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Anthony J Kim,
Nicholas J Boylan,
Jung Soo Suk,
Minyoung Hwangbo,
Tao Yu,
Benjamin S Schuster,
Liudimila Cebotaru,
Wojciech G Lesniak,
Joon Seok Oh,
Pichet Adstamongkonkul,
Ashley Y Choi,
Rangaramanujam M Kannan,
Justin Hanes
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ABSTRACT: Mucus-penetrating DNA nanoparticles: A novel synthetic strategy was used to achieve a dense PEG coating on the surface of cationic polymer-based DNA nanoparticles. The dense PEG coating (blue in scheme) endows the nanoparticles with a muco-inert surface, which enables their rapid mucus penetration (trajectory indicated by gray line) and provides efficient gene transfer in various cell types.
Angewandte Chemie International Edition 03/2013; · 13.45 Impact Factor
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ABSTRACT: Our goal was to enhance ultrasound (US)-targeted skeletal muscle transfection through the use of poly(ethyleneglycol) (PEG)/polyethylenimine (PEI) nanocomplex gene carriers and adjustments to US and microbubble (MB) parameters. C57BL/6 mice received an intravenous infusion of MBs and either "naked" luciferase plasmid or luciferase plasmid condensed in PEG/PEI nanocomplexes. Pulsed ultrasound (1MHz; 0.6MPa or 0.8MPa) was applied to the right hindlimb for 12min. Luciferase activity in both hindlimbs was assessed at 3, 5, 7, and 10days post-treatment by bioluminescent imaging. When targeted to hindlimb using unsorted MBs and 0.6MPa US, 7days after treatment, we observed a >60-fold increase in luciferase activity in PEG/PEI nanocomplex-treated muscles over muscles treated with "naked" plasmid DNA. Luciferase activity was consistently greater after treatment with PEG/PEI nanocomplexes at 0.6MPa as compared to 0.8MPa. The combination of small diameter MBs and 0.6MPa US also resulted in significantly greater gene expression when compared to concentration matched intramuscular injections, a control condition in which considerably more PEG/PEI nanocomplexes were present in tissue. This result suggests that, in addition to facilitating PEG/PEI nanocomplex delivery from the bloodstream to tissue, US enhances transfection via one or more secondary mechanisms, including increased cellular uptake and/or trafficking to the nucleus of PEG/PEI nanocomplexes. We conclude that PEG/PEI nanocomplexes may be used to markedly enhance the amplitude of US-MB-targeted skeletal muscle transfection and that activating "small" MBs with a moderate level (0.6MPa) of acoustic pressure can further enhance these effects.
Journal of Controlled Release 07/2012; 162(2):414-21. · 5.73 Impact Factor
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ABSTRACT: Highly compacted DNA nanoparticles, composed of single molecules of plasmid DNA compacted with block copolymers of polyethylene glycol and poly-L-lysine (PEG-CK(30)), have shown considerable promise in human gene therapy clinical trials in the nares, but may be less capable of transfecting cells that lack surface nucleolin. To address this potential shortcoming, we formulated pH-responsive DNA nanoparticles that mediate gene transfer via a nucleolin-independent pathway. Poly-L-histidine was inserted between PEG and poly-L-lysine to form a triblock copolymer system, PEG-CH(12)K(18). Inclusion of poly-L-histidine increased the buffering capacity of PEG-CH(12)K(18) to levels comparable with branched polyethyleneimine. PEG-CH(12)K(18) compacted DNA into rod-shaped DNA nanoparticles with similar morphology and colloidal stability as PEG-CK(30) DNA nanoparticles. PEG-CH(12)K(18) DNA nanoparticles entered human bronchial epithelial cells (BEAS-2B) that lack surface nucleolin by a clathrin-dependent endocytic mechanism followed by endo-lysosomal processing. Despite trafficking through the degradative endo-lysosomal pathway, PEG-CH(12)K(18) DNA nanoparticles improved the in vitro gene transfer by ~20-fold over PEG-CK(30) DNA nanoparticles, and in vivo gene transfer to lung airways in BALB/c mice by ~3-fold, while maintaining a favorable toxicity profile. These results represent an important step toward the rational development of an efficient gene delivery platform for the lungs based on highly compacted DNA nanoparticles.
Biomaterials 12/2011; 33(7):2361-71. · 7.40 Impact Factor
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ABSTRACT: Highly compacted DNA nanoparticles (DNPs) composed of polyethylene glycol linked to a 30-mer of poly-l-lysine via a single cysteine residue (CK(30)PEG) have previously been shown to provide efficient gene delivery to the brain, eyes and lungs. In this study, we used a combination of flow cytometry, high-resolution live-cell confocal microscopy, and multiple particle tracking (MPT) to investigate the intracellular trafficking of highly compacted CK(30)PEG DNPs made using two different molecular weights of PEG, CK(30)PEG(10k) and CK(30)PEG(5k). We found that PEG MW did not have a major effect on particle morphology nor nanoparticle intracellular transport. CK(30)PEG(10k) and CK(30)PEG(5k) DNPs both entered human bronchial epithelial (BEAS-2B) cells via a caveolae-mediated pathway, bypassing degradative endolysosomal trafficking. Both nanoparticle formulations were found to rapidly accumulate in the perinuclear region of cells within 2h, 37±19% and 47±8% for CK(30)PEG(10k) and CK(30)PEG(5k), respectively. CK(30)PEG(10k) and CK(30)PEG(5k) DNPs moved within live cells at average velocities of 0.09±0.04μm/s and 0.11±0.04μm/s, respectively, in good agreement with reported values for caveolae. These findings show that highly compacted DNPs employ highly regulated trafficking mechanisms similar to biological pathogens to target specific intracellular compartments.
Journal of Controlled Release 10/2011; 158(1):102-7. · 5.73 Impact Factor
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Virgil Percec,
Daniela A Wilson,
Pawaret Leowanawat,
Christopher J Wilson,
Andrew D Hughes,
Mark S Kaucher,
Daniel A Hammer,
Dalia H Levine, Anthony J Kim,
Frank S Bates, [......],
Timothy P Lodge,
Michael L Klein,
Russell H DeVane,
Emad Aqad,
Brad M Rosen,
Andreea O Argintaru,
Monika J Sienkowska,
Kari Rissanen,
Sami Nummelin,
Jarmo Ropponen
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ABSTRACT: Self-assembled nanostructures obtained from natural and synthetic amphiphiles serve as mimics of biological membranes and enable the delivery of drugs, proteins, genes, and imaging agents. Yet the precise molecular arrangements demanded by these functions are difficult to achieve. Libraries of amphiphilic Janus dendrimers, prepared by facile coupling of tailored hydrophilic and hydrophobic branched segments, have been screened by cryogenic transmission electron microscopy, revealing a rich palette of morphologies in water, including vesicles, denoted dendrimersomes, cubosomes, disks, tubular vesicles, and helical ribbons. Dendrimersomes marry the stability and mechanical strength obtainable from polymersomes with the biological function of stabilized phospholipid liposomes, plus superior uniformity of size, ease of formation, and chemical functionalization. This modular synthesis strategy provides access to systematic tuning of molecular structure and of self-assembled architecture.
Science 05/2010; 328(5981):1009-14. · 31.20 Impact Factor
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Advanced Functional Materials 08/2009; 19(18):2930 - 2936. · 10.18 Impact Factor
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ABSTRACT: DNA is the premier material for directing nanoscale self-assembly, having been used to produce many complex forms. Recently, DNA has been used to direct colloids and nanoparticles into novel crystalline structures, providing a potential route to fabricating meta-materials with unique optical properties. Although theory has sought the crystal phases that minimize total free energy, kinetic barriers remain essentially unstudied. Here we study interfacial equilibration in a DNA-directed microsphere self-assembly system and carry out corresponding detailed simulations. We introduce a single-nucleotide difference in the DNA strands on two mixed microsphere species, which generates a free-energy penalty for inserting 'impurity' spheres into a 'host' sphere crystal, resulting in a reproducible segregation coefficient. Comparison with simulation reveals that, under our experimental conditions, particles can equilibrate only with a few nearest neighbours before burial by the growth front, posing a potential impediment to the growth of complex structures.
Nature Material 12/2008; 8(1):52-5. · 32.84 Impact Factor
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ABSTRACT: We probe DNA hybridization kinetics by measuring the lifetime distribution of single 16-bp duplexes under thermal dissociation. Our unique approach, based on two DNA-coated microspheres in an extended optical tweezer, allows the study of single duplex DNA molecules under negligible molecular tension. In contrast to earlier experiments, we find a stretched exponential lifetime distribution, which is likely due to dissociation proceeding via a number of competing pathways between highly force-sensitive intermediate states. Similar measurements of microspheres linked by multiple DNA bridges find they have unexpected short bound lifetimes, also consistent with force sensitivity.
Biophysical Journal 03/2008; 94(3):891-6. · 3.65 Impact Factor
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Journal of the American Chemical Society 10/2007; 129(38):11698-9. · 9.91 Impact Factor
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ABSTRACT: DNA is a powerful and versatile tool for nanoscale self-assembly. Several researchers have assembled nanoparticles and colloids into a variety of structures using the sequence-specific binding properties of DNA. Until recently, however, all of the reported structures were disordered, even in systems where ordered colloidal crystals might be expected. We detail the experimental approach and surface preparation that we used to form the first DNA-mediated colloidal crystals, using 1 mum diameter polystyrene particles. Control experiments based on the depletion interaction clearly indicate that two standard methods for grafting biomolecules to colloidal particles (biotin/avidin and water-soluble carbodiimide) do not lead to ordered structures, even when blockers are employed that yield nominally stable, reversibly aggregating dispersions. In contrast, a swelling/deswelling-based method with poly(ethylene glycol) spacers resulted in particles that readily formed ordered crystals. The sequence specificity of the interaction is demonstrated by the crystal excluding particles bearing a noninteracting sequence. The temperature dependence of gelation and crystallization agree well with a simple thermodynamic model and a more detailed model of the effective colloidal pair interaction potential. We hypothesize that the surfaces yielded by the first two chemistries somehow hinder the particle-particle rolling required for annealing ordered structures, while at the same time not inducing a significant mean-force interaction that would alter the self-assembly phase diagram. Finally, we observe that particle crystallization kinetics become faster as the grafted-DNA density is increased, consistent with the particle-particle binding process being reaction, rather than diffusion limited.
Langmuir 03/2006; 22(5):1991-2001. · 4.19 Impact Factor
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ABSTRACT: The specific binding of complementary DNA strands has been suggested as an ideal method for directing the controlled self-assembly of microscopic objects. We report the first direct measurements of such DNA-induced interactions between colloidal microspheres, as well as the first colloidal crystals assembled using them. The interactions measured with our optical tweezer method can be modeled in detail by well-known statistical physics and chemistry, boding well for their application to directed self-assembly. The microspheres' binding dynamics, however, have a surprising power-law scaling that can significantly slow annealing and crystallization.
Physical Review Letters 03/2005; 94(5):058302. · 7.37 Impact Factor
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ABSTRACT: We describe a swelling-based method to prepare sterically stabilized polymer colloids with different functional groups or biomolecules attached to their surface. It should be applicable to a variety of polymeric colloids, including magnetic particles, fluorescent particles, polystyrene particles, PMMA particles, and so forth. The resulting particles are more stable in the presence of monovalent and divalent salt than existing functionalized colloids, even in the absence of any surfactant or protein blocker. While we use a PEG polymer brush here, the method should enable the use of a variety of polymer chemistries and molecular weights.
Journal of the American Chemical Society 03/2005; 127(6):1592-3. · 9.91 Impact Factor
