͼ50 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.

ung cancer, with a mortality of ϳ90%, is the leading fects in diffusely damaged bronchial epithelium that Lcause of cancer-related deaths in the United States.1 ultimately underlie all lung cancers,6 a failure that Survival in the past 20 years has improved by only 2%, underscores the real need for gene therapy against lung and to this day it remains dismal.2–4 cancers. One reason for this poor survival is that the three Consequently, others and we have devoted much traditional modalities of treatment have failed to treat effort to devising effective approaches to the gene ther- lung cancer in its early stages or to adequately target apy of early lung cancers. To our advantage, recent lung cancer cells. Systemic chemotherapy has been used advances in understanding the molecular genetics of with little success because most drugs delivered in this lung cancer have made it possible to diagnose lung way are immediately destroyed or inactivated by liver cancers even earlier than before7,8 Because the genetic and blood components; as a result, not enough drug lesion originates on the bronchoalveolar epithelium, the reaches the lung tumor. Those drug molecules that do lesion, even though it can be diffuse, is usually superﬁ- reach tumor cells then must confront the intracellular cially localized on the surface of the pulmonary airway. obstacle of multiple drug resistance. Drugs delivered Thus, the best chance for gene therapy of early lung systemically may also cause life-threatening systemic cancers seems to us to be airway administration, because toxicity. As for radiation therapy, it does not distinguish this would most easily allow therapeutic agents to reach between tumor cells and normal cells and is not sensitive and penetrate most malignant cells. to lung cancer.5 Surgery, meanwhile, is useless for In general, researchers in the ﬁeld have been consid- removing the whole pulmonary tree, most metastases, ering two types of therapeutic gene delivery: viral and and most invisible tumors. Worse yet, none of these nonviral. Use of an adenoviral vector carrying a p53 gene modalities can correct the genetic alterations and de- has already been approved for clinical trial,9–15 but the potential immunogenicity, high toxicity (including per- manent damage to the genome), and limited methods of 16–20 Received April 29, 1999; accepted September 6, 1999. administration could limit its application. In con- Address correspondence and reprint requests to Dr. Roman Perez-Soler, trast, most of the nonviral gene delivery systems that are 21 Rita J. Stanley Kaplan Comprehensive Cancer Center, New York Univer- being considered have no immunogenicity. In our own sity School of Medicine, 550 First Avenue, New York, NY 10016. work, we have focused on nonviral systems and have

Cancer Gene Therapy, Vol 7, No 5, 2000: pp 683–696 683 684 ZOU, ZONG, LING, ET AL: NONVIRAL GENE DELIVERY SYSTEM FOR LUNG CANCER determined that an effective nonviral gene therapy sys- Expression plasmids tem would include a gene delivery vector, administration Several expression plasmids were used. pC53SN is an 8.3-kb method, and therapeutic gene. plasmid containing wt human p53 cDNA under the control of Among the nonviral gene delivery systems so far a cytomegalovirus (CMV) promoter. Empty vector plasmid proposed by others and by us, the cationic liposome is was constructed by deleting p53 cDNA from the pC53SN the best candidate because of its low toxicity.22,23 The plasmid. wwp-luc is a 7.9-kb plasmid containing the luciferase feasibility of its use in the airways has already been gene under the control of a p21 promoter, which is activated demonstrated in different animals,23–32 and studies of specifically by wt p53. pC53SN and wwp-luc plasmids were gifts liposome-gene complexes administered by intranasal33 of Dr. Bert Vogelstein at John Hopkins University (Baltimore, 34 Md). ␤-galactosidase (␤-gal) is an 8.5-kb plasmid containing and aerosol routes in cystic fibrosis patients have the lacZ gene (Escherichia coli lacZ gene encoding ␤-gal, a revealed preliminary evidence of effective transfection of commonly used reporter gene) under the control of a CMV the bronchial epithelium. We ourselves have hypothe- promoter. The pC53SN vector (p53 gene removed) was used as sized that the intratracheal or aerosol administration of a nonexpression plasmid control (Vp). liposome-tumor suppressor gene complexes for early lung cancer gene therapy would be more effective and Preparation of liposomes practical than intravenous chemotherapy and intratu- moral viral gene therapy, especially for liposomes, be- All lipids were obtained from Avanti Polar Lipids (Alabaster, Ala). Large liposomes (400–660 nm and 800-1200 nm) were cause attacks from macrophages, high-density lipopro- prepared by hydration and centrifugation (1000 ϫ g) methods. teins, and phospholipases would be presumably less Medium liposomes (170–240 nm) were prepared by detergent fierce in the bronchial airway than in the liver and removal plus extrusion methods as described below. Small 35 bloodstream. liposomes (20–40 nm and 60–110 nm) were prepared by In previous studies, we developed an orthotopic ani- sonicating large or medium liposomes. Hydration was per- mal model to mimic human early lung cancer and then formed with a rotary evaporator for 2 hours at 5°C above the used it to prove our hypothesis by showing that intratra- phase transition temperature of the cationic lipids at 50 rpm followed by five freeze-thaw cycles. A set of extrusion mem- cheally injected liposome-p53 gene complexes could ␮ ␮ inhibit orthotopic human lung tumor growth and pro- branes with different pore sizes ranging from 0.1 mto1.0 m long the life span of the tumor-bearing mice.36 We used (Gelman Sciences, Ann Arbor, Mich) was used to regulate the liposome particle size based on the requirements of the p53 as our prototype gene because of its proven tumor experiments. Each formulation was passed through at least two suppressor function37–39 and frequent (50–70%) muta- 7,40,41 extrusion membranes with three filtration circles on each, first tion in lung cancer. a larger pore size and then a smaller pore size at the same In this light, we therefore focused our present work on temperature as that used for the hydration phase. Liposomes optimizing our liposome-p53 gene formulation and im- containing stearylamine (ST) were prepared using the same proving its therapeutic potential by pharmaceutical method. The G67 liposomes (provided by Genzyme, Cam- means. Because nonviral gene delivery systems are bridge, Mass) were also treated by the extrusion method if mainly hampered by their low delivery efficiency, our required by the experimental design. The liposome particle immediate goal was first to identify those factors that size was measured by a multiple-angle dynamic light-scattering most affected our system’s ability to transfect cells and method using a Nicomp Submicron Particle Sizer 370 (Nicomp Particle Sizing System, Santa Barbara, Calif). produce therapeutic results and then to modify them so as to optimize our liposome formulation for the delivery Preparation of cationic lipid micelles of therapeutic genes via the airway as a treatment for early lung cancers. 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 polyoxyethyl- ene20 sorbitan monolaurate (Tween 20; Sigma, St. Louis, Mo) at room temperature. The phospholipid/surfactant ratios are MATERIALS AND METHODS listed in Table 1. The hydrated micelle suspensions were spun Cell lines and cell culture at maximum power in a vortex two times for 2 minutes each time. The particle size was measured as described above. H358 (human non-small cell lung carcinoma cells (NSCLC) with a p53 deletion mutation, developed from human bronchial Liposome-DNA or micelle-DNA complex formation epithelium), H322 (human NSCLC cells with a p53 mutation at codon 248, developed from human bronchial epithelium), The liposome-DNA or micelle-DNA complexes were formed Saos2 (human osteogenic sarcoma cells with a p53 deletion), by mixing the preformed cationic liposomes or micelles with HeLa (human cervical carcinoma cells with wild-type (wt) plasmids in Opti-Mem (Life Technologies, Gaithersburg, Md) p53), A549 (human lung adenocarcinoma cells with mutant at a weight ratio specified for each experiment and then p53), and NIH/3T3 (murine embryo cells with wt p53) were incubating the mixture at 37°C for 20 minutes. All complexes purchased from the American Type Culture Collection (Man- were used immediately after the incubation. Opti-Mem is a cell assas, Va). Cells were cultured in a cell culture incubator at a culture medium that contains the basic nutrients needed for constant temperature (37°C) and humidity in filtered air cell growth and a minimal amount of the serum proteins that containing 5% CO2. Only the first five passages of each cell line can inhibit liposome-mediated gene transfection. The particle were used. size of the complexes was measured immediately after incuba-

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

% Transfection Name Composition Lipid ratio (mol) Size (mean Ϯ SD, nm) in 3T3 cells

Micelle D1 DODAP/De 7:1 6.5 Ϯ 3.2 0.1 Ϯ 0.2 Micelle D2 DODAP/De 10:1 8.2 Ϯ 4.5 0.8 Ϯ 0.3 Micelle D3 DODAP/De 20:1 8.9 Ϯ 3.3 0.2 Ϯ 0.1

Micelle T1 DOTAP/De 7:1 6.6 Ϯ 3.7 0.1 Ϯ 0.3 Micelle T2 DOTAP/De 10:1 7.6 Ϯ 3.5 1.4 Ϯ 0.7 Micelle T3 DOTAP/De 20:1 7.8 Ϯ 3.9 0.2 Ϯ 0.3

Micelle L1 DLEP/De 7:1 5.7 Ϯ 3.1 0.2 Ϯ 0.2 Micelle L2 DLEP/De 10:1 6.8 Ϯ 3.4 1.6 Ϯ 0.5 Micelle L3 DLEP/DOPE 20:1 7.2 Ϯ 4.1 0.3 Ϯ 0.2

Micelle M1 DMEP/DOPE 7:1 7.1 Ϯ 3.0 0.5 Ϯ 0.3 Micelle M2 DMEP/DOPE 10:1 7.5 Ϯ 3.6 3.1 Ϯ 1.4 Micelle M3 DMEP/DOPE 20:1 8.3 Ϯ 4.5 2.2 Ϯ 0.9

Micelle P1 DPEP/DOPE 7:1 7.4 Ϯ 3.8 0.3 Ϯ 0.2 Micelle P2 DPEP/DOPE 2:1 7.9 Ϯ 4.0 3.3 Ϯ 0.6 Micelle P3 DPEP/DOPE 3:1 8.1 Ϯ 5.4 1.7 Ϯ 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 Ϯ SD of three independent experiments carried out with duplicate samples.

tion by the dynamic light-scattering method using the Nicomp number of viable nontreated cells, and Td equals number of Sizer. viable liposome-p53-treated cells.

In vitro cell transfection p53 function assay Cells were plated in 6-well tissue culture plates overnight at p53 function was determined by cotransfecting the cells with 0.5–1 ϫ 106 cells/well and then exposed to lipid-DNA com- the p53 gene and wwp-luc. In this system, measured luciferase plexes (DNA dose 2 ␮g/106 cells) in 1 mL of Opti-Mem for 6 activity is proportional to the cellular p53 function. A total of 6 hours. Next, an equal amount of RPMI 1640 medium contain- 2 ϫ 10 cells were plated in 90-mm culture dishes. After 24 ing 20% fetal bovine sera (Life Technologies) was added, and hours, the cells were transfected with liposome-DNA com- the cells were cultured for another 24 hours. Different lipid/ plexes containing equal amounts of three plasmids: wwp-luc, 6 DNA ratios and DNA doses were tested. Transfection was ␤-gal, and pC53SN (2 ␮g DNA/10 cells). Controls included terminated by replacing the medium with fresh RPMI 1640 cells transfected with DP3-DNA containing equal amounts of medium containing 10% fetal bovine serum. All transfec- wwp-luc, ␤-gal, and empty vector plasmid (Vp, without p53), tions were performed at 37°C in an atmosphere of 5% CO2 cells transfected with pC53SN alone, and cells treated with and 95% air. either liposomes or luminometry reaction buffer (100 mM K2PO4, 5 mM adenosine triphosphate, 15 mM MgSO4, and 1 Determination of transfection efficiency and mM dithiothreitol (pH 7.8)). Cells were incubated at 37°C for killing efficiency 36 hours in fresh medium after transfection, washed with 4°C phosphate-buffered saline (PBS), and harvested with a cell The ␤-gal staining method was used to determine transfection scraper in 1.0 mL of luminometry reaction buffer at 4°C. Cells efficiency after 48 hours of transfection as described above. In were then carefully resuspended, sonicated for 2 minutes on brief, cells were fixed and stained with 5-bromo-4-chloro-3- ice, and centrifuged at 1000 ϫ g for 5 minutes. A total of 25 ␮L indolyl ␤-D-galactoside (X-Gal) for 5 hours. For each kind of of the supernatant was mixed with 325 ␮L of the reaction treatment, the total cell numbers from two parallel samples buffer and 100 ␮L of luciferin solution (0.3 mg/mL) on ice, and (two wells) were counted using a hemocytometer just before all the mixture was measured immediately in a luminometer cells were fixed. Transfection efficiency was determined by (TD20/20; Promega, Madison, Wis) at a wavelength of 550– counting the blue cells in six randomly selected fields for at 570 nm. The ␤-gal activity in 25 ␮L of the supernatant was least 200 cells each under a microscope and was subsequently measured as described previously.42,43 Briefly, a 25-mL sample expressed as the percentage of blue cells per total cells. was diluted to 990 mL with a tris(hydroxymethyl)aminometh- p53 transfection caused significant lung cancer cell death in ane-HCl buffer (pH 7.5) and then mixed with 10 mL of tissue culture. To account for this, the percentage of killed dimethylsulfoxide solution containing resorufin (7.5 mg/mL). cells, or killing efficiency, was calculated as follows: % Kill- After the cells had been incubated for 30 minutes at 30°C, the ϭ Ϫ ϫ ing (Tl/C Td/C) 100%, where Tl equals the number of optical density (OD) of the treated samples was measured with viable cells treated with liposome alone, C equals the total a spectrometer at 558 nm. Transfection efficiency was mea-

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sured by ␤-gal activity. p53 function was expressed as “correct- displayed multiple visible bilateral lung tumor nodules. By ed luminometry units,” which were obtained by dividing the week 10–16, the mice died of multiple bilateral lung tumors luminometry units by the OD values of ␤-gal activity to factor without distant metastases. out uneven transfection efficiency. Antitumoral activity of liposome-p53 in mice bearing Apoptosis assays early endobronchial H358 and H322 tumors The percentage of apoptotic cells was determined by a termi- In all therapeutic experiments, treatment was started early (at nal deoxynucleotidyl transferase-mediated deoxyuridine day 4 postinoculation) because our main objective was to 5-triphosphate nick end-labeling (TUNEL) reaction assay ac- investigate the efficacy of liposome-p53 in the treatment of cording to the manufacturer’s recommendation (Boehringer endobronchial lesions before they had invaded the lung paren- Mannheim, Indianapolis, Ind). Cells (106 cells/well) in 1.0 mL chyma. A multiple dose schedule was used because under of Opti-Mem were plated in 6-well plates, grown for 24 hours optimal conditions a single transfection with DP3-p53 causes ϳ at 37°C in a 5% CO2 atmosphere, and transfected with apoptosis in only 40% of cells in vitro (see Results). A dose DP3-p53 as described above. Untreated cells and cells exposed was given every 4 days; however, the number of doses was to DP3 alone were used as controls. After transfection, cells limited to five because of the technical difficulties involved in were washed with PBS containing 1% bovine serum albumin, repeatedly cannulating the trachea through the mouse’s fixed with 4% paraformaldehyde in PBS at room temperature mouth. 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 Tumor growth inhibition (TGI) experiments ␮ then washed once with PBS and incubated in 50 L of TUNEL 6 reaction mixture containing 2 U of terminal transferase and Male nu/nu mice were each inoculated intratracheally with 10 fluorescein-dUTP at 37°C for 60 minutes. After being washed H358 cells and divided into eight groups with five mice in each three times with PBS, the fluorescently labeled cells were group. One group was left untreated. The other seven groups assessed by fluorescence microscopy and flow cytometry (Epics were treated intratracheally with pC53SN plasmids alone, ST Profile Analyzer; Coulter, Hialeah, Fla). liposomes alone, G67 liposomes alone, DP3 liposomes alone, or ST-p53, G67-p53, or DP3-p53 complexes on days 4, 8, 12, 16, Endobronchial H358 and H322 lung tumor models and 20 after H358 cell inoculation, respectively. The dose was 2 ␮g of DNA per administration. The liposome size for all Male nu/nu mice (7–8 weeks of age, 18–22 g each; Harlan formulations was 60–110 nm. The lipid/DNA ratio was 6:1 for Sprague-Dawley, Indianapolis, Ind) were inoculated intratra- DP3-p53 and ST-p53 and 2:1 for G67-p53. All mice were killed cheally with H358 or H322 cells (1–2 ϫ 106 cells/mouse). on the day the first untreated tumor-bearing mouse became Inoculated cells initially attached to the surface of the bron- moribund (day 74–77 in this study). The lungs were resected chial epithelium. By week 2–4, multiple microscopic tumors and weighed. The percentage of tumor growth inhibition arising in the bronchial epithelium and in connection with the (%TGI) was calculated as follows: %TGI ϭ (1 Ϫ [lung weight bronchial lumen were well-established. By week 7–9, the mice difference between each p53-treated group and healthy group]/

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

% Transfection Name Composition Lipid ratio (mol) Size (mean Ϯ SD, nm) in 3T3 cells

Liposome D1 DODAP/DOPE 1:3 83.6 Ϯ 37.8 0.6 Ϯ 0.3 Liposome D2 DODAP/DOPE 1:1 88.6 Ϯ 42.1 0.3 Ϯ 0.2 Liposome D3 DODAP/DOPE 3:1 78.3 Ϯ 45.2 0.2 Ϯ 0.3

Liposome T1 DOTAP/DOPE 1:3 70.9 Ϯ 38.1 5.6 Ϯ 1.0 Liposome T2 DOTAP/DOPE 1:1 66.9 Ϯ 34.9 5.4 Ϯ 1.1 Liposome T3 DOTAP/DOPE 3:1 59.2 Ϯ 33.9 1.7 Ϯ 0.4

Liposome L1 DLEP/DOPE 1:1 457 Ϯ 273 3.7 Ϯ 1.9 Liposome L2 DLEP/DOPE 2:1 230 Ϯ 175 3.8 Ϯ 1.5 Liposome L3 DLEP/DOPE 3:1 146 Ϯ 85 4.7 Ϯ 1.4

Liposome M1 DMEP/DOPE 1:1 1573 Ϯ 446 4.1 Ϯ 1.8 Liposome M2 DMEP/DOPE 2:1 1000 Ϯ 410 7.4 Ϯ 1.4 Liposome M3 DMEP/DOPE 3:1 1250 Ϯ 317 11.4 Ϯ 5.2

Liposome DP1 DMEP/DOPE 1:1 1354 Ϯ 772 3.7 Ϯ 1.1 Liposome DP2 DPEP/DOPE 2:1 1250 Ϯ 782 3.1 Ϯ 0.9 Liposome DP3 DPEP/DOPE 3:1 80.0 Ϯ 25.2 11.2 Ϯ 6.6

G67 L#67/DOPE 1:2 380 Ϯ 280 8.5 Ϯ 2.1 *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 Ϯ 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 2:1 3:1 6:1 12:1 24:1

Liposome ST 0.4 Ϯ 0.1 0.6 Ϯ 0.2 2.9 Ϯ 0.1 6.2 Ϯ 0.2 0 Ϯ 0 Liposome T1 ND† 0.3 Ϯ 0.1 3.2 Ϯ 0.4 5.6 Ϯ 1.0 ND Liposome M3 ND 4.8 Ϯ 1.1 9.2 Ϯ 2.1 11.4 Ϯ 5.2 1.4 Ϯ 0.3 Liposome DP3 2.0 Ϯ 0.4 5.4 Ϯ 1.0 10.1 Ϯ 3.5 11.2 Ϯ 6.6 9.5 Ϯ 3.1 G67 6.8 Ϯ 1.7 8.5 Ϯ 2.1 4.3 Ϯ 1.4 0 Ϯ 00Ϯ 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 Ϯ SD of three independent experiments carried out with duplicate samples. † ND, transfection efﬁciency not detected.

[lung weight difference between the untreated tumor-bearing formulations made of various lipid components in vari- group and healthy group]) ϫ 100%. ous ratios. A wide variety of lipids were used to prepare micelles Survival experiments and liposomes. Tables 1 and 2 show the lipid composi- Both p53-null (H358, p53 deletion) and p53-mutant (H322, p53 tion, lipid ratio, particle size, and transfection efficiency 248 codon point mutation) tumor-bearing mice were used in for each micelle and liposome formulation used in the survival experiments. Animals were inoculated intratrache- NIH/3T3 cells. For formulations of the same lipid com- ally with 1–2 ϫ 106 H358 or H322 cells/mouse. Treatment consisted of five intratracheal doses given on days 4, 8, 12, 16, position, the three with the highest transfection efficien- ␮ cies were listed. Transfection efficiency was measured by and 20. The dose was 2–8 g of DNA per administration. ␤ Increased life span (%ILS) was calculated as follows: %ILS ϭ -gal transfection and X-Gal staining. ([average survival days of the treatment group/average survival The data in these tables show that although micelles days of the control group] Ϫ 1) ϫ 100%. successfully mediated transfection in vitro, they were in Differences in survival among groups were analyzed for statis- general much less effective than the liposomes made of tical significance by the two-sided log-rank test. All statistical the same phospholipids. The reason for this was not tests used in this study were two-sided statistical tests. clear, but two of the main factors were particle size and membrane structure. The relationship between transfec- RESULTS tion efficiency and the size of liposome-DNA complexes was investigated later. Liposomes were generally more effective than micelles A similar result was also confirmed in vivo. The in vitro and in vivo micelle formulation (micelle P2) with the highest trans- Many different cationic liposome formulations have fection efficiency (3.3.%) among the micelle formula- been tested for their ability to mediate transfection. In tions was tested for TGI (see Fig 5A). The result was contrast, the ability of micelles to mediate transfection parallel with the in vitro transfection: micelle-p53 had has not been investigated extensively. It is not clear much less of an effect on tumor inhibition than the whether the liposome bilayer is necessary for transfec- liposome-p53 formulations tested. It seems that the tion. To answer this question, we compared the trans- micelle may not be able to transfect enough of the tumor fection efficiency of Ͼ50 different micelle and liposome 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 1250-900 710-500 430-320 280-190 150-110

Liposome ST 2.3 Ϯ 1.2 2.1 Ϯ 0.8 5.2 Ϯ 1.5 4.8 Ϯ 1.1 0.9 Ϯ 0.5 Liposome T1 0.3 Ϯ 0.1 1.8 Ϯ 0.2 2.3 Ϯ 0.2 6.2 Ϯ 0.4 6.1 Ϯ 0.4 Liposome M3 0.8 Ϯ 0.4 4.2 Ϯ 1.0 5.9 Ϯ 1.2 11.2 Ϯ 1.7 7.6 Ϯ 1.1 Liposome DP3 0.9 Ϯ 0.5 2.1 Ϯ 0.4 4.1 Ϯ 0.7 10.6 Ϯ 1.2 8.5 Ϯ 1.1 G67 1.9 Ϯ 1.1 3.2 Ϯ 0.5 6.0 Ϯ 1.3 7.2 Ϯ 1.2 0.8 Ϯ 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 Ͼ99.9% of particles (by number) in each formulation. Data represent transfection efﬁciency for each complex, which represents the mean Ϯ 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 Ϯ 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 Different lipid/DNA weight ratios were also tested for lipid composition and lipid/DNA ratio the micelle and liposome formulations whose composi- As Tables 1 and 2 also show, transfection efficiencies tions corresponded to the highest transfection efficiency. ␮ varied greatly according to the lipid compositions of The DNA amount was fixed at 2 g of DNA/well. The different micelles or liposomes, suggesting that transfec- experimental conditions and the determination of trans- tion efficiency was highly dependent upon lipid compo- fection efficiency were the same as described above. As sition. Of all of the micelle formulations tested, micelle Table 3 shows, transfection efficiency could be optimized P2 had the highest transfection efficiency (3.3%) (Table by altering the lipid/DNA ratios, and different liposomes 1); liposomes M3 and DP3 had the highest transfection had a different optimal lipid/DNA ratio. Other than efficiencies (11.4% and 11.2%, respectively) of all of the micelle P2 and G67, which have optimal lipid/DNA liposome formulations tested (Table 2). Liposomes M3 ratios of 2:1 or 3:1, respectively, the optimal lipid-DNA and DP3 both performed better than the control lipo- ratio for the rest of the liposome formulations was 12:1 some G67 under these experimental conditions. 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

NIH/3T3 Bronchial Saos2 A549 lung H358 lung H322 lung Formulation mouse embryo epithelial osteosarcoma carcinoma carcinoma carcinoma

Liposome ST 5.2 Ϯ 1.5 1.7 Ϯ 0.8 3.2 Ϯ 1.4 1.1 Ϯ 0.6 1.2 Ϯ 0.8 0.8 Ϯ 0.3 Liposome T1 6.1 Ϯ 0.4 5.1 Ϯ 1.7 5.5 Ϯ 2.1 2.0 Ϯ 0.8 1.7 Ϯ 1.1 0.5 Ϯ 0.2 Liposome DP3 13.8 Ϯ 3.4 9.4 Ϯ 2.3 4.7 Ϯ 1.4 3.9 Ϯ 1.5 10.7 Ϯ 3.6 5.8 Ϯ 2.1 G67 9.2 Ϯ 2.4 7.4 Ϯ 1.7 13.4 Ϯ 3.5 4.5 Ϯ 1.2 7.2 Ϯ 2.8 4.1 Ϯ 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 Ϯ SD of three independent experiments carried out with duplicate samples. led to a very good transfection efficiency that was only heterogeneously sized liposomes. Therefore, it is not slightly lower than that given by the 12:1 ratio. Because clear whether such differences were due to the special of its relatively low toxicity, the 6:1 ratio seemed more liposome-DNA size preferences of different cell lines or suitable for in vivo experiments. to the lipid compositions of the liposomes. To determine whether different cell lines need different optimal sizes Transfection efficiency was highly dependent upon the of liposome-DNA complexes to achieve the highest size of liposome-DNA complexes transfection efficiency, three groups of DP3-␤-gal complexes with size ranges of 110–150 nm (small), 190–280 As shown in Tables 1 and 2, the liposome formulations nm (medium), and 900-1250 nm (large) were used to had a wide particle size distribution and different aver- transfect two human NSCLC cell lines (H358 and H322) age sizes before the size selection. Therefore, the parti- as described above. The lipid/DNA ratio was 12:1, and cle sizes of the liposome-DNA complexes formed by the DNA dosage was 2, 6, or 10 ␮g of DNA/well. those liposomes with DNA were also different. Suspect- Our results, as shown in Figure 1, led us to two ing that transfection efficiency may also be affected by conclusions: First, the transfection efficiency was not the size of the liposome-DNA complexes, we subse- significantly changed by increasing the DNA dose from 2 quently tested the effect of size on transfection efficiency to 10 ␮g of DNA/106 cells. Second, even when using the (Table 4). same liposome-DNA, the optimal complex size for H358 The size ranges shown in Table 4 encompassed Ͼ99% cells was 190–280 nm, whereas the optimal size for H322 of the liposome-DNA complexes in a sample measured cells was 900-1250 nm. Therefore, for different cell lines, with a Nicomp Submicron Particle Sizer 370. Transfec- the optimal sizes of liposome-DNA complexes differed tion efficiency data are the mean Ϯ SD of three different even for liposome-DNA with the same lipid composition experiments. The DNA (␤-gal) dose was 2 ␮gofDNA/ and the same DNA construction. well. The results showed that transfection efficiency was also highly dependent upon the particle size of the Different cell lines could have different optimal liposome-DNA complexes, consistent with the idea that lipid compositions the right size range of the liposome-DNA complexes is crucial for achieving high transfection efficiency. Inter- To determine whether different cell lines needed differ- estingly, for all liposomes, the highest transfection effi- ent optimal lipid compositions to achieve the highest ciency in NIH/3T3 cells was obtained with liposomes- transfection efficiency, we constructed different lipo- DNA in the same size range, 190–280 nm. The only some-DNA complexes with a fixed size range of 190–280 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 liposome- Table 6. Effect of Adding ST to Liposome on Transfection DNA complexes was not lipid composition-dependent. Efficiency In Vitro* Whether it was cell line-dependent was our next question. Lipid ratio (mol) Size % Transfection Name (DPPC†/CHOL‡/ST) (mean Ϯ SD, nm) (in 3T3 cells) Different cell lines could have different optimal sizes of liposome-DNA complexes Liposome ST1 2:2:1 216 Ϯ 145 1.3 Ϯ 0.8 Liposome ST2 3:3:2 204 Ϯ 136 0.7 Ϯ 0.6 It has been shown that the transfection efficiency of the Liposome ST3 7:4:5 164 Ϯ 88 8.2 Ϯ 2.5 same liposome formulation can differ in different cell 44 *Liposomes containing ST of optimal size were prepared by hydration lines (i.e., can be cell line-dependent). Except for followed by extrusion through a 0.2-␮m membrane. The lipid/DNA ratio varying characteristics of different cell lines, the formu- was 12:1. The data for each formulation represent the mean Ϯ SD of three lation factors will also significantly affect the transfection independent experiments carried out with duplicate samples. efficiency. However, almost all of the results from pre- †DPPC, dipalmitoyl phosphatidylcholine. vious studies were obtained by using a mixture of ‡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 Ϯ 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 supplemented with ST. We selected dipalmitoyl phos- lines. The results are shown in Table 5. The DNA dosage phatidylcholine and cholesterol because neither has a was 2 ␮g of DNA/well. The experimental conditions and charge and because the combination easily forms a methods were the same as those described previously. relatively stable liposome in vivo. Table 6 shows the Under these experimental conditions, liposome DP3 was results. When the lipid ratio was appropriate (e.g., the best formulation for transfecting NIH/3T3 cells, 7:4:5), the ST liposome was capable of initiating and normal bronchial epithelial cells, H358 cells, and H322 carrying out transfection with a high degree of efficiency. cells; liposome G67 was the best formulation for trans- These results demonstrated that the phospholipid chem- fecting Saos2 cells and A549 cells. Therefore, when the ical structure may not be critical for the transfection, and liposomes size was fixed, transfection efficiency was that lipid exchange may not be the main pathway for the dependent upon both cell line and lipid composition. liposome-mediated transfection. Because of its relatively high toxicity (our unpublished data), we did not use ST ST liposome-mediated transfection efficiency in the survival experiments. ST is a small cationic molecule with a single-carbon H358 cells were effectively transfected via liposome- frame. It is not a phospholipid, but its positive charge p53 complexes in vitro and surfactant properties are similar to those of cationic phospholipids used in liposomes. ST is easy to insert into We subsequently tested the efficacy of several liposome lipid bilayers to make different liposomes; its positive- formulations at delivering p53 into H358 cells in vitro.To charge portion will remain on the liposome surface. ascertain whether p53 plasmid could be successfully Therefore, liposomes made of neutral phospholipids and delivered into the cells and functional p53 protein could ST will have a positive charge from the amino group of be expressed, liposomes ST, G67, and DP3 were used to ST but not from cationic phospholipids. Furthermore, form liposome-p53 complexes with three plasmids, because the relative amount of phospholipid will be pC53SN (containing a CMV promoter-driven human wt reduced by inserting ST into the liposomes, the ST p53 gene), wwp-luc (containing a p21 promoter-driven liposomes will theoretically have less lipid exchange with luciferase gene), and ␤-gal (containing the CMV pro- cells than the liposomes made of cationic phospholipids. moter driven lacZ gene), to transfect H358 cells. Theo- ST has not been investigated extensively for mediating retically, if p53 plasmid is delivered into the cells and is transfection.45 To understand whether phospholipid expressed, the functional p53 protein will activate the structure affects transfection and whether reducing lipid p21 promoter and induce the expression of luciferase. exchange affects transfection efficiency, we tested several Luciferase and ␤-gal activities were measured at 48 liposome formulations made with two neutral lipids and 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 Ϯ 95% confidence intervals of three independent experiments carried out with duplicate samples. activities, which were corrected for transfection effi- the p53 killing efficiency was 30–40%. One possible ciency by measuring the ␤-gal activity, showed a 60-, reason we considered was that the ␤-gal assay was not 120-, or 130-fold induction in cells transfected with sensitive enough, so the transfection efficiency was actu- liposome ST-p53, G67-p53, or DP3-p53 complexes, re- ally much higher than that measured by ␤-gal staining. spectively, compared with the normal saline controls, but This possibility was later confirmed using a more sensi- no induction in cells transfected with pC53SN plasmid tive green fluorescent protein (GFP) plasmid as the alone, liposome ST-vector plasmid, G67-vector plasmid, reporter gene (see Discussion). Another possibility was or DP3-vector plasmid (Fig 2). Thus, cells transfected that a “bystander effect” might exist. with any of the three liposome-p53 complexes expressed To demonstrate that the cell death seen after DP3-p53 functional p53 protein, and DP3 was slightly better than transfection was due to apoptosis, TUNEL assays were G67 at delivering p53 plasmids in vitro. 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. Figure 4. Lung biopsies of mice bearing orthotopic H358 human ϫ 6 By comparing Figures 1 and 3, it can be seen that NSCLC. Male nude mice were inoculated with 1–2 10 H358 cells/mouse intratracheally. After 3 weeks, the lungs were resected, DP3-p53 effectively killed both p53-null and p53-mutant fixed with 10% formalin, and embedded in paraffin. The embedded lung cancer cells. However, the p53 killing efficiency was lung tissue was sectioned and stained with hematoxylin and eosin. much higher than the transfection efficiency under opti- Bronchial lumina and lung tumors are labeled 1 and 2, respectively. mal transfection conditions for both the H358 and H322 As shown in this figure, H358 tumors grew orthotopically in the cell lines. The transfection efficiency measured by ␤-gal lungs of nude mice in a multinodular pattern without evidence of staining was ϳ10% for H358 and H322 cells, whereas 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 ϭ .009; liposome-p53 versus no treatment, P ϭ .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 ϭ .008; liposome-p53 versus no treatment, P ϭ .004.

fluorescent signal, indicating the presence of DNA frag- carcinoma and caused death by local growth without mentation in the apoptotic cells (data not shown). evidence of metastatic spread. Apoptotic cells were examined by flow cytometry, and To determine whether our liposome-p53 formulations the results showed that at 30 hours after DP3-p53 would inhibit tumor growth in vivo, lung TGI experi- transfection, 85% of the detached cells and 15% of the ments were performed. As Figure 5A shows, both DP3- attached cells were apoptotic, indicating that DP3-p53 p53 and G67-p53 treatment significantly inhibited tumor transfection had induced apoptosis in H358 cells. Apo- growth by day 77; the %TGI was 97% for DP3-p53 and ptosis in the transfected cells was also confirmed by 94% for G67-p53 (P Ͼ .05). There were no visible agarose gel electrophoresis (data not shown). tumors in the lung tissues of G67-p53-and DP3-p53- treated mice, and all five mice in these two groups were DP3-p53 and G67-p53 administered intratracheally alive until the day they were killed. The best micelle were both effective at inhibiting lung tumor formulation (micelle P2-p53) had little antitumoral ac- growth in vivo tivity compared with the liposome formulations; perhaps To determine the antitumoral efficacy of liposome-p53 the micelles were not able to transfect enough tumor treatment in vivo, we developed a human lung cancer cells. Groups treated with either p53 DNA alone or model in nude mice that mimics human bronchial ma- empty liposome alone showed limited tumor inhibition in lignancy and early lung cancer. Repeated experiments both tumor models (%TGI of Ͻ27%). Most of the lung showed that human lung cancer cells implanted intratra- parenchymas in these animals were replaced by tumors by cheally in mouse bronchi attached to the bronchial day 77. The average lung/body weight ratios of the five epithelium and gave rise to multiple tumors, and that the control groups (untreated and those treated with pC53SN, life spans of these mice correlated with the number of LST, G67, or DP3 liposome) were 6- to 13-fold higher than cancer cells inoculated. Figure 4 shows a mouse lung those of the three liposome-p53-treated groups. There was biopsy at 3 weeks after intratracheal H358 tumor inoc- no significant difference between the average lung/body ulation. The results indicate that H358 cells grew ortho- weight ratios of G67-p53- or DP3-p53-treated mice and topically in the lungs of nude mice in a multinodular normal mice not inoculated with tumor (8.0 vs. 7.3 mg/g, pattern similar to that seen for human bronchioalveolar n ϭ 13, P Ͼ .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 ␮gor8␮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, able, or chronic, administration. Repeatable administra- and similar results were obtained (Fig 5B). tion is necessary for treating early lung cancers in the bronchial epithelium for three reasons. First, just one Dp3-p53 and G67-p53 administered intratracheally administration of either liposome-gene or virus-gene significantly prolonged the life of lung particles may not penetrate deeply enough into the tumor-bearing mice bronchial epithelium to reach all malignant cells, even The optimal liposome-p53 complex sizes (190–280 nm though in most cases bronchial premalignant and early for H358 treatment and 900-1250 nm for H322 treat- malignant lesions are superficial and limited in thickness ment) and lipid/DNA ratios (6:1 for DP3-p53 and 2:1 for to a few layers of cells. With repeated administrations, G67-p53) were used. The DNA dose was 2 ␮g and 8 ␮g however, surface malignant cells can be killed first and of p53 DNA per mouse per injection for H358 and H322 then deeper layers of malignant cells can be exposed and treatment, respectively. In the H358 survival experi- subsequently killed. Second, the cancer cells remaining ments, the mean median survival of the three control alive after the first administration will continue to grow groups (no treatment, p53 alone, DP3 alone) was 107 and develop tumors. Third, some patients most likely days compared with 179 and 167 days of mean survival will need repeated administrations if they continue to be for the DP3-p53- and G67-p53-treated groups (Fig 6A). exposed to airborne carcinogens such as cigarette 46 In the H322 survival experiments, the mean median smoke, asbestos, ground radon, and certain chemicals. survival of the three control groups was 93 days com- Compared with a viral delivery system, a cationic pared with 219 and 199 days for the DP3-p53- and liposome delivery system has the major disadvantage of G67-p53-treated groups (Fig 6B). Both liposome-p53 its low transfection efficiency. Indeed, in our experi- formulations significantly prolonged the lives of the ments, the in vitro transfection efficiencies for cationic tumor-bearing mice by ϳ1.6- to 2.4-fold (P Ͻ .009 by liposome-DNA complexes were generally ϳ10% as log-rank test) after only five doses. DP3-p53 was slightly measured by the ␤-gal staining method. However, the more or at least equally as effective as G67-p53. p53 killing efficiency (i.e., the number of cells undergo- ing apoptosis after a single transfection with DP3-p53) was ϳ40–50%. Possible reasons for this discrepancy DISCUSSION include the low sensitivity of ␤-gal staining in determining transfection efficiency and/or the existence of a bystander effect, as described previously for adenoviral- A cationic liposome delivery system has distinct advan- p53 transfection.47–49 To assess transfection efficiency tages over a viral delivery system. The main advantages more accurately, we therefore used the GFP plasmid as are minimal toxicity, no immunogenicity, and repeat- a reporter plasmid, because GFP expression is much

Cancer Gene Therapy, Vol 7, No 5, 2000 694 ZOU, ZONG, LING, ET AL: NONVIRAL GENE DELIVERY SYSTEM FOR LUNG CANCER easier to detect and much more sensitive in measuring composition are used. Because the optimal complex size transfection efficiency. As a result, we found that the needed to transfect a particular cell line was the same for transfection efficiency of DP3-GFP in H358 or H322 different liposome-DNA formulations, we also con- cells in vitro is constantly ϳ40% (data not shown). cluded that it is best to find the optimal liposome-DNA Therefore, we now think the discrepancy between trans- size first and then optimize its lipid composition and fection efficiency and p53 killing efficiency was most lipid/DNA ratio using homogeneous liposomes within likely due to the insensitivity of the ␤-gal staining the optimal size range. method rather to a bystander effect. In fact, in the case We also hypothesized that, in the case of treating early of DP3-p53, there was little difference between its p53 lung cancer in bronchi, the functions of p53 in liposome- killing efficiency and its transfection efficiency as mea- p53 formulations in vitro correlate positively with their sured by GFP, which raises the question of whether the TGI effects in vivo. Our rationale was that, compared bystander effect of p53 truly exists. with physical environment in the circulatory system, the For future development of an effective nonviral gene physical environment in the bronchi is relatively simple36 therapy for treating early lung cancer, optimization of a and similar to that in the culture dish. The bronchial liposome-gene delivery system will be crucial. This idea environment also contains fewer substances, such as drove our present study and led us to explore two high-density lipoprotein and phospholipase, that can important ways to improve cationic lipid gene delivery destroy liposomes immediately and contains too little systems: selection of the best cationic material and liquid to significantly dilute the liposome-DNA concen- optimization of the formulations. We identified several tration or change the physical properties of the complex. important factors that significantly affect gene transfec- Therefore, we reasoned that liposome-DNA complexes tion. First, we noted that liposomes made of cationic delivered intratracheally to the bronchi would more phospholipids were more effective than liposomes made likely retain their physical properties and their transduc- of ST, and that liposomes were more effective than ing ability. The opposite assumption was that liposome- micelles made of the same cationic phospholipids. In DNA complexes administered intravenously or intratu- micelle structures, the hydrophobic portion of the lipids morally would be less effective because blood and tissue associates to form regions from which water is excluded, fluids might change their physical properties and there- whereas the hydrophilic head groups remain on the fore their ability to induce transfection. Furthermore, we outer surface to maximize their interaction with the assumed that certain enzymes and proteins in the blood water and oppositely charged ions. Unlike liposomes, and in tissue fluids would destroy most liposome-DNA micelle particles made of phospholipids usually are complexes, further decreasing the in vivo transfection smaller and have no lipid bilayer structures. However, it efficiency. In those situations, the transfection efficiency was not clear before our study whether the liposome of liposome-DNA complexes in vitro most likely will not bilayer was necessary for transfection. Nevertheless, our correlate with their transfection efficiency in vivo.In study partially answered the question: cationic phospho- short, our data supported our hypothesis and showed lipid micelles do have the potential to mediate transfec- that those formulations with higher in vitro p53 function tion, and a bilayer structure is not necessary. Our results (Fig 2) also had higher in vivo antitumoral effects (Figs also hinted that the differences between liposome-medi- 5 and 6). ated transfection and micelle-mediated transfection may We used p53 as prototype gene because its tumor reveal certain aspects of the mechanism of the transfec- suppressor function has been solidly confirmed,37–39 and tion process. Second, our results implied that the lipo- because its mutation is frequent in lung cancer cases some-DNA complex was a suitable form for delivering (50–70%).7,40,41 Theoretically, the technique we devel- DNA into cells and that, during transfection, bilayer oped in this study also can be used to deliver any other structure may play an important role. However, the therapeutic genes by intratracheal or aerosol adminis- nonphospholipid ST was not effective as cationic phos- tration after minor modification. pholipid. This can be explained by the fact that cationic G67 has been shown previously to be highly effec- phospholipid and cell phospholipid exchange is also tive in delivering plasmid into mouse lungs at levels important for transfection. comparable with those attained with recombinant Cell lines differ in their cell size and possibly in their adenovirus vectors at multiplicities of infection rang- cell membrane lipid compositions and surface protein ing from 1 to 20.29,50 Through this study, we have distribution. Thus, we hypothesized that the optimal size developed and tested a novel cationic liposome for- of liposome-DNA complexes may be cell line-depen- mulation, DP3, that is at least as effective as G67 for dent. In other words, liposome-DNA complexes with the the treatment of early lung cancer both in vitro and in optimal size might attach more easily and tightly to vivo and is a promising gene delivery system for lung certain types of cell surfaces than to others, so as to cancer gene therapy. induce endocytosis or create the right surface tension needed for optimal fusion and lipid exchange with the cell membrane. Our results confirmed this hypothesis ACKNOWLEDGMENTS and showed that the optimal size of liposome-DNA complexes for different cell lines may differ even when We thank Dr. Bert Vogelstein for providing the pC53SN and the same liposomes, the same DNA, and the same lipid wwp-luc plasmid. This work was supported by a research grant

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