Neomycin-based DNA nanocarriers as gene delivery and antimicrobial agents: synthesis, characterization and validation

Introduction Since the first attempts to delivery exogenous DNA to cells by means of cationic polymers1 and lipids2, gene delivery vectors have made significant strides forward. However, although non-viral gene delivery strategies are emerging as a valid alternative to conventional therapeutic approaches, their clinical application is still hampered by their poor efficacy. In this light, the development of multifunctional vectors specifically decorated with functional moieties holds promises for improving the gene delivery behavior of the resulting DNA-containing particles. We herein propose the design, synthesis and characterization of an array of novel transfectants built on (lipid) tetramino-tetrahexyloxycalix[4]arenes (4A4Hex-calix[4]) scaffolds and generation 2 polyamidoamine (PAMAM) dendrimers, tethered with multivalent aminoglycoside Neomycin moieties (Neo, a naturally occurring antibiotic especially effective against Gram-negative bacteria3), as promising gene delivery tools with inherent antibacterial properties. Experimental Methods Starting from 4A4Hex-calix[4] scaffolds and PAMAM, AGs-based conjugates were synthetized using isothiocyanate/amine click-chemistry reactions (Fig. 1A). 4A4Hex-calix[4]-Neo and PAMAM-Neo were then diluted in deionized water, and complexed with plasmid DNAs (encoding the firefly modified luciferase, pGL3) at different transfectant nitrogen-to-nucleic acid phosphate (N/P) ratios to give rise to nanoparticles called lipoplexes and polyplexes, respectively. The physico-chemical properties of such assemblies (size and surface charge) were evaluated by means of Dynamic Light Scattering (DLS), while their DNA complexation ability was assessed by fluorophore titration assay. In vitro cell transfections were performed on HeLa, U87-MG, COS7 cell lines. Briefly, 2 x 104 cells/cm2 were challenged with complexes (DNA dose: 0.16 µg/cm2) for up to 48 hrs, then cytotoxicity was evaluated by AlamarBlue® assay and transfection efficiency by Luciferase Assay System. The antimicrobial activity of 4A4Hex-calix[4]-Neo and PAMAM-Neo, used either as aqueous solutions and in the form of suspension of DNA-containing nanoparticles, was evaluated against Gram negative Escherichia coli (E. coli) JM109 bacteria. Briefly, bacteria were inoculated (1.5 x 105 cells/cm2) with transfectant solutions or complexes suspension for 24 hrs, then the antibacterial efficacy of every compound was evaluated by means of the OD600nm measurements4. Results and Discussion Herein, we reported the development of new classes of AG-based transfectants built on the 4A4Hex-calix[4] scaffold5 and PAMAM6. AG-based transfectants exhibited greater DNA packing ability than the gold standard transfectant 25 kDa bPEI1 (maximal complexation at N/P≥1.5 vs. N/P 3) due to the inherent multivalency of Neo, which displays hexavalent binding sites for anions (ie., DNA). DNA complexation with 4A4Hex-calix[4]-Neo and PAMAM-Neo led to the formation of nanocomplexes (150–300 nm in size), with a slightly positive surface charge (+20–35 mV), that is in the range of good colloidal stability. DNA-containing nanoparticles, everyone tested at the optimal N/P, invariably showed better transfection efficiency than 25 kDa bPEI, along with negligible cytotoxicity in HeLa, U87-MG and COS-7 cells (Fig. 1B). Interestingly, PAMAM−Neo complexes at any N/P tested displayed a ≈ 10-fold increase in the Luc expression than the pristine PAMAM. Besides, due to the grafting of Neo, 4A4Hex-calix[4]-Neo and PAMAM-Neo derivatives exhibited remarkable antimicrobial activity. At optimal N/P, 4A4Hex-calix[4]-Neo-based lipoplexes displayed an antibacterial efficiency of ≈100%, whereas PAMAM-Neo-based polyplexes inhibited the bacterial growth of ≈70%. Notably, the antibacterial efficiencies of 4A4Hex-calix[4]-Neo and PAMAM-Neo complexed with pDNA were even greater than the same derivatives in solution, but were slightly less active than the corresponding free AGs. Based on these findings, we can speculate that the antimicrobial effect of our derivatives specifically rely on the grafting of the pristine antibiotic moiety on the 4A4Hex-calix[4] and PAMAM. Conclusion Altogether, these findings highlight the potential of Neo-based derivatives as efficient multifunctional carries capable of delivering nucleic acids and blunting Gram-negative bacterial infections at once.