protein transport to the nucleus by means of passive

protein as comparedto the PEI-siRNA, at a weight ratio of 3:1 the ND-PEI800:siRNA complex show analmost twofold higher knockdown in cellular green fluorescent proteinexpression.         In presence of serum in the treatmentmedium, the ND-PEI800:siRNA complex showed better knockdown in greenfluorescent protein expression as well as lower cytotoxicity than LipofectamineTM,which is a gold standard for in vitro delivery of nucleicacids. Therefore, NDs have the capacity to improve the transfection abilityof polymers while remaining biocompatible with the cell lines. Genedelivery is the introduction of genetic material or gene therapeutics intocells, aiming to exchange the ‘impaired’gene to regain biological function or add a new gene to trigger additionalfunctions207.

Long agoviruses have been discovered as primitiveand smart enough to interchange their genetic material into a genome of cells.Since the delivery of genetic material via viruses (viralvectors) has been broadly followed in clinics to irreversibly changecell functions—a permanent transfection.Although viral vectors have high gene transfection effectiveness, they giverise to serious safety concerns. That’s why non-viral delivery is also activelypursued. Non-viral strategies are good to deliver genetic material exclusivelyto cytoplasm-transient transfection.

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The transiently transfected genetic material occupyincytoplasm, does not replicate, and is gradually lost when the cells divide.Transposing DNA in chromosome by non-viral vectors is much less well organizedcompared to viral vectors. Genetic material can be transport to the nucleus bymeans of passive diffusion of non-viral vectors (nanoparticles)through the nuclear pore complex (NPC).The passive diffusion through NPC strongly depends on the net size of thevector and genetic cargo (preferentially less than 5 nm indiameter)208. Many differentnanocarriers are studied as gene delivery agents, comprises gold and magneticiron oxide nanoparticles (magnetofection). ND-basedgene delivery platforms are attractivebecause NDs are biocompatible,and have a rich surface chemistry,amenable to different modifications to help cell entry and ferrya gene.

ND particle size (2 to 5 nm in diameter)meets the criteria for a passive diffusion into the nucleus. Unprecedentedexamples of ND particle nuclear entry have been express a few years ago withFenton treated ND. The Fenton oxidationleads to ND free of amorphous carbon and of much smaller size (onaverage 4.4 nm after the oxidation of 7 nm NDs contained in the soot),small enough that it can passively penetrate into HeLa cells nuclei. Thereported capability of ND to easily escape from the endosomes is also essentialfor delivery of genetic material into nuclei209. Fastescape from endosome helps to protect genetic material from digesting enzymes.

Perhaps,the most studied application of ND for gene delivery is based on thenon-covalent integration of poly-cationic molecules onto the ND surfacefollowed by interconnection withnegatively charged nucleic acids. For example, pEGFPLuc plasmids encodingLuciferase and green fluorescent protein (GFP)have been successfully delivered in cytoplasm by means of aa ND-PEI vector.Positively charged PEI-ND have significantly improved transfection ascompared to PEI or ND alone, probably, due to a faster endosomal liberation.Since high molecular weight cationic vectors show high cellular toxicity, theauthors have optimized the ratio PEI:ND:DNA to attain high transcription rates, while minimizing toxicity.

It is important to sustain the right balance between the quantity of DNA on thesurface of the vector and the DNA-induced reduction of the positive charge ofthe structure, which is needed for the efficient endosomal libration andtranscription of genetic material210.It was measured that 4.1 nm ND particle binds on average 70 branched 800 Da PEImolecules. Studies have explained that siRNA and ND-PEI ratios (1to 75 w/ w siRNA to ND-PEI, respectively)can be tuned to knock down GFP and EWS-Fli1 genes more efficientlythan the well-known liposomal vector Lipofectamine.        The potential of ND-PEI to liberate siRNA inthe cytoplasm much faster than other vectors (includingother ND-polycationic complexes) is due to the fast endosomalrelease of the vector verified by TEM . The large number of primary andtertiary amino groups (at least 216 ?molg?1)on the ND-PEI surface results in osmotic influxof counter-ions through the endosome membrane to protonated ND-PEI complexleading to endosome swelling and disruption.

In addition to ND-PEI complexes, hydrogenateddetonation NDs with zeta potential +55mV have been studied to electrostatically bind the negatively charged siRNA.The approximte number of siRNA molecules is 37 per one 7 nm ND particle. Theair oxidized ND-COOH (zeta potential –50mV) can not exhibit any non-specificbinding of siRNA as expected. Carboxylated derivatives of larger, 20 nm HPHTNDshave been exploited in a covalent reaction with amino-modified nucleic acid through EDC/NHSchemistry211.

Anothercovalent DNA binding technique has been recently demonstrated via a copper-freecoupling of dibenzocy-clooctin-modifiednucleic acid to azido-functionalized 100 nm HPHTND212,213.