Charge-neutral DNA nanoparticles have been developed in which single molecules of DNA are compacted to their minimal possible size. We speculated that the small size of these DNA nanoparticles may facilitate gene transfer in postmitotic cells, permitting nuclear uptake across the 25-nm nuclear membrane pore. To determine whether DNA nanoparticles can transfect nondividing cells, growth-arrested neuroblastoma and hepatoma cells were transfected with DNA/liposome mixtures encoding luciferase. In both models, growth-arrested cells were robustly transfected by compacted DNA (6,900 -360-fold more than naked DNA). To evaluate mechanisms responsible for enhanced transfection, HuH-7 cells were microinjected with naked or compacted plasmids encoding enhanced green fluorescent protein. Cytoplasmic microinjection of DNA nanoparticles generated a ϳ10-fold improvement in transgene expression as compared with naked DNA; this enhancement was reversed by the nuclear pore inhibitor, wheat germ agglutinin. To determine the upper size limit for gene transfer, DNA nanoparticles of various sizes were microinjected into the cytoplasm. A marked decrease in transgene expression was observed as the minor ellipsoidal diameter approached 25 nm. In summary, suitably sized DNA nanoparticles productively transfect growth arrested cells by traversing the nuclear membrane pore.Although nonviral gene transfer methods transfect dividing cells, these technologies fail to transfect most postmitotic cells (1-10), with the principal exceptions of naked DNA gene transfer into muscle (11) and large volume hydrodynamic gene transfer into liver (12, 13). In dividing cells, nuclear membrane disintegration during mitosis allows plasmid DNA to enter the nucleus prior to membrane reformation. Otherwise, the intact nuclear membrane restricts transfer of naked DNA into the nucleus. The nuclear membrane pore (NMP) 1 has an internal channel diameter of 25 nm (14, 15) and does not permit naked DNA to effectively cross into the nucleus, probably due to the extended size of hydrated DNA and its negative charge density (4,16,17). The NMP does permit passive transfer of gold particles less than 9 -10 nm in diameter and linear DNA fragments up to ϳ300 bp (18 -22) as well as facilitated transport of proteins and small DNA segments (up to ϳ1 kbp) having nuclear localization signals (7,(22)(23)(24)(25)(26)(27)(28). The relative inefficiency of naked DNA, liposome-DNA complexes, and protein-and polymer-based DNA conjugates to transfect nondividing cells productively remains a significant barrier for in vivo gene therapy. Electrostatic interactions between polycationic polymers and DNA can result in conjugates consisting of one or more molecules of DNA and a sufficient number of polycations to produce a nearly charge-neutral complex (29 -31). The ratio of positive to negative charges, buffer components, polycation counterion, DNA concentration, and pH, among other variables, influence the composition, size, and shape of these DNA conjugates (29,32). Based on specific fo...

DNA can be compacted using polyethylene glycol-substituted poly-L-lysine into discrete unimolecular (with respect to DNA) nanoparticles with minor diameter < 20 nm that are stable in normal saline for at least 23 months at 4 degrees C. We compared the activity of firefly luciferase in lungs of C57BL/6 mice that received 100 microg compacted plasmid in 25 microl saline (shown to be the optimal dose) via intratracheal or intranasal instillation with levels in animals given 100 microg naked plasmid or in untreated mice. Mice dosed with compacted DNA nanoparticles had peak activity of luciferase in lung at 2 days postinstillation, which declined in log-linear fashion with a half-life of 1.4 days. Luciferase activity in animals dosed with naked DNA was 200-fold less. Addition of polyethylene glycol to the complex was necessary for efficient gene transfer and animals that received DNA compacted with unmodified poly-L-lysine did not exhibit luciferase activity above background. Immunohistochemical staining for bacterial beta-galactosidase 2 days after administration of a compacted lacZ expression plasmid (n = 8) revealed expression predominantly in the dependent portions of the right lungs of mice, in alveolar and airway epithelial cells, though macrophages and sometimes endothelial cells also were transfected. No staining for beta-galactosidase was observed in uninjected animals (n = 4) or those dosed with naked lacZ plasmid (n = 7). Tissue survey for transgene expression shows expression only in lung and trachea following intranasal administration. Stable compacted DNA nanoparticles transfer exogenous genes to airway epithelium and show promise for lung gene therapy.

Summary Background Reoperation rates are high after surgery for hip fractures. We investigated the effect of a sliding hip screw versus cancellous screws on the risk of reoperation and other key outcomes. Methods For this international, multicentre, allocation concealed randomised controlled trial, we enrolled patients aged 50 years or older with a low-energy hip fracture requiring fracture fixation from 81 clinical centres in eight countries. Patients were assigned by minimisation with a centralised computer system to receive a single large-diameter screw with a side-plate (sliding hip screw) or the present standard of care, multiple small-diameter cancellous screws. Surgeons and patients were not blinded but the data analyst, while doing the analyses, remained blinded to treatment groups. The primary outcome was hip reoperation within 24 months after initial surgery to promote fracture healing, relieve pain, treat infection, or improve function. Analyses followed the intention-to-treat principle. This study was registered with ClinicalTrials.gov, number NCT00761813. Findings Between March 3, 2008, and March 31, 2014, we randomly assigned 1108 patients to receive a sliding hip screw (n=557) or cancellous screws (n=551). Reoperations within 24 months did not differ by type of surgical fixation in those included in the primary analysis: 107 (20%) of 542 patients in the sliding hip screw group versus 117 (22%) of 537 patients in the cancellous screws group (hazard ratio [HR] 0.83, 95% CI 0.63–1.09; p=0.18). Avascular necrosis was more common in the sliding hip screw group than in the cancellous screws group (50 patients [9%] vs 28 patients [5%]; HR 1.91, 1.06–3.44; p=0.0319). However, no significant difference was found between the number of medically related adverse events between groups (p=0.82; appendix); these events included pulmonary embolism (two patients [<1%] vs four [1%] patients; p=0.41) and sepsis (seven [1%] vs six [1%]; p=0.79). Interpretation In terms of reoperation rates the sliding hip screw shows no advantage, but some groups of patients (smokers and those with displaced or base of neck fractures) might do better with a sliding hip screw than with cancellous screws. Funding National Institutes of Health, Canadian Institutes of Health Research, Stichting NutsOhra, Netherlands Organisation for Health Research and Development, Physicians’ Services Incorporated.

Background:Foxp3+ regulatory T cells (Tregs) play a vital role in preventing autoimmunity, but also suppress antitumour immune responses. Tumour infiltration by Tregs has strong prognostic significance in colorectal cancer, and accumulating evidence suggests that chemotherapy and radiotherapy efficacy has an immune-mediated component. Whether Tregs play an inhibitory role in chemoradiotherapy (CRT) response in rectal cancer remains unknown.Methods:Foxp3+, CD3+, CD4+, CD8+ and IL-17+ cell density in post-CRT surgical samples from 128 patients with rectal cancer was assessed by immunohistochemistry. The relationship between T-cell subset densities and clinical outcome (tumour regression and survival) was evaluated.Results:Stromal Foxp3+ cell density was strongly associated with tumour regression grade (P=0.0006). A low stromal Foxp3+ cell density was observed in 84% of patients who had a pathologic complete response (pCR) compared with 41% of patients who did not (OR: 7.56, P=0.0005; OR: 5.27, P=0.006 after adjustment for presurgery clinical factors). Low stromal Foxp3+ cell density was also associated with improved recurrence-free survival (HR: 0.46, P=0.03), although not independent of tumour regression grade.Conclusions:Regulatory T cells in the tumour microenvironment may inhibit response to neoadjuvant CRT and may represent a therapeutic target in rectal cancer.