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

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.

π-π-Stacked Poly(ε-caprolactone)-b-poly(ethylene glycol) Micelles Loaded with a Photosensitizer for Photodynamic Therapy.

To improve the in vivo stability of poly(-caprolactone)-b-poly(ethylene glycol) (PCL-PEG)-based micelles and cargo retention by π-π stacking interactions, pendant aromatic rings were introduced by copolymerization of -caprolactone with benzyl 5-methyl-2-oxo-1,3-dioxane-5-carboxylate (TMC-Bz). It was shown that the incorporation of aromatic rings yielded smaller micelles (18-30 nm) with better colloidal stability in PBS than micelles without aromatic groups. The circulation time of i.v. injected micelles containing multiple pendant aromatic groups was longer (t½-α: ~0.7 h; t½-ß: 2.9 h) than that of micelles with a single terminal aromatic group (t½ < 0.3 h). In addition, the in vitro partitioning of the encapsulated photosensitizer (meta-tetra(hydroxyphenyl)chlorin, mTHPC) between micelles and human plasma was favored towards micelles for those that contained the pendant aromatic groups. However, this was not sufficient to fully retain mTHPC in the micelles in vivo, as indicated by similar biodistribution patterns of micellar mTHPC compared to free mTHPC, and unequal biodistribution patterns of mTHPC and the host micelles. Our study points out that more detailed in vitro methods are necessary to more reliably predict in vivo outcomes. Furthermore, additional measures beyond π-π stacking are needed to stably incorporate mTHPC in micelles in order to benefit from the use of micelles as targeted delivery systems.