Role of Generation on Successful DNA Delivery of PAMAM–(Guanidino)Neomycin Conjugates

Abstract

The decoration of polyamidoamine (PAMAM) dendrimers with some functional moieties holds promises for improving the gene-delivery behavior of the resulting polyplexes. Herein, we reported the synthesis and the development of nonviral gene vectors built on an array of PAMAMs, spanning generation 2 (G2), G4, and G7, specifically tethered with multivalent neomycin (Neo) or guanidinoneomycin (GNeo). Following a thorough chemical characterization by means of NMR and MALDI analyses, the ability of PAMAM−(G)Neo conjugates to serve as transfectants was investigated. First, we demonstrated that every PAMAM, whether or not tethered with Neo (or GNeo), exhibited greater DNA packing ability than the gold standard transfectant 25 kDa bPEI. Measurements of the colloidal properties showed that PAMAMs, when mixed with plasmid DNA (pDNA) as a function of the nitrogen-to-phosphate ratio (N/P), allowed preparing complexes of ≈150−300 nm in diameter with a positive surface charge of ≈+20−35 mV, strictly dependent on the PAMAM generation. The conjugation with Neo affected the DNA release behavior from PAMAM-based polyplexes and their ultimate transfection efficiency. When low generation PAMAM G2 was tethered with Neo, we obtained the most efficient transfectants, with remarkable antibacterial activity against Gram negative bacteria. It is worth noting that pDNA/PAMAM−(G)Neo nanoassemblies, which were tested at the optimal N/P, invariably showed better transfection efficiency than 25 kDa bPEI, along with low cytotoxicity in both HeLa and COS-7 cells. Altogether, these results underline the potential of such PAMAM−(G)Neo dendrimers as promising vehicles for efficient gene delivery into cells.

Non-Viral in Vitro Gene Delivery: It is Now Time to Set the Bar!

“…Some authors have indeed reported that the transfection efficiency of bPEI in vitro increased with increasing the M W of the CP (in the range from ≈1.8 to ≈70 kDa) [62,114], while others have highlighted that the lower the M W of the branched polymer (≈12 kDa), the greater the effectiveness [115]. Further, we and some others have pointed out the different pDNA complexation ability of lPEI and bPEI and have found that, irrespective of the M W of the CP taken into account, lPEIs complex best at N/P 5, while bPEIs at N/P 3 [57,62,64,116,117]. This different behavior may be ascribed to the different structures of the PEI isomers: of note, lPEI possesses only secondary amines, while bPEI has primary, secondary, and tertiary amines depending on the polymerization degree [118].…”

“…Because they are relatively safe, display easily tunable physico-chemical properties, can be produced in large quantities with high reproducibility and affordable costs, and show unlimited ferrying capacity [53][54][55], non-viral vectors are nowadays at the forefront of gene delivery [1]. The two major approaches to non-viral gene delivery involve the combination of NAs with transfection molecules, that is, cationic lipids (CLs; e.g., DOTAP, DOTMA) [56][57][58][59][60][61] in different forms, and cationic polymers (CPs; e.g., poly-L-lysines, polyethyleneimines (PEIs), poly(amidoamine)s, chitosans) [62][63][64][65][66][67]. The beauty of this approach lies in its simplicity, yet in its effectiveness.…”

“…Besides, they can display different chemistries and architectures [93]. CPs include commercially sourced materials ( Figure 2), such as poly-L-lysines (PLLs) [94,95], poly(ethyleneimines) (PEIs) [96,97], poly(amidoamines) (PAMAMs) [64,[98][99][100][101], poly [2-(dimethyamino)ethyl methacrylates] (PDMAEMs) [102], and chitosans (CSs) [103][104][105][106], used as received or reacted and functionalized [63,[107][108][109][110], and those purposely synthesized [111]. Of note, the efficacy of a transfectant strongly depends on some chemical and geometrical features.…”

“…Yet, the way polyplexes are added to cells may also dramatically affect the transfection efficiency. By and large, the delivery of complexes to cells is obtained through the addition of the transfection suspension to the cells cultured in the culture medium [62,96], or the culture vessel in which the cells were plated is emptied, polyplexes are first pre-diluted in the culture medium and then added to the cells [57,64]. As a general observation, lPEI-polyplexes prepared in 10 mM Hepes have smaller D H and are more effective when directly added to the transfection well, while bigger particles (e.g., those prepared in 150 mM NaCl) benefit from the pre-dilution step in cell culture medium before they are incubated with cells (Figure 6b).…”

Aminoglycosides: From Antibiotics to Building Blocks for the Synthesis and Development of Gene Delivery Vehicles

“…In particular, PAMAM G4-Paro 57 resulted in being more performant than bPEI, even in the presence of 10% and 50% in volume of fetal bovine serum (FBS). Afterward, we decided to also investigate the role of the PAMAM generation [ 73 ]. Accordingly, with the same synthetic strategy and a slightly different linker, we prepared four new conjugates starting from PAMAM dendrimers of different generations, namely PAMAM G2-Neo 59 , PAMAM G4-Neo 60 , and PAMAM G7-Neo 62 , bearing 13, 40, and 307 neomycin moieties, respectively ( Figure 17 b) [ 73 ].…”