Bioconjugate Chem. 2005, 16, 1390−1398
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Biophysical and Transfection Studies of an Amine-Modified Poly(vinyl alcohol) for Gene Delivery Matthias Wittmar,† James S. Ellis,§ Frank Morell,† Florian Unger,† Jeanette Christine Schumacher,† Clive J. Roberts,§ Saul J. B. Tendler,§ Martyn C. Davies,§ and Thomas Kissel*,† Philipps University Marburg, Department of Pharmaceutics and Biopharmacy, Ketzerbach 63, D-35032 Marburg, Germany, and Laboratory of Biophysics and Surface Analysis, School of Pharmacy, The University of Nottingham, Nottingham, NG7 2RD, UK. Received March 31, 2005; Revised Manuscript Received October 4, 2005
Novel, multifunctional polymers remain an attractive objective for drug delivery, especially for hydrophilic macromolecular drugs candidates such as peptides, proteins, RNA, and DNA. To facilitate intracellular delivery of DNA, new amine-modified poly(vinyl alcohol)s (PVAs) were synthesized by a two-step process using carbonyl diimidazole activated diamines to produce PVAs with different degrees of amine substitution. The resulting polymers were characterized using NMR, thermogravimetric analysis (TGA), and gelpermation chromatography (GPC). Atomic force microscopy (AFM), dynamic light scattering photon correlation spectroscopy (PCS), and zeta-potential were used to investigate polyplexes of DNA with PVA copolymers. These studies suggest an influence of the polycation structure on the morphology of condensed DNA in polyplexes. Significant differences were observed by changing both the degrees of amine substitution and the structure of the PVA backbone, demonstrating that both electrostatic and hydrophobic interactions affect DNA condensation. DNA condensation measured by an ethidium bromide intercalation assay showed a higher degree of condensation with pDNA with increasing degrees of amine substitution and more hydrophobic functional groups. These findings are in line with transfection experiments, in which a good uptake of these polymer DNA complexes was noted, unfortunately, with little endosomal escape. Co-administration of chloroquine resulted in increased endosomal escape and higher transfection efficiencies, due to disruption of the endosomal membrane. In this study, the structural requirements for DNA complexation and condensation were characterized to provide a basis for rational design of nonviral gene delivery systems.
INTRODUCTION
Delivery of hydrophilic molecules such as proteins and DNA is generally considered as the Achilles heel for their therapeutic application (1-3). These molecules are rapidly degraded by enzymes found under in vivo conditions both intracellularly at the site of application as well as in the general circulation, causing low bioavailabilities and requiring frequent injections (4-6). Because of their size and lability, uptake through the epithelium of the gastrointestinal tract is low and thus necessitates parenteral administration (7, 8). To overcome these problems, carrier systems are necessary to allow efficient, safe, and convenient delivery to patients (9-20). In this context, nanoscale carriers such as nanoparticles and nanocomplexes have reached increasing attention, since they can be administered by various routes, including the intravenous and intranasal routes (9, 21, 22). Controlled and sustained release of these drug candidates can be accomplished using microspheres and implants from biodegradable polymers (1, 14, 20, 23). The classic copolyesters of lactic and glycolic acid (PLGA) are not ideal for protein and DNA delivery since inactivation and uncontrolled release is a consequence of poor compatibility between lipophilic polymers and hydrophilic * To whom correspondence should be addressed. Tel.: ++496421-2825881. Fax: +49-6421-2827016. E-mail: kissel@ staff.uni-marburg.de. † Philipps University Marburg. § The University of Nottingham.
drug candidates (7, 24-27). This is especially the case for DNA, where the complexation capabilities and protecting abilities of the carrier substance are very important. We hypothesized that comblike polyesters consisting of both lipophilic and hydrophilic components could be beneficial for drug delivery. We selected PVA with a molecular weight of 15 000 g/mol, which is considered biocompatible and can be eliminated from the body by renal excretion (28-30). To this polymer backbone, amine groups were covalently coupled in a polymer-analogous reaction using carbonyl diimidazole (CDI) to introduce cationic charges under physiological conditions (31-33). This modification was thought to affect colloidal stability of carrier systems by imparting positive surface charges on one hand (34) and increasing the protein or DNA loading by electrostatic interactions on the other hand (35, 36). As the secondary and tertiary amino-groups functions possess lower cytotoxicity, diamines, and PVA were coupled via the hydrolytically stable urethane bond (38). The resulting PVA could be used in different ratios to complex DNA. EXPERIMENTAL SECTION
Materials. Diethylaminoethylamine (purum, >98%), Diethylaminopropylamine (purum, >98%), Dimethylaminopropylamine (purum, >98%), PVA (MW 15000; degree of polymerization 300 (P ) 300); degree of hydrolysis 8689%), CDI (purum, ∼97%), N-methyl pyrrolidone (NMP) (absolute), dimethylacetamide (DMAc) (for HPLC, 99.8% and 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone
10.1021/bc0500995 CCC: $30.25 © 2005 American Chemical Society Published on Web 10/29/2005
Bioconjugate Chem., Vol. 16, No. 6, 2005 1391
Amine-Modified PVA for Gene Delivery Table 1. Backbone Polymers: Synthesis and Molar Mass polymer
feedinga
Mnb g/mol
DMAPA(7) DMAPA(13) DMAPA(21) DMAPA(32) DMAPA(69) DMAPA(105) DMAEA(55) DEAEA(70) DEAPA(68) DMABA(99) DMAHA(81)
12.00/1.20/0.07/4 12.00/2.39/0.17/4 12.00/4.81/0.31/4 12.00/11.99/0.79/4 11.00/23.09/1.51/4 4.50/15.00/0.85/4 7.00/14.35/0.85/4 10.00/22.48/1.37/4 10.00/23.12/1.32/4 2.67/9.04/0.55/3.75 2.48/2.75/0.15/3.75
15320 15890 16750 17880 22420 20880 27400 23630 23910 27560 28000
Mnc g/mol
Mwc g/mol
Mn/ Mwc
9895 12070 11770 12170 15590 n.d. n.d. 29580 33590
11710 14460 13690 14410 20060 n.d. n.d. 32780 67790
1.2 1.2 1.2 1.2 1.3 n.d. n.d. 1.1 2.0
a Feed (mass/g): m(PVA)/m(amine-CI)/m(DMPU)/days of reaction. b Calculated by 1H NMR depending on the degree of polymerization of PVA (P ) 300). c Measured by GPC-MALLS.
(DMPU) (puriss., absolute, over molecular sieve) were purchased from Fluka GmbH (Germany) and used as received. Lithium bromide (extra pure) (Merck) was used as received. Tetrahydrofuran (THF) (BASF, Germany) was dried over sodium and distilled under nitrogen before use. All other chemicals were used as received without further purification. Synthesis of Amine Carbonylimidazoles. A total of 90 mL of dry THF was distilled under nitrogen into a rigorously dried 100 mL round-bottomed flask equipped with gas inlet, a septum cap, and a magnetic stir bar. Then, 16.72 g (0.10 mol) of CDI was dissolved, and the diamine (at a 1:1 molar ratio) was injected by syringe, while the temperature in the flask was monitored to ensure that it did not exceed 55 °C (31). After stirring of the sample for 16 h at room temperature, the resulting imidazole/amine-carbonylimidazole solution was isolated by evaporation of THF. The resulting oily, slight yellow mixture was used without further purification after the amount of amine-CI was quantitated by 1H NMR spectroscopy. Yields: >90%. Synthesis of Amine-Modified PVAs. Poly(vinyl (dialkylamino)alkylcarbamate-co-vinyl acetate-co-vinyl alcohol): A 250 mL round-bottomed flask with gas inlet and magnetic stirring bar was rigorously dried and filled with PVA and anhydrous NMP. During heating to 80 °C, the PVA was dissolved. After complete dissolution (dialkylamino)alkyl)-1H-imidazole-1-carboxamide was added, and finally DMPU was injected. The mixture was heated to 80 °C under stirring for 4.5 days (Table 1). The resulting amine-modified PVA was purified by ultrafiltration using an YM1 membrane (cut off 1000 g/mol, Millipore). During filtration, the solvent was substituted by demineralized water. After filtration of 2.5 L water, the volume in the cell was reduced to 50 to 100 mL. The solution was frozen at -20 °C and dried by lyophilization (Edward Freeze Dryer Modulyo, standard conditions). The polymers were milled and stored until use at 40 °C in a vacuum to minimize water uptake during storage. The polymers are obtained as slightly yellowish hygroscopic powders. Yields: ∼83%. Nomenclature. The source-based IUPAC nomenclature for these polymers lead to the designation: poly(vinyl 3-(alkylamino)alkylcarbamate-co-vinyl acetate-covinyl alcohol). As an abbreviation the short form of the amine followed by a number in brackets was used. (e.g., DEAPA(x) ) 3-diethylaminopropylamine; while x the number displays the modified vinyl alcohol monomers in the chain, for example, DMAPA(13), etc.) Sample Characterization. 1H and 13C NMR spectroscopic data were collected using a JEOL Eclipse+ 500 and a Joel GX 400 D at a frequency of 500 respective
Table 2. TGA Measurements Degradation Caused by Amine Substitution polymer
amine substitutiona
mass lossb/%
calc mass lossc/%
DMAPA(7) DMAPA(13) DMAPA(21) DMAPA(32) DMAPA(69) DMAPA(105) DMAEA(55) DMABA(99) DMAH(81) DEAEA(70) DEAPA(68)
2.3 4.4 7.1 10.8 23.0 33.2 18.4 32.2 27.0 23.3 22.7
4.8 11.8 20.0 36.9 60.7 78.2 48.7 79.9 71.2 54.8 58.5
7.6 14.1 21.9 31.1 53.1 65.9 41.7 67.2 62.1 55.1 57.0
a Degree of amine substitution/% calculated form 1H NMR measurements. b Mass loss during the first degradation step. c Mass loss calculated from NMR data.
400 MHz for 1H NMR and 126 respective 101 MHz for C NMR at 50 °C in DMSO-d6 (euriso-top, 10 µg/mL (Figure 7). This toxicity seems to be independent of the amine type and is only dependent on DS. Apparently, charge density is the decisive factor in determining toxicity, which does not appear to be affected by the means in which it is introduced to the cell. Toxicity is therefore unspecific and a function of charge and not of structure (58). Investigation of the transfection abilities of these polymers are shown in Figure 8. Increasing DS of amine leads to an overall better transfection in comparison to lower-substituted polymers. Increasing the N/P ratio leads to increasing transfection in the presence of chloroquine. Without addition of chloroquine, there is no detectable transfection. In contrast, CLSM images demonstrate an uptake of polymer/DNA particles into the cell but no release into the cytoplasm. The particles stay intact in the endosomes but they cannot leave them without the addition of chloroquine. Positive charges of polymers enable particles formed by these substances and DNA to enter cells. Indeed, this uptake process is only a part of the whole transfection mechanism. It is necessary that something enables the particles to leave the endosomes and to reach the cytoplasm and lastly the nucleus. In the literature, it is discussed that polymers such as poly(ethylene imine) (PEI) open the endosomes by a socalled proton sponge effect (40). This effect is based on the buffering capacity of the polymer. Because of its high density of amino groups, PEI has a huge buffer capacity and leads to an increasing influx of protons and water into the endosomes. This process finally results in a breakdown of the vesicles and release of its content into the cytoplasm. The low amine density in the synthesized PVA results in insufficient proton buffering. As a result, polyplexes are not released from the endosomes, resulting in very poor transfection. This result is not unexpected and in line with transfection efficiencies observed with other polycations such as poly-L-lysine (59). Further modifications of the PVA backbone using PLGA grafting have led to surprisingly efficient gene delivery systems reported elsewhere (60). In conclusion, this study underscores the necessity to establish more detailed structural-activity relationships between polycations as demonstrated here for the aminemodified PVA structure and DNA morphology. Such a relationship was highlighted using PCS, AFM, and zetapotential. Our transfection data demonstrate that the rate-limiting step for gene expression seems to be associated with endosomal escape of PVA-DNA polyplexes. The addition of chloroquine appears to circumvent this process and achieves superior luciferase expression. The results of this study clarifies that an effective transfection device needs more functionality than simply positive charges. It must also be able to leave the endosomes
Wittmar et al.
without addition of further substances. As demonstrated by Oster et al., the grafting of these PVA will lead to transfection efficiencies even higher than with PEI/DNA complexes (60). ACKNOWLEDGMENT
We gratefully acknowledge financial support of this work by the Deutsche Forschungsgemeinschaft (DFG). We thank Wyatt Technology Europe GmbH and Dr. A. Theisen. James Ellis would like to thank the BBSRC for funding and Professor X. Chen for his help and advice. Supporting Information Available: NMR data of amine-carbonylimidazole; CHN analysis and NMR data of amine-modified poly(vinyl alcohol); figure of FT-IR and 1 H-NMR of amine-modified PVA assignment. This material is available free of charge via the Internet at http:// pubs.acs.org. LITERATURE CITED (1) Lengsfeld, C. S., Manning, M. C., and Randolph, T. W. (2002) Encapsulating DNA within biodegradable polymeric microparticles. Curr. Pharm. Biotechnol. 3, 227-235. (2) Gulati, M., Grover, M., Singh, S., and Singh, M. (1998) Lipophilic drug derivatives in liposomes. Int. J. Pharm. 165, 129-168. (3) Drin, G., Rousselle, C., Scherrmann, J.-M., Rees, A. R., and Temsamani, J. (2002) Peptide delivery to the brain via adsorptive-mediated endocytosis: advances with SynB vectors. AAPS Pharm. Sci. 4, 26. (4) Evans, R. K., Zheng Xu, Bohannon, K. E., Wang, B., Bruner, M. W., and Volkin, D. B. (2000) Evaluation of degradation pathways for plasmid DNA in pharmaceutical formulations via accelerated stability studies. J. Pharm. Sci. 89, 76-87. (5) Uchida, T., Yagi, A., Oda, Y., Nakada, Y., and Goto, S. (1996) Instability of bovine insulin in poly(lactide-co-glycolide) (PLGA) microspheres. Chem. Pharm. Bull. (Tokyo) 44, 235-236. (6) Pillai, O., and Panchagnula, R. (2001) Insulin therapies past, present and future. Drug Discovery Today 6, 10561061. (7) van de Weert, M., Hennink, W. E., and Jiskoot, W. (2000) Protein instability in poly(lactic-co-glycolic acid) microparticles. Pharm. Res. 17, 1159-1167. (8) Fasano, A. (1998) Innovative strategies for the oral delivery of drugs and peptides. Trends Biotechnol. 16, 152-157. (9) Akiyoshi, K., Kobayashi, S., Shichibe, S., Mix, D., Baudys, M., Kim, S. W., and Sunamoto, J. (1998) Self-assembled hydrogel nanoparticle of cholesterol-bearing pullulan as a carrier of protein drugs: Complexation and stabilization of insulin. J. Controlled Release 54, 313-320. (10) Bernkop-Schnurch, A., Scholler, S., and Biebel, R. G. (2000) Development of controlled drug release systems based on thiolated polymers. J. Controlled Release 66, 39-48. (11) Brazel, C. S., and Peppas, N. A. (2000) Modeling of drug release from swellable polymers. Eur. J. Pharm. Biopharm. 49, 47-58. (12) Breitenbach, A., Li, Y. X., and Kissel, T. (2000) Branched biodegradable polyesters for parenteral drug delivery systems. J. Controlled Release 64, 167-178. (13) Fischer, D., Bieber, T., Li, Y., Elsasser, H. P., and Kissel, T. (1999) A novel non-viral vector for DNA delivery based on low molecular weight, branched polyethylenimine: effect of molecular weight on transfection efficiency and cytotoxicity. Pharm. Res. 16, 1273-1279. (14) Higaki, M., Azechi, Y., Takase, T., Igarashi, R., Nagahara, S., Sano, A., Fujioka, K., Nakagawa, N., Aizawa, C., and Mizushima, Y. (2001) Collagen minipellet as a controlled release delivery system for tetanus and diphtheria toxoid. Vaccine 19, 3091-3096. (15) Jones, D. H., Corris, S., McDonald, S., Clegg, J. C., and Farrar, G. H. (1997) Poly(DL-lactide-co-glycolide)-encapsulated
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