Metal-free magnetic resonance imaging (MRI) agents could overcome the established toxicity associated with metal-based agents in some patient populations and enable new modes of functional MRI in vivo. Herein, we report nitroxide-functionalized brush-arm star polymer organic radical contrast agents (BASP-ORCAs) that overcome the low contrast and poor in vivo stability associated with nitroxide-based MRI contrast agents. As a consequence of their unique nanoarchitectures, BASP-ORCAs possess per-nitroxide transverse relaxivities up to ∼44-fold greater than common nitroxides, exceptional stability in highly reducing environments, and low toxicity. These features combine to provide for accumulation of a sufficient concentration of BASP-ORCA in murine subcutaneous tumors up to 20 h following systemic administration such that MRI contrast on par with metal-based agents is observed. BASP-ORCAs are, to our knowledge, the first nitroxide MRI contrast agents capable of tumor imaging over long time periods using clinical high-field 1H MRI techniques.

Surface modification by poly(ethylene glycol) (PEG) onto gene carrier prepared through the electrostatic assembly of pDNA and polycation (polyplex) is a widely acknowledged strategy to advance their systemic application. In this regard, PEG crowdedness on the polyplex surface should give important contribution in determining blood circulation property; however its accurate quantification has never been demonstrated. We report here the first successful determination of PEG crowdedness for PEGylated polyplexes (polyplex micelle) formed from PEG–poly(l-lysine) block copolymers (PEG–PLys) and plasmid DNA (pDNA). Tethered PEG chains were found to adopt mushroom and even squeezed conformation by modulating PEG crowdedness through PLys segment length. Energetic analysis was conducted on the polyplex micelle to elucidate effect of PEG crowdedness on shape and clarify its essential role in regulating packaging structure of pDNA within the polyplex micelle. Furthermore, the PEG crowdedness significantly correlated to blood retention profile, approving its critical role on both shape and systemic circulation property.

Adequate retention in systemic circulation is the preliminary requirement for systemic gene delivery to afford high bioavailability into the targeted site. Polyplex micelle formulated through self-assembly of oppositely-charged poly(ethylene glycol) (PEG)-polycation block copolymer and plasmid DNA has gained tempting perspective upon its advantageous core-shell architecture, where outer hydrophilic PEG shell offers superior stealth behaviors. Aiming to promote these potential characters toward systemic applications, we strategically introduced hydrophobic cholesteryl moiety at the ω-terminus of block copolymer, anticipating to promote not only the stability of polyplex structure but also the tethered PEG crowdedness. Moreover, Mw of PEG in the PEGylated polyplex micelle was elongated up to 20 kDa for expecting further enhancement in PEG crowdedness. Furthermore, cyclic RGD peptide as ligand molecule to integrin receptors was installed at the distal end of PEG in order for facilitating targeted delivery to the tumor site as well as promoting cellular uptake and intracellular trafficking behaviors. Thus constructed cRGD conjugated polyplex micelle with the elevated PEG shielding was challenged to a modeled intractable pancreatic cancer in mice, achieving potent tumor growth suppression by efficient gene expression of antiangiogenic protein (sFlt-1) at the tumor site.

No abstract

Poly(ethylene glycol) (PEG) modification onto a gene delivery carrier for systemic application results in a trade-off between prolonged blood circulation and promoted transfection because high PEG shielding is advantageous in prolonging blood retention, while it is disadvantageous with regard to obtaining efficient transfection owing to hampered cellular uptake. To tackle this challenging issue, the present investigation focused on the structure of polyplex micelles (PMs) obtained from PEG-poly(l-lysine) (PEG-PLys) block copolymers characterized as rod-shaped structures to seek the most appreciable formulation. Comprehensive investigations conducted with particular focus on stability, PEG crowdedness, and rod length, controlled by varying PLys segment length, clarified the effect of these structural features, with particular emphasis on rod length as a critical parameter in promoting cellular uptake. PMs with rod length regulated below the critical threshold length of 200 nm fully exploited the benefits of cross-linking and the cyclic RGD ligand, consequently, exhibiting remarkable transfection efficiency comparable with that of ExGen 500 and Lipofectamine(®) LTX with PLUS™ even though PMs were PEG shielded. The identified PMs exhibited significant antitumor efficacy in systemic treatment of pancreatic adenocarcinoma, whereas PMs with rod length above 200 nm exhibited negligible antitumor efficacy despite a superior blood circulation property, thereby highlighting the significance of controlling the rod length of PMs to promote gene transduction.