Characterization of a cyclodextrinoligonucleotide complex by capillary electrophoresis using laser-induced fluorescence.
OBJECTIVE
Cyclodextrins (CDs) have been identified as a viable alternative to viral vectors for use in therapeutic applications. Here, the stability of the complex formed between the a multiply charged, cationic, fully substituted heptakis-(6-amino-2-galactosyl)cyclodextrin (BCDX12) with a multiply charged 12-mer hexachlorofluorescein tagged arabinopolynucleotide (Hex-PAH) have been evaluated.


METHODS
The stability of complexes of Hex-PAH and BCD-X12 was studied with respect to mole ratio (1:1, 1:2, and 1:5 Hex-PAH:BCD-X12), pH, buffer concentration, temperature, and agitation using capillary electrophoresis with laser induced fluorescence detection (CE/LIF). Two neutral CDs and an additional cationic CD were also tested under the same analytical conditions to determine their ability to form complexes.


RESULTS
Hex-PAH:BCDX12 complexes at mole ratios of 1:2 were stable in 10 mM (160 mM total borate concentration) sodium tetraborate buffer at pH 7.5 and at temperatures of 4 degrees C and 25 degrees C over 48 hours. However, the Hex-PAH:BCD-X12 complex was less stable at 37 degrees C and at higher buffer concentrations and pH values. Strong vortex mixing prior to analysis was found to disrupt the complex. Of the four CDs tested for their ability to complex with Hex-PAH, only BCDX12 formed stable complexes with Hex-PAH under the test conditions.


CONCLUSIONS
Capillary electrophoresis was found to be well suited to test the stability of cyclodextrin-nucleotide complexes. CE/LIF indicated that only a single Hex- PAH:BCD-X12 complex was formed at all formulation ratios, and that the complexes were electrophoretically identical to each other, and increasing the molar ratio beyond 1:2 did not contribute measurably to complex stability. Storage temperature and agitation conditions were found to influence complex stability. Since no stable complexes were formed with neutral cyclodextrins, the results support the hypothesis of a 'charge associated' complex rather than an inclusion complex, although inclusion complexes cannot be excluded on the basis of these studies.
Guthrie
Jeffrey W
JW
Department of Laboratory Medicine and Pathology, Faculty of Medicine and Dentistry and School of Public Health, University of Alberta, Edmonton, Alberta, Canada.
Ryu
Jang-Ha
JH
Le
X Chris
XC
Wiebe
Leonard I
LI
D001088:Arabinonucleotides / Q000737:chemistry*; D001881:Borates / Q000737:chemistry; D019075:Electrophoresis, Capillary; D005452:Fluoresceins / Q000737:chemistry; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D007834:Lasers; D009841:Oligonucleotides / Q000737:chemistry*; D013696:Temperature; D047392:beta-Cyclodextrins / Q000737:chemistry*
D001088:Arabinonucleotides; D001881:Borates; D005452:Fluoresceins; D009841:Oligonucleotides; D047392:beta-Cyclodextrins; C010634:sodium borate