Abstract
Purpose
To investigate the moisture-induced aggregation (i.e., a loss of solubility in water) of DNA in a solid state and to develop rational strategies for its prevention.
Methods
Lyophilized calf thymus DNA was exposed to relative humidity (RH) levels from 11% to 96% at 55°C. Following a 24-h incubation under these stressed conditions, the solubility of DNA in different aqueous solutions and the water uptake of DNA were determined. The effects of solution pH and NaCl concentration and the presence of excipients (dextran and sucrose) on the subsequent moisture-induced aggregation of DNA were examined. The extent of this aggregation was compared with that of a supercoiled plasmid DNA.
Results
Upon a 24-h incubation at 55°C, calf thymus DNA underwent a major moisture-induced aggregation reaching a maximum at a 60% RH; in contrast, the single-stranded DNA exhibited the maximal aggregation at a 96% RH. Moisture uptake and aqueous solubility studies revealed that the aggregation was primarily due to formation of inter-strand hydrogen bonds. Aggregation of DNA also proceeded at 37°C, albeit at a slower rate. Solution pH and NaCl concentration affected DNA aggregation only at higher RH levels. This aggregation was markedly reduced by co-lyophilization with dextran or sucrose (but not with PEG). The aggregation pattern of a supercoiled plasmid DNA was similar to that of its linear calf thymus counterpart.
Conclusions
The moisture-induced aggregation of lyophilized DNA is caused mainly by non-covalent cross-links between disordered, single-stranded regions of DNA. At high RH levels, renaturation and aggregation of DNA compete with each other. The aggregation is minimized at low RH levels, at optimal solution pH and salt concentration prior to lyophilization, and by co-lyophilizing with excipients capable of forming multiple hydrogen bonds, e.g., dextran and sucrose.
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References
http://www.wiley.co.uk/genetherapy/clinical/ (accessed 03/15/06).
R. C. Mulligan. The basic science of gene therapy. Sci. 260:926–932 (1993).
L. Wang, K. Takabe, S. M. Bidlingmaier, R. Charles, and I. M. Verma. Sustained correction of bleeding disorder in hemophilia B mice by gene therapy. Proc. Natl. Acad. Sci. USA 96:3906–3910 (1999).
F. McCormick. Cancer gene therapy: fringe or cutting edge? Nature Revs. 1:130–141 (2001).
D. R. Shaw and T. V. Strong. DNA vaccines for cancer. Front. Biosci. 11:1189–1198 (2006).
A. M. Abdelnoor. Plasmid DNA vaccines. Current Drug Targets: Immune Endocrine and Metabolic Disorders 1:79–92 (2001).
J. Schultz, G. Dollenmaier, and K. Molling. Update on antiviral DNA vaccine research (1998–2000). Intervirology 43:197–217 (2001).
C. R. Middaugh, R. K. Evans, D. L. Montgomery, and D. R. Casimiro. Analysis of plasmid DNA from a pharmaceutical perspective. J. Pharm. Sci. 87:130–146 (1998).
R. Thomas. The denaturation of DNA. Gene 135:77–79 (1993).
C. S. Lengsfeld, and T. J. Anchordoquy. Shear-induced degradation of plasmid DNA. J. Pharm. Sci. 91:1581–1589 (2002).
R. Liu, R. Langer, and A. M. Klibanov. Moisture-induced aggregation of lyophilized proteins in the solid state. Biotechnol. Bioeng. 37:177–184 (1991).
H. R. Costantino, R. Langer, and A. M. Klibanov. Moisture-induced aggregation of lyophilized insulin. Pharm. Res. 11:21–29 (1994).
A. M. Klibanov and J. A. Schefiliti. On the relationship between conformation and stability in solid pharmaceutical protein formulations. Biotechnol. Lett. 26:1103–1106 (2004).
M. Falk, K. A. Hartman, Jr., and R. C. Lord. Hydration of deoxyribonucleic acid. III. A spectroscopic study of the effect of hydration on the structure of deoxyribonucleic acid. J. Am. Chem. Soc. 85:391–394 (1963).
S. L. Lee, P. G. Debenedetti, J. R. Errington, B. A. Pethica, and D. J. Moore. A calorimetric and spectroscopic study of DNA at low hydration. J. Phys. Chem. B. 108:3098–3106 (2004).
M. H. Zehfus and W. C. Johnson, Jr. Conformation of P-form DNA. Biopolymers 23:1269–1281 (1984).
N. Lavalle, S. A. Lee, and A. Rupprecht. Counterion effects on the physical properties and the A to B transition of calf thymus DNA films. Biopolymers 30:877–887 (1990).
S. W. Poxon and J. A. Hughes. The effect of lyophilization on plasmid DNA activity. Pharm. Dev. Technol. 5:115–122 (2000).
I. A. Novikov and B. I. Sukhorukov. On the role of water in the thermal instability of DNA. Molekulyarnaya Biologiya 11:521–530 (1977).
J. F. Young. Humidity control in the laboratory using salt solutions — a review. J. Appl. Chem. 17:241–245 (1967).
D. Voet and J. G. Voet. Biochemistry Wiley, New York, 1995, pp. 848–914.
O. L. Vaveliouk, G. I. Tseretely, and T. V. Belopolskaya. Thermal stability of DNA and its association with the process of vitrification. Tsitologiya 41:958–965 (1999).
M. Bastos, V. Castro, G. Mrevlishvili, and J. Teixeira. Hydration of ds-DNA and ss-DNA by neutron quasielastic scattering. Biophys. J. 86:3822–3827 (2004).
M. A. Semenov and T. V. Bol’bukh. Study of conformational-dependent isotherms of DNA hydration. Biofizika 29:377–382 (1984).
J. Hong, M. W. Capp, C. F. Anderson, R. M. Saecker, D. J. Felitsky, M. W. Anderson, and M. T. Record, Jr. Preferential intercations of glycine betaine and of urea with DNA: Implications for DNA hydration and for effects of these solutes on DNA stability. Biochemistry 43:14744–14758 (2004).
G. K. Helmkamp and P. O. P. Ts’o. The secondary structures of nucleic acids in organic solvents. J. Am. Chem. Soc. 83:138–142 (1960).
N. B. Bam, J. L. Cleland, J. Yang, M. C. Manning, J. F. Carpenter, R. F. Kelly, and T. W. Randolph. Tween protects recombinant human growth hormone against agitation-induced damage via hydrophobic interactions. J. Pharm. Sci. 87:1554–1559 (1998).
H. Tabor. The protective effect of spermine and other polyamines against heat denaturation of deoxyribonucleic acid. Biochemistry 3:496–501 (1962).
R. Mandel and G. D. Fasman. Chromatin models. Interaction between DNA and polypeptides containing L-lysine and L-valine: circular dichroism and thermal denaturation studies. Biochemistry 15:3122–3130 (1976).
R. K. Agarwal and A. Peri. PCR amplification of highly GC-rich DNA template after denaturation by NaOH. Nucleic Acids Res. 21:5283–5284 (1993).
A. J. E. Colvill and D. O. Jordan. Influence of ionic strength on the reversibility of the denaturation of DNA in dilute solution. J. Mol. Biol. 7:700–709 (1963).
T. O’Connor, S. Mansy, M. Bina, D. R. McMilin, M. A. Bruck, and R. S. Tobias. The pH-dependent structure of calf thymus DNA studied by Raman spectroscopy. Biophys. Chem. 15:53–64 (1982).
P. D. Lawley. Interaction studies with deoxyribonucleic acid. III. Effect of changes in sodium-ion concentration, pH, and temperature on the ultraviolet absorption spectrum of sodium thymonucleate. Biochim. Biophys. Acta. 21:481–488 (1958).
Y. Mi and G. Wood. The application and mechanisms of polyethylene glycol 8000 on stabilizing lactate dehydrogenase during lyophilization. PDA J. Pharm. Sci. Technol. 58:191–202 (2004).
T. J. Anchordoquy, K.-I. Izutsu, T. W. Randolph, and J. F. Carpenter. Maintenance of quaternary structure in the frozen state stabilizes lactate dehydrogenase during freeze-drying. Arch. Biochem. Biophys. 390:35–41 (2001).
L. Kreilgaard, S. Frokjaer, J. M. Flink, T. W. Randolph, and J. F. Carpenter. Effects of additives on the stability of recombinant human factor VIII during freeze-drying and storage in the dried solid. Arch. Biochem. Biophys. 360:121–134 (1998).
T. J. Anchordoquy, T. K. Armstrong, and M. C. Molina. Low molecular weight dextrans stabilize nonviral vectors during lyophilization at low osmolalities: concentrating suspensions by rehydration to reduced volumes. J. Pharm. Sci. 94:1226–1236 (2005).
S. D. Allison and T. J. Anchordoquy. Mechanisms of protection of cationic lipid–DNA complexes during lyophilization. J. Pharm. Sci. 89:682–691 (2000).
J. Cherng, P. V. D. Wetering, H. Talsma, D. J. A. Crommelin, and W. E. Hennink. Freeze-drying of poly((2-dimethylamino)ethyl methacrylate)-based gene delivery systems. Pharm. Res. 14:1838–1841 (1997).
Acknowledgment
This work was financially supported by Grant GM26698 from the National Institutes of Health.
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Sharma, V.K., Klibanov, A.M. Moisture-Induced Aggregation of Lyophilized DNA and its Prevention. Pharm Res 24, 168–175 (2007). https://doi.org/10.1007/s11095-006-9138-7
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DOI: https://doi.org/10.1007/s11095-006-9138-7