Synthesis of Cholesteryl Polyamine Carbamates:  pK_a Studies and Condensation of Calf Thymus DNA

Abstract

Novel polyamine carbamates have been designed and prepared from cholesterol. Our synthesis uses an orthogonal protection strategy based upon trifluoroacetyl and Boc-protecting groups. These unsymmetrical polyamine carbamates have been prepared from symmetrical (e.g., spermine and thermine) polyamines. Detailed interpretations of (1)H and (13)C NMR spectroscopic data led to the unambiguous assignment of these polyamine carbamates. These target conjugates contain a variety of positive charges distributed along methylene chains. Their pK(a)s have been determined potentiometrically for conjugates substituted with up to five amino functional groups. Condensation of calf thymus DNA into particles was monitored using light scattering at 320 nm. Salt-dependent binding affinity for calf thymus DNA was determined using an ethidium bromide fluorescence quenching assay. These cholesteryl polyamine carbamates are models for lipoplex formation with respect to gene delivery (lipofection), a key first step in gene therapy.

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

“…7) (66). This result is in agreement with other physical studies on DNA particle size, when completely condensed at the nanoscale level (35,36).…”

“…The HEPES buffer was filtered through a 0.45-mm membrane before use. N 4 ,N 9 -Dioleoylspermine and N 1 -cholesteryl spermine carbamate were synthesized as we have previously reported (35,36).…”

“…Both are designed to incorporate a spermine backbone conjugated with a lipophilic moiety, i.e., the oleoyl groups [two amide links; Fig. 5 (36). Both our novel DNA vectors show effective condensation (i.e., yielding 90% fluorescence reduction in the EthBr fluorometric assay at N/P charge ratios as low as 1.5Y2.0) and high transfection efficiency.…”

Cationic corticosteroid for nonviral gene delivery

“…The mass ratio of 1:2 of DS:DOPE (yellow bars, Figure 2a) provided a high EGFP expression, while minimizing the use of the DS conjugate and minimizing the total lipid load below 10 mg total lipid/mg DNA. In a separate set of experiments maintaining the DS:DOPE mass ratio constant at 1:2, the amount of DS relative to DNA was systematically varied from 1 to 10 charge equivalents (0.9 mg DS/mg DNA), assuming an average charge of the spermine of DS to be 3.8/molecule, 11 and no net charge contribution from DOPE. The optimal lipofection plateaued at a charge ratio of 6 DS:1 DNA, giving a more than 10-fold increase in the amount of transgene expression relative to Lipofectamine reagent.…”

“…Bovine aortic endothelial cells (BAECs, passages [4][5][6][7][8][9][10][11][12][13] were passed at a 1:3 split to 24-well culture plates, and then grown to dense confluence before lipofection. The growth medium was Dulbeccos's modified Eagle's medium containing 10% heat-inactivated charcoal-filtered (to remove steroid hormones) fetal calf serum (Hyclone), 0.30 mg/ml glutamine, 150 U/ml penicillin, and 0.15 mg/ml streptomycin (Gibco).…”