Limiting the level of tertiary amines on polyamines leads to biocompatible nucleic acid vectors.

We have designed an efficient, synthetic nucleic acid vector, which is relatively non-toxic. [N-(2-ethylamino)-6-O-glycolchitosan - EAGC] polymers were 10-50 fold less toxic than Lipofectamine 2000, able to complex DNA, mRNA and siRNA into positively charged (zeta potential=+40 - 50mV), 50-450nm nanoparticles. The level of tertiary amine N-2-ethylamino substitution (DStert) was inversely proportional to the IC50 of the EAGC polymers in the A431 cell line: IC50=6.18DStert-0.9, r2=0.9991. EAGC polyplexes were stable against a heparin challenge, able to protect the nucleic acids from nuclease degradation and achieve levels of transfection comparable to Lipofectamine 2000 formulations. The relative biocompatibility of the vector allowed 10 fold higher doses of DNA (1µg compared to 0.1µg per well with Lipofectamine 2000) and siRNA (10.7µg per well vs 1.3µg with Lipofectamine 2000) to be applied to cells, when compared to Lipofectamine 2000. Finally intranasal application of EAGC - siRNA complexes resulted in siRNA transfer to the neurons of the olfactory bulb.

Physical characterisation and long-term stability studies on quaternary ammonium palmitoyl glycol chitosan (GCPQ)--a new drug delivery polymer.

N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan (GCPQ) is a self-assembling polymer, which enables the oral bioavailability of peptide and hydrophobic drugs. In preparation for clinical testing, here we examine GCPQ's synthesis reproducibility, pKa, thermal, and rheological properties. GCPQ was synthesised by acid degradation of glycol chitosan (GC), reaction with palmitic acid N-hydroxysuccinimide (PNS) and methylation. A GC monomer, PNS molar feed ratio of 0.92 together with a gravimetric feed ratio for N-palmitoyl-6-O-glycolchitosan, methyl iodide of 3.3, reproducibly produces GCPQ48 (Mw = 19.9 ± 9.9 kDa, Mn = 13.1 ± 2.4 kDa, mol % palmitoylation = 23 ± 2.7, mol % quaternisation = 10 ± 0.23, n = 56). GCPQ48 decomposes at 218 ± 4.3 °C, is glassy at room temperature (Tg = 164.4 ± 8.5 °C), is a weak base (pKa = 5.99 ± 0.15), and produces micellar dispersions at neutral pH. Below a concentration of 0.07 g mL(-1) , GCPQ48 dispersions showed Newtonian rheological behaviour but at higher concentrations, the polymer undergoes shear thinning because of the chain disentanglement at high shear rates. GCPQ48 forms a network of micelles and concentrated (0.09 g mL(-1) ) dispersions are viscoelastic, with the storage modulus exceeding the loss modulus at high frequencies. Solid GCPQ48 was stable when stored at room temperature for 18 months.