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Patent Issued for DNA Loaded Supported Gold Nanoparticles, Process for the Preparation and Use

July 8, 2014



By a News Reporter-Staff News Editor at Life Science Weekly -- From Alexandria, Virginia, NewsRx journalists report that a patent by the inventors Prasad, L.V. Bhagavatula (Maharashtra, IN); Periyasamy, Shanmugham Vijaykumar (Maharashtra, IN); Othalathara, Usharraj Abhilash (Maharashtra, IN); Bashir, Mohammad Khan (Maharashtra, IN), filed on August 3, 2010, was published online on June 24, 2014 (see also Council of Scientific & Industrial Research).

The patent's assignee for patent number 8759054 is Council of Scientific & Industrial Research (New Delhi, IN).

News editors obtained the following quote from the background information supplied by the inventors: "Genetic manipulation to display the desired trait in biological components has opened up many new avenues. Viral vectors have been identified as successful gene delivery vehicle. Since they have the limitations like acute toxicity, cellular immune response, oncogenicity due to insertional mutagenesis, limited cargo capacity, resistance to repeated infection and production and quality control alternatives, non viral vectors like lipids, polymeric compounds, carbohydrate dendrimers and polypeptide based systems have been demonstrated to be better alternatives. Nanoparticles, especially carbon nanostructures are being studied for gene delivery applications. However, most of the studies are restricted to the animal system or the cell lines. On the other hand non viral vectors for plant systems are relatively underdeveloped, except for a novel method developed by Sanford et al as recited in Nature, 1987 327, 70-73, using a particle gun that employs accelerated DNA coated micro gold projectiles, that deliver DNA into intact plant cells. As an improvement to the micro gold structure, Wang and co-workers synthesized mesoporous silica loaded with nano gold for multiplex gene delivery as published in Nature Nanotech., 2007, 2, 295-300. While the low density mesoporous silica covered with gold was shown to penetrate soft maize embryo, there was no demonstrable data to show penetration of hard embryo like the one of woody tree species.

"Recently, a tetrapod sharp structure was reported to deliver DNA into the human cell line mimicking viral vector capsid where the tetrapod's sharp tip carrying the plasmid enters into the cell bringing out necessary transfection; in an article titled 'Three dimensional functionalized tetrapodlike ZnO nanostructures for plasmid DNA delivery' by Leng Nie et al in the journal, 'Small' 2006, 2, 621-625. Similarly, it is also reported that silicon nano needles (of 200-300 nm diameter) required a force of 0.7-2.0 nN) as compared to a carbon nanotube (of 30-40 nm diameter) that required a lower force (0.1-0.2 nN) to penetrate the plasma membrane by Vakarelski et al in Langmuir, 2007, 23, 10893-10896, that once again highlighted the utility of sharp objects for gene delivery type applications.

"It is noteworthy to mention here that ample literature is available on the synthesis of metal nanoparticles by microbes, both intracellularly and extracellularly (reference is drawn to Narayanan, K. B. and Sakthivel, N. 2010. Advances in Colloids and Interface Science. 156 [1-2]: 1-13). However, no report on gene delivery through nanoparticles loaded on sharp edged supports is available, which can not only facilitate DNA transfer to soft tissues but also hard tissues.

"Thus, the prior art survey reveals a need for a carrier of genetic material with sharp edges. Further, the carrier should also have adequate capacity to carry the genetic material and the sharpness to penetrate to hard material, but with less damage. The process of preparation of said carriers should be a simple, easy to implement and still fill the gaps in prior arts and needs in the art."

As a supplement to the background information on this patent, NewsRx correspondents also obtained the inventors' summary information for this patent: "Envisaging that the combination of a carrier with sharp edges and nano gold could be an important breakthrough in gene gun based transformation, herein we describe the preparation of carbon supported gold nanoparticles using a combination of biochemical/physical/chemical transformations. The particles are prepared by inert heating of the intracellular gold nanoparticles, in situ synthesized by fungus Aspergillus ochraceus ITCC 6436 corresponding to ATCC 18500. The intracellular gold nanoparticles heat treated at 600.degree. C. are denoted as HTC-600 Au further in the text for brevity. This matrix supports gold nanoparticles giving the platform for the DNA to bind and provide minimum density for the carrier, to gain threshold velocity in the gene gun to pierce the hard cell wall in plant systems. The Au nanoparticles embedded in the carbon matrix provide the platform to which the DNA can bind. The carbon support clamped the Au nanoparticles and prevented them from leaching during sonication, a step required in DNA coating procedure. On the other hand, in the pure carbon material, the DNA could not be bonded as no gold was present.

"Accordingly, the present invention provides DNA loaded supported gold nanoparticles characterized in that the DNA loaded supported nano gold particles having sharp edges as shown in FIG. 2A, curve 2.

"The present invention further provides a process for the preparation of DNA loaded supported gold nanoparticles, wherein the steps comprising: [a] inoculating a microbial culture capable of synthesizing intracellular gold nanoparticles in a nutrient medium and culturing to obtain a biomass; [b] harvesting the biomass obtained in step [a] followed by washing the biomass with sterilized distilled water under aseptic conditions; [c] suspending the washed biomass obtained in step [b] in HAuCl.sub.4 solution and incubating for 2-3 days followed by washing the biomass with sterilized distilled water under aseptic conditions; [d] heat treating the washed biomass obtained in step [c] for 6-8 hours in a tubular furnace under inert conditions to obtain the heat treated carbon gold HTC-600 Au; [e] washing the HTC-600 Au as obtained in step [d] with ethanol; [f] mixing the ethanol-washed, sterilized HTC-Au-600 with DNA suspended in the XHO buffer (Tris.Cl, NaC-based buffer); [g] equilibrating the mixture as obtained in step [f] with spermidine and PEG in sequence followed by sonication in 2.5 M CaCl.sub.2; [h] centrifuging the sonicated mixture as obtained in step [g] at 12000 rpm and washing the pellets twice using absolute alcohol and finally resuspending in the required volume of ethanol to obtain DNA loaded supported gold nanoparticles."

For additional information on this patent, see: Prasad, L.V. Bhagavatula; Periyasamy, Shanmugham Vijaykumar; Othalathara, Usharraj Abhilash; Bashir, Mohammad Khan. DNA Loaded Supported Gold Nanoparticles, Process for the Preparation and Use. U.S. Patent Number 8759054, filed August 3, 2010, and published online on June 24, 2014. Patent URL: http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO1&Sect2=HITOFF&d=PALL&p=1&u=%2Fnetahtml%2FPTO%2Fsrchnum.htm&r=1&f=G&l=50&s1=8759054.PN.&OS=PN/8759054RS=PN/8759054

Keywords for this news article include: DNA Research, Nanoparticle, Nanostructural, Nanostructures, Nanotechnology, Emerging Technologies.

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Source: Life Science Weekly


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