Structure and Dynamics of Thermosensitive pDNA Polyplexes Studied by Time-Resolved Fluorescence Spectroscopy

Abstract

Combining multiple stimuli-responsive functionalities into the polymer design is an attractive approach to improve nucleic acid delivery. However, more in-depth fundamental understanding how the multiple functionalities in the polymer structures are influencing polyplex formation and stability is essential for the rational development of such delivery systems. Therefore, in this study the structure and dynamics of thermosensitive polyplexes were investigated by tracking the behavior of labeled plasmid DNA (pDNA) and polymer with time-resolved fluorescence spectroscopy using fluorescence resonance energy transfer (FRET). The successful synthesis of a heterofunctional poly(ethylene glycol) (PEG) macroinitiator containing both an atom transfer radical polymerization (ATRP) and reversible addition-fragmentation chain-transfer (RAFT) initiator is reported. The use of this novel PEG macroinitiator allows for the controlled polymerization of cationic and thermosensitive linear triblock copolymers and labeling of the chain-end with a fluorescent dye by maleimide-thiol chemistry. The polymers consisted of a thermosensitive poly(N-isopropylacrylamide) (PNIPAM, N), hydrophilic PEG (P), and cationic poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA, D) block, further referred to as NPD. Polymer block D chain-ends were labeled with Cy3, while pDNA was labeled with FITC. The thermosensitive NPD polymers were used to prepare pDNA polyplexes, and the effect of the N/P charge ratio, temperature, and composition of the triblock copolymer on the polyplex properties were investigated, taking nonthermosensitive PD polymers as the control. FRET was observed both at 4 and 37 °C, indicating that the introduction of the thermosensitive PNIPAM block did not compromise the polyplex structure even above the polymer's cloud point. Furthermore, FRET results showed that the NPD- and PD-based polyplexes have a less dense core compared to polyplexes based on cationic homopolymers (such as PEI) as reported before. The polyplexes showed to have a dynamic character meaning that the polymer chains can exchange between the polyplex core and shell. Mobility of the polymers allow their uniform redistribution within the polyplex and this feature has been reported to be favorable in the context of pDNA release and subsequent improved transfection efficiency, compared to nondynamic formulations.

Scheme 1. Two-Step Synthesis Route Yielding a Heterofunctional PEG Macroinitiator with Both an Atom Transfer Radical Polymerization (ATRP) Initiator and a Chain-Transfer Agent (CTA) for Subsequent Reversible Addition–Fragmentation Chain-Transfer (RAFT) Polymerization

Scheme 2. Schematic Overview of the Synthesis Route of NPD Triblock Copolymers Using the Heterofunctional PEG Macroinitiator (P Polymer)

Atom transfer radical polymerization (ATRP) is used in the first step to polymerize N-isopropylacrylamide (NIPAM, N) yielding the intermediate NP polymer. Next, 2-(dimethylamino)ethyl methacrylate (DMAEMA, D) is polymerized by reversible addition–fragmentation chain-transfer (RAFT) polymerization to obtain the final NPD polymer.

Scheme 3. Schematic Overview of Experimental Design for Time-Resolved Fluorescence Spectroscopic Analysis of Polyplexes Based on pDNA and NP Diblock and NPD Triblock Copolymers

(A) Fixed temperature measurements: polyplexes were prepared with pDNA-FITC and unlabeled polymer up to N/P 2, followed by addition of either more unlabeled polymer (a, unlabeled) or Cy3-labeled polymer (b, mixed) up to N/P 10. Alternatively, polyplexes were prepared with pDNA-FITC and Cy3-labeled polymer for all N/P ratios (c, fully labeled). Preparation of the polyplexes and measurements were performed at either 4 or 37 °C. (B) Temperature cycle measurements: polyplexes were prepared with pDNA-FITC and unlabeled polymer (N/P 10) at either 4 or 37 °C. Subsequently, polyplexes were subjected to a temperature cycle, including cooling and heating series as described in detail in section 3.3.3.

Figure 1

(A) GPC chromatograms (RI-detection) of the starting compound NH₂-PEG-OH (solid line), intermediate product Br-C(CH₃)₂-CO-NH-PEG-OH (dashed line) and final PEG macroinitiator Br-C(CH₃)₂-CO-NH-PEG-CTA (dotted line). (B) GPC analysis with dual RI (black) and UV–vis (at 310 nm, yellow) detection of final PEG macroinitiator (solid line) and CTA compound (dashed line).

Figure 2

(A) Chemical structure of NPD triblock polymer consisting of a PEG midblock (P), flanked by blocks of PNIPAM (N) and PDMAEMA (D). (B) Chemical structure of PD diblock polymer consisting of a PEG block (P) and a PDMAEMA (D) block.

Figure 3

(A) UV–vis spectra of NPD polymer before (solid line) and after (dotted line) aminolysis with n-butylamine for 24 h at RT. (B) GPC analysis with dual RI (black) and UV/vis (at 550 nm, pink) detection of Cy3-labeled NPD polymer (solid line) and free maleimide-Cy3 dye (dashed line).

Figure 4

Fluorescence lifetime ratio of pDNA-FITC as a function of N/P ratio in the presence of unlabeled and/or labeled NPD polymer (A,B) and PD polymer (C,D) at 4 and 37 °C. The fluorescence lifetime of pDNA-FITC (τ₀) was used to calculate the fluorescence lifetime ratio (τ₀/⟨τ⟩).

Figure 5

Suggested polyplex structure for studied NPD and PD polymers (A) compared to the structure of the polyplex formed by PEI and PLL (B) as revealed earlier. The pDNA is depicted in blue, core polymers in yellow, and shell polymers in red.

Figure 6

In vitro evaluation of polyplexes on HeLa cells. Cells were transfected with pDNA (0.50 μg/well) formulated in NPD and PD polyplexes at different N/P ratios in serum supplemented culture medium for 6 h. A formulation with l-PEI, 25 kDa (N/P 6) was added as a control. Transfection efficiency was determined by a luciferase reporter assay (A), and cell viability was determined by an MTS assay (B).

Figure 7

Mean amplitude weighted fluorescence lifetime (⟨τ⟩) of pDNA-FITC as a function of temperature in the presence of unlabeled NPD polymer (A,B, blue) and PD polymer (C,D, blue) at N/P 10. The arrows indicate the direction of the temperature change and points marked with a star (bright blue) indicate changes in the polyplex structure. The mean amplitude weighted fluorescence lifetimes obtained before the temperature change are shown in red dots.

Figure 8

In vitro evaluation of polyplexes before and after the temperature cycle on HeLa cells. Cells were treated with pDNA (0.50 μg/well) formulated in NPD and PD polyplexes (N/P 10) in serum supplemented culture medium for 6 h. The polyplexes were prepared at either 4 or 37 °C and subsequently subjected to a temperature cycle (4 → 37 → 4 °C and 37 → 4 → 37 °C, respectively). A formulation with l-PEI, 25 kDa (N/P 6) and naked pDNA were added as positive and negative controls, respectively. Transfection efficiency was determined by a luciferase reporter assay (A), and cell viability was determined by an MTS assay (B).