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Lipopolyamine-Mediated Single Nanoparticle Formation of Calf Thymus DNA Analyzed by Fluorescence Correlation Spectroscopy

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Purpose

The aim of this study is to analyze linear calf thymus DNA (ct DNA) nanoparticle formation with N 4,N 9-dioleoylspermine and N 1-cholesteryl spermine carbamate.

Methods

Fluorescence correlation spectroscopy (FCS) was used to determine the quality of ct DNA condensed by lipopolyamines. ct DNA was prelabeled with PicoGreen (PG) to allow fluorescence intensity fluctuation measurement and analysis.

Results

N 4,N 9-Dioleoylspermine efficiently condensed ct DNA into point-like molecules with diffusion coefficient (D) = 1.8 × 10−12 m2/s and particle number (PN) = 0.7 [at ammonium/phosphate (N/P) charge ratio=1.0–1.5]. The determined PN values are close to the theoretical value of 0.6, providing evidence that the DNA conformation has been fully transformed, and thus a single nanoparticle has been detected. N 1-Cholesteryl spermine carbamate showed (slightly) poorer DNA condensation efficiency, even at higher N/P ratios (N/P = 1.5–2.5) with D = 1.3 × 10−12 m2/s and PN value of 5.2. N 4,N 9-Dioleoylspermine is a more efficient DNA-condensing agent than N 1-cholesteryl spermine carbamate.

Conclusions

FCS measurement using PG as the probe is a novel analytical method to detect single nanoparticles of condensed DNA in nonviral gene therapy formulation studies.

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Abbreviations

A :

correction factor

AF488:

Alexa Fluor-488

C :

molarity

CR:

count rate

ct DNA:

calf thymus DNA

D :

diffusion coefficient

DNase:

nuclease

dsDNA:

double-stranded DNA

D w :

diffusion coefficient in water

EthBr:

ethidium bromide

FCS:

fluorescence correlation spectroscopy

G(τ):

autocorrelation function

HEPES:

4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid

I :

fluorescence intensity signal

k B :

Boltzmann constant

M :

molecular mass

N/P:

ammonium/phosphate ratio

N A :

Avogadro's number

NPC:

nuclear pore complex

NVGT:

nonviral gene therapy

PG:

PicoGreen®

PN:

particle number

r h :

hydrodynamic radius

R6G:

Rhodamine-6G

SMS:

single-molecule spectroscopy

ssDNA:

single-stranded DNA

T :

triplet fraction

T c :

thermodynamic temperature

V :

confocal volume

η :

dynamic viscosity

τ :

correlation time

τ tr :

triplet decay time

τ D :

diffusion time

ω 1 :

lateral radii of detection volume

ω 2 :

axial radii of detection volume

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Acknowledgments

We thank the Universities UK for an ORS award (partial studentship support to N.A.), the Grant Agency of the Academy of the Sciences of the Czech Republic (grant A400400621 to T.K.), and the Grant Agency of the Czech Republic (GACR grant 203/05/2308 to M.H.).

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  1. Department of Pharmacy and Pharmacology, University of Bath, Bath, BA2 7AY, UK

    Noppadon Adjimatera & Ian S. Blagbrough

  2. J. Heyrovský Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, Dolejškova 3, 182 23, Prague 8, Czech Republic

    Teresa Kral & Martin Hof

  3. Department of Physics and Biophysics, Agricultural University, Norwida 25, 50-375, Wrocław, Poland

    Teresa Kral

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  1. Noppadon Adjimatera
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  2. Teresa Kral
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  3. Martin Hof
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  4. Ian S. Blagbrough
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Correspondence to Ian S. Blagbrough.

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Adjimatera, N., Kral, T., Hof, M. et al. Lipopolyamine-Mediated Single Nanoparticle Formation of Calf Thymus DNA Analyzed by Fluorescence Correlation Spectroscopy. Pharm Res 23, 1564–1573 (2006). https://doi.org/10.1007/s11095-006-0278-6

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  • DOI: https://doi.org/10.1007/s11095-006-0278-6

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