Clinically Available Endosomolytic Agent for Gene Delivery - American

Chapter 11

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Clinically Available Endosomolytic Agent for Gene Delivery 1

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K. Itaka , K. Miyata , A. Harada , H. Kawaguchi , K. Nakamura , andK.Kataoka * 1,

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Department of Materials Science and Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan *Corresponding author: email: [email protected]

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The ability of hydroxychloroquine, a chloroquine derivative, to improve the gene transfection efficiency of a carrier system based on non-viral delivery was investigated. Hydroxychloro quine gave enhanced gene expression and low cytotoxicity in an in vitro transfection study. In particular, prolonged incubation for 24 hours with 100 μΜ hydroxychloroquine significantly enhanced the expression using a polyion complex (PIC) micelle system with excellent stability in serum-containing medium. Therefore, appropriate endosomo lytic agents should be useful for in vivo gene delivery using non-viral delivery systems, with the FDA-approved hydroxy chloroquine as one of the most promising candidates for this purpose.

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© 2004 American Chemical Society

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Introduction Chloroquine has widely been used for the enhancement of DNA/polycation complex based gene transfer. (1-3) Chloroquine was originally discovered as an anti-malaria drug, and its anti-inflammatory effects are well known. The mechanism of increasing the transfection efficiency assumes that choloroquine is a weak base, and that its consequent endosomotropism contributes to its activity by interfering with lysosomal degradation and enhancing the release of DNA into the cytoplasm. (4) Chloroquine, however, shows cytotoxicity and there still remain some difficulties with its application in clinical gene therapy. One of the chloroquine derivatives, hydroxychloroquine, in which one of the N-ethyl substituents of chloroquine is B-hydroxylated, is considered to be as effective as the parent molecule but with lower toxicity. (J) In fact, hydroxy chloroquine is a FDA-approved drug, and high doses have been clinically used for the treatment of rheumatoid arthritis and lupus erythematosus. (6-8) In this study, we evaluate the ability of hydroxychloroquine to enhance transfection efficiency and discuss its feasibility as a clinically available endosomolytic agent.

Materials and Methods Chemicals. Poly(L-lysine) with a degree of polymerization of 80, and poly (ethylene glycol-L-lysine) block copolymer (PEG-PLL; PEG Mw 12,000 g/mol; 71 PLL segments) were synthesized as reported previously. (9) The LipofectAMINE™ reagent and fetal bovine serum (FBS) were purchased from GIBCO BRL. Chloroquine and hydroxychloroquine were purchased from Sigma and Acros Organics, respectively. Plasmid DNA. pGL3-Luc (Promega) was used in all experiments. This plasmid was amplified in competent DH5a Escherichia coli and purified using EndoFree™ Plasmid Maxi or Mega Kits (QIAGEN), The DNA concentration was determined by its UV absorbance measured at 260 nm. Formation of DNA-loaded complex particles. PEG-PLL block copolymer and pDNA were separately dissolved in 10 mM of Tris-HCl buffer at pH 7.4. Polyion complex (PIC) micelles were formed by mixing both solutions at various charge ratios, based on the residual molar amount of the lysine unit to nucleotide. The poly(L-lysine)/DNA complex (Plys-polyplex) was similarly


156 prepared by mixing the poly(L-lysine) and pDNA solutions. The polycationie lipid/DNA complex (lipoplex) was prepared by mixing the pDNA solution and LipofectAMINE™ reagent following the manufacturer's protocol. In all cases, the final DNA concentration was adjusted to 30 |Lig/ml. Transfection. 293T cells were seeded in 24-well culture plates. After 24hours incubation in the culture medium (Dulbecco's modified eagle medium; Sigma), the cells were rinsed, and 250 pi medium containing chloroquine or hydroxychloroquine was added to each well. 25 pi of the DNA-loaded complex solution was then applied to each well. For the transfection efficiency study (Figure 1), the cells were incubated for 4 hours. For the prolonged incubation study (Figure 3), the transfection time was varied from 3 to 24 hours. After removal of the transfection medium, the cells were further incubated in fresh culture medium. At 48 hours after the initial application of the DNA-loaded complexes, the luciferase gene expression was measured. Throughout this procedure, 10% FBS was included in the culture medium. Cytotoxicity Assay. The cytotoxicity of chloroquine and hydroxy chloroquine was evaluated by an MTT assay (Cell Counting Kit: Dojindo). 293T cells were plated into 96-well tissue culture plates. The medium was removed 24 hours later, and 100 pi DMEM (with 10% FBS) containing chloro quine or hydroxychloroquine (10-100 pM) was added to each well. After several hours of incubation, the viability of the cells in each well was measured following the manufacturer's protocol. To compare the cytotoxicity between the two agents, the distribution of values was first analyzed in each group using the F-test. Between groups with normal distributions, the parametrical analysis using the Student's t-test (StatView-J 4.5; Stat View, Berkeley) was performed.

Results and Discussion The presence of hydroxychloroquine in the transfection medium enhanced the gene expression of Plys-polyplex and PIC micelles (Figure 1). The results were concentration-dependent up to 100 pM, and almost equivalent to those found for chloroquine, indicating that hydroxychloroquine can be used under similar conditions as chloroquine. However, gene expression decreased for concentrations greater than 150 pM due to cytotoxicity. This observation is in accordance with reports showing 100 pM chloroquine as the optimum condition for in vitro transfection. (1-3) Conversely, both agents acted negatively on the

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transfection by lipoplex, presumably caused not only by the severe cytotoxicity in the co-presence of both lipoplex and the endosomolytic agent, but also the differences in the intracellular routing between lipoplex and complexes based on cationic polymers.

Figure 1. Transfection efficiency to 293T cells by Plys-polyplex, PIC micell and lipoplex in the presence of chloroquine or hydroxychloroquine (10-20 pM) in the culture medium, (n = 4; ± S.E.j. Cytotoxicity may be one of the most serious problems for the clinical application of endosomolytic agents. Indeed, we have observed that 100 fiM chloroquine caused a serious cytotoxocity for most types of cells, especially in


158 culture media without serum. However, the viability considerably improved in media containing 10% serum. Figure 2 represents the viability of 293T cells in the presence of chloroquine or hydroxychloroquine in serum-containing medium. The cells could survive and proliferate even in 100 pM concentration for 24 hours. Similar results were obtained for other kinds of cell lines, although the degree of toxicity varied among these lines. It should be noted that the cytotoxicity of hydroxychloroquine was lower than that of chloroquine. For example, the number of live cells after 8 hours (p = 0.002) and 24 hours (p < 0.001) of incubation was significantly higher in the presence of 100 pM hydroxychloroquine than in the presence of 100 pM chloroquine.

Figure 2. Viability of 293T cells in the presence of chloroquine or hydroxy chloroquine (10-100 pM) in the culture medium, (n = 8; ±S.E.). The effect of prolonged incubation was investigated next. The gene expression showed a remarkable increase with longer incubation in the copresence of DNA-loaded complexes and 100 pM of hydroxychloroquine (Figure 3). This phenomenon was not observed in the absence of hydroxychloroquine (data not shown). Interestingly, the time-dependent increase in gene expression was more obvious for PIC micelles compared to Plys-polyplex, resulting in higher gene expression of the former after 24-hour incubation. This unique gene expression profile of PIC micelles may be explained by their excellent

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stability in the serum-containing medium as well as their appreciably lower cytotoxicity compared to Plys-polyplex. (10,11)

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Figure 3. Transfection efficiency by prolonged incubation in the transfecti medium (Plys-polyplex, PIC micelles) with 100 pM hydroxychloroquine, ( 4;±S.E.). As reported in the literature, the steady-state blood concentration of hydroxychloroquine in patients with rheumatoid arthritis (daily dosis 400 mg) was 870.3 ± 329.3 ng/ml, equivalent to 2 pM. (12) Thus, if a somewhat higher concentration of hydroxychloroquine is permissible locally and temporarily, these conditions will effectively work for increasing the transfection efficiency. Moreover, DNA-loaded complexes and endosomolytic agents should not necessarily be used simultaneously, rather could be used separately or in a different way of administration. Although the conditions between in vitro and in vivo studies are quite different, and the ability of hydroxychloroquine to increase the transfection efficiency in vivo is not known, the results of this study indicate that the administration of hydroxychloroquine via a suitable method may be worth considering for clinical gene therapy with non-viral delivery systems.

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Conclusions


The enhanced gene expression found for Plys-polyplex and PIC micelles with hydroxychloroquine present in the transfection medium, and the comparatively low cytotoxicity of hydroxychloroquine suggest that clinically available endosomolytic agents may improve effective in vivo gene delivery when used in combination with an appropriate delivery system possessing a considerable tolerability and low toxicity under physiological conditions, such as PIC micelles.

Acknowledgments This work was financially supported by Grants-in-Aid for Scientific Research (No. 11167210 to K.K and No. 12877221 to H.K.) and Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology of Japan as well as by the Core Research Program for Evolutional Science and Technology (CREST) from the Japan Science and Technology Corporation (JST).

References 1. Luthman, H.; Magnusson, G. Nucleic Acids Res. 1983, 11, 1295-308. 2. Erbacher, P.; Roche, A.C.; Monsigny, M.; Midoux, P. Exp Cell Res. 1996, 225, 186-94. 3. Wolfert, M.A.; Seymour, L.W. Gene Ther. 1998, 5, 409-14. 4. Wagner, E. J. Control. Release 1998, 53, 155-8. 5. The Pharmacological Basis of Therapeutics, 10th edition Hardman, J.G.; Limbird, L.E.; Gilman, A.G., Eds.; McGraw-Hill: Columbus, OH, 2001; pp 1077-1080. 6. Hamilton, E.B.; Scott, J.T. Arthritis Rheum. 1962, 5, 502-512. 7. Clark, P.; Casas, E.; Tugwell, P.; Medina,C.;Gheno,C.;Tenorio, G.; Orozco, J.A. Ann. Intern. Med. 1993,119,1067-71. 8. HERA Study Group, Am. J. Med. 1995, 98, 156-68. 9. Harada, A.; Kataoka, K. Macromolecules 1995, 28, 5294-5299. 10. Itaka, K.; Harada, A.; Nakamura, K.; Kawaguchi, H.; Kataoka, K. Biomacromol. 2002, 3, 841-5. 11. Itaka, K.; Yamauchi, K.; Harada, A.; Nakamura, K.; Kawaguchi, H.; Kataoka,K.,Unpublished. 12. Tett, S.E.; Cutler, D.J.; Beck,C.;Day, R.O. J. Rheumatol. 2000, 27, 165660.

Clinically Available Endosomolytic Agent for Gene Delivery - American

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