Successive outermost-to-core shell directionality of the protonation of poly(propyl ether imine) dendritic gene delivery vectors

Abirami Lakshminarayanan, Narayanaswamy Jayaraman
2017 Canadian journal of chemistry (Print)  
The protonation behaviour of polycationic compounds have direct relevance to their 1 ability to condense and deliver nucleic acids. This report pertains to a study of the protonation 2 behaviour of polycationic poly(propyl ether imine) (PETIM) dendritic gene delivery vectors, that 3 are constituted with tertiary amine core moiety and branch sites, n-propyl ether linkages and 4 primary amine peripheries. The ability of this series of dendrimers to condense nucleic acids and 5 mediate endosomal
more » ... mediate endosomal escape was studied by unravelling the protonation behaviour of the 6 dendrimers aided by pH-metric titrations, 1 H and 15 N NMR spectroscopies. The results 7 demonstrate protonation of the primary and tertiary amines of outermost-to-core shells occurring 8 in a successive step-wise fashion, in contrast to other polycationic vectors. Theoretical 9 calculations based on the Ising model rationalize further the finer details of protonation at each 10 shell. The protonation pattern correlates with the endosomal buffering and nucleic acid 11 condensation properties of this PETIM based dendritic gene delivery vectors. The study 12 establishes that the protonation behaviour is a critical and essential parameter in order to assess 13 the gene condensation and delivery vector properties of a polycationic compound. Synthetic vectors are important tools in order to achieve successful gene delivery, by 2 overcoming various physiological barriers. 1 Nucleic acid condensation by a vector and 3 subsequent endosomal escape of the complex are tunable through site-specific modifications of 4 the synthetic vector. Few examples are the cyclodextrins, 2 triazines, 3 histidine, imidazole, 4 poly-5 lysine 5 and cations 6 that facilitate increased nucleic acid condensation and endosomal escape. 6 Determining the effects of chemical modifications of a synthetic vector is optimal and provides 7 invaluable information about the gene transfection properties of a synthetic vector. Identifying 8 the protonation behavior of a cationic vector is important in this context. Whereas theoretical 9 studies based on molecular dynamic simulations have highlighted the importance of protonation 10 behavior, 7,8 only few reports concerning the development of synthetic polycationic vectors for 11 gene delivery consider this important aspect. Earlier studies established the protonation of 12 polycationic poly(amidoamine) (PAMAM) and poly(propylene imine) (PPI) dendrimers, 9,10 as 13 well as, linear and branched polyethylene imine (PEI), 9 chitosan, 11 poly(acrylic acid) (PAA) and 14 poly(methylacrylic acid) (PMAA) polymers. 12 The studies demonstrate that protonation of 15 cationic sites influence polymer properties, such as, protein aggregation, 13 host-guest 16 interactions, gene delivery, 10,12 catalysis, 14 molecular sensing and controlled drug release. 15,16 17 The PETIM series of dendrimers is characterized by tertiary amine branch sites, ether linkages, 18 n-propyl spacer and primary amine at their peripheries. 17,18 Systematic molecular dynamics 19 simulation studies of the structure of PETIM dendrimers illustrated a flexible structure, due to 20 the presence of n-propyl spacers with ether linkages. 19,20 Furthermore, PETIM dendrimers 21 exhibit profound gene vector abilities 21-23 and hence, a study of the protonation behavior of this 22 series of dendrimers is important. An assessment of the protonation pattern in PETIM 23 Page 3 of 28 Canadian Journal of Chemistry 12 pH titrations. The pH titration of the dendrimer solutions with aq. HCl was carried out using a 13 glass electrode, Ag/AgCl reference electrode with a salt bridge. Prior to the titration 14 experiments, the instrument was calibrated using standard buffer solutions (pH 4.008, 50.0 mM 15 K 2 HPO 4 ; pH 10.012, 25.0 mM Na 2 CO 3 plus 25.0 mM NaHCO 3 ). All the titrations were carried 16 out at 25 ± 0.2 o C. Aq. dendrimer solutions at a final concentration of 16 mM, in the presence of 17 150 mM NaCl, were prepared and the initial pH of the solution was recorded. The solutions were 18 then titrated with aq. HCl solution (10 µL) and pH was recorded after 60 s equilibration of the 19 solutions. The titration was continued until pH 3. The readings obtained were analysed using 20 the software Origin 8. All the titrations were carried out in triplicates and back titrations were 21 performed, in order to validate the reproducibility of the pH measurements. 22 Page 4 of 28 Canadian Journal of Chemistry D 2 O (450 µL) and 1 H NMR spectra were recorded at 400 MHz with 1s delay and accumulation 2 of 4 scans per sample. The dendrimer solutions were then titrated DCl (10 N), such that the 3 incremental acid concentration was 0.03 moles per titration. The solutions were homogenized 4 and equilibrated for 60 s prior to recording of 1 H NMR spectra. 15 N NMR spectra were recorded 5 at 40.5 MHz using a 45 s delay time. Due to the low abundance of 15 N nuclei (<0.3%), 10000 6 scans for G3(NH 2 ) 24 and 3000 scans for G1(NH 2 ) 6 were accumulated for each sample. CH 3 NO 2 7 was used as an external reference for calibration of the 15 N NMR spectra, which was inserted as 8 a sealed sample in the NMR tube. Chemical shift of the nitrogen atom of CH 3 NO 2 (δ = 380.23 9 ppm) was used to calibrate the spectra. 24 10 11 Results 12 pH Titrations. PETIM dendrimers, constituted with primary amine functionalities at the 13 peripheries, were synthesized by two iterative Michael addition reactions and two iterative 14 functional group reductions, as described in Scheme 1. 22 A pH titration of the amine 15 functionalized dendrimers G0(NH 2 ) 3 , G1(NH 2 ) 6 , G2(NH 2 ) 12 and G3(NH 2 ) 24 (Figure 1a ) was 16 undertaken to determine the pH required for complete protonation of the dendrimer amines. 17 Aqueous solutions of the dendrimer(16 mM) in aq. NaCl (150 mM) were titrated with aq. HCl 18 (0.3 M) and the pH of the solution was measured after 60 s equilibration (Figure 1b) . A first 19 order analysis of the titration curves indicated the equivalence points to be 6.2, 6.0, 6.5 and 5.8 20 for dendrimers of generations 0, 1, 2, and 3, respectively. The observed changes in pH of the 21 dendrimer solution, are reversed fully by performing a back-titration with aq. NaOH (0.3 M) and 22 Page 5 of 28
doi:10.1139/cjc-2017-0153 fatcat:niauov6mkjd6fmkws6dyemsis4