Poly(β-amino ester)–DNA complexes: Time-resolved fluorescence and cellular transfection studies

Abstract

A large number of different polymers have been developed and studied for application as DNA carriers for non-viral gene delivery, but the DNA binding properties are not understood. This study describes the efficiency of nanoparticle formation by time-resolved fluorescence measurements for poly(β-amino esters), cationic biodegradable polymers with DNA complexation and transfection capability. From the large library of poly(β-amino esters) ten polymers with different transfection efficacies were chosen for this study. The binding constants for nanoparticle formation were determined and compared to polyethylene imines with the same method. Although the DNA binding efficiency of the amine groups are similar for both types of polymers, the overall binding constants are an order of magnitude smaller for poly(β-amino esters) than for 25 kDa polyethylenimines, but yet poly(β-amino esters) show comparable DNA transfection efficacy with polyethyleneimines. Within this series of polymers the transfection efficacy showed increasing trend in association with relative efficiency of nanoparticle formation.

The study of polyplex formation and stability by time-resolved fluorescence spectroscopy of SYBR Green I-stained DNA

“…14,16 Unfortunately, possibly owing to the too low signal of EtBr, fluorescence measurements had to be performed at a very high DNA concentration (300 μM, corresponding to ≈0.1 µg µL −1 ) far above those generally used in vitro (on the order of 1-10 ng µL −1 of DNA or even lower), 10,18 and obviously in vivo, in gene delivery assays, inasmuch as certain suspensions were reported to be cloudy. 14,16 Unfortunately, possibly owing to the too low signal of EtBr, fluorescence measurements had to be performed at a very high DNA concentration (300 μM, corresponding to ≈0.1 µg µL −1 ) far above those generally used in vitro (on the order of 1-10 ng µL −1 of DNA or even lower), 10,18 and obviously in vivo, in gene delivery assays, inasmuch as certain suspensions were reported to be cloudy.…”

“…Free SYBR Green I displayed an extremely low signal and a short temporal decay, below the temporal resolution of the system (≈200 ps). This choice was motivated by the experimental evidence that unlike EtBr that showed temporal and spectral vari- ations following DNA condensation by polymers, 14 no change occurred in the emission spectrum of SYBR Green I upon variation of the N/P (not shown). 19 Instead, no significant increase of the fluorescence signal was detected upon mixing SYBR Green I with each polymer investigated (data not shown).…”

“…[14][15][16][17] Time-resolved techniques indeed allow disentangling the fluorescence amplitude and lifetime, thus providing additional information with respect to popular steady-state analyses. [14][15][16][17] Time-resolved techniques indeed allow disentangling the fluorescence amplitude and lifetime, thus providing additional information with respect to popular steady-state analyses.…”

Enhancing the In Vitro and In Vivo Stabilities of Polymeric Nucleic Acid Delivery Nanosystems

“…44–46 Every polymer, with its unique features, has a different optimal N/P ratio, and these ratios may vary by several orders of magnitude. 47 Typically, a polymer with a higher charge density and molecular weight tends to be more stable and requires a lower N/P ratio for efficient gene delivery.…”

Synthesis and Characterization of Homopolymers Bearing Acid‐Cleavable Cationic Side‐Chains for pH‐Modulated Release of DNA

“…Generally, major categories of gene delivery vectors include viral vectors and nonviral vectors . Compared to viral vectors, nonviral vectors based on cationic polymers offer many potential advantages, such as low immunogenicity, large loading capacity of DNA of any size, better protection of DNA, flexibility of design, and easy large‐scale production . Typical cationic polymers for gene delivery are polyethylenimine (PEI), poly[2‐(dimethylamino)ethyl methacrylate] (PDMAEMA), poly(β‐amino ester)s, PAMAM dendrimers, and so forth .…”