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

© 2000 Nature America, Inc. 0929-1903/00/$15.00/ϩ0 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 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

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