Nanotechnology refers to research and technology development at the atomic, molecular and macromolecular scale leading to the controlled manipulation and study of structures and devices with the length scale in the 1 to 100 nanometers range ( McNeil 2005 ). It is also defined as the interdisciplinary science involved in diagnosis, treatment, monitoring and control of biological systems ( Fortina et al., 2006 ). Nanosized materials have been investigated as the potential medicines since several decades ( Garnett and Kallinteri, 2006, Gwinn and Vallyathan, 2006 ) for many chronic diseases as well as disorders, nano medicines offers the potential and hope for a cure and promises to give improved therapy and diagnostic ( Gabriel and Silva, 2004 ).

Nanoparticles can perform the tedious molecular tasks in a simple way to enhance or correct the physiological system at the cellular and sub cellular level that advanced biotechnologists cannot accomplish(Mcneil, 2005 ). A combination of concepts of engineering, physics and molecular biology form the core of nano research ( Fortina et al., 2006 ). A change in the properties of substances at nano level is the main feature of utilization of the nano material in various biological and medical disciplines. Engineers or physicist will be interested in the shrinking dimensions of fabricated structures. In another perspective, molecular biologists would like to study domain of molecular or cellular level. The joining of these two paths of research has opened up new possibilities in biological, physical, chemical and engineering research.

Development in DNA vaccines, novel proteins and peptides based antigens produced by recombinant technology should open a new frontier for nanocarrier based vaccine delivery ( Shahiwala et al., 2007 ). The fact that nanocarriers can easily modified for active targeting, i.e. tissue specific delivery to local lymph nodes, cell specific targeting to antigen presenting cells or targeting to sub cellular compartments like nucleus for DNA vaccines. Additional emphasis need to be placed on the development of efficient target specific nanocarriers that can preferentially interact with the antigen presenting cells upon mucosal administration. In addition, development of novel materials used for the nanocarrier design could be synthesized to include potent adjuvant effects because the discovery of safer adjuvant may allow for development of better prophylactic and therapeutic vaccines against chronic diseases.

Construction of a gene having an appropriate promoter and its easy passage to the genome of the designated source followed by proper folding of the protein coded by the incorporated gene are the focused areas in molecular biology. The concentration of nanotechnology is focused on preparation of nanochannels, nanopores, quantum dots, nanotubes, nanowires and nano capacitors. The outstanding possibility of joining DNA fragments or proteins to nanoparticles and resulting flow or direction of this combined entity have added a new tool to the kits of molecular biologists7 and have added to the identification of DNA hybridization event by nanosize electrodes. Accurate prediction of DNA sequence or protein folding once it is delivered inside cell will help to know the expression of the integrated gene with the help of nanoparticles.

Most molecular recognition techniques rely on a binding event and subsequent interrogation of the optical, electrochemical or magnetic tag carried out by the molecule involved in the binding ( Kane and Stroock, 2007 ).Use of nanoparticle can remove tagging step and would depend on detection of the change of an important property of the analyte or the molecular aggregates formed upon binding. Nanochannels have capacity to stretch out the DNA molecule and simplify the sequencing. This will greatly accurate the sequencing data. The crucial criteria for the protein nanoparticle interaction are ability of the protein structure to sustain its conjugation with nanoparticle and retain its surface chemistry structure, activity and stability. In this direction, antigen-antibody conjugating with the nanoparticles has been reported ( Shenton et al., 1999, Yang et al., 2004 ). Lieber and coworkers 11 ( Hu et al., 1999, Wang et al., 2005 ) have used nanowires based sensors for the label free detection of small molecule-protein interaction and for the ultra sensitive detection of DNA and DNA sequence variations. Protein interaction with the carbon nanotubes has been used for sensing both small molecules and proteins (Chen et al., 2003 ). According to Kane and Stroock ( 2007 ), it is possible to use both proteins to organize nanomaterials and nanomaterials to organize proteins and further, interaction of proteins and virus particles with nanoparticles has been demonstrated. The viral antigen optimization and expression levels in plants are necessary to use as a commercially viable production systems. Attachment of antigen to nanoparticles and their entry into plant cell might be useful in post translational process and protein organization ( Shenton et al. 1999 ). Nanoparticles will facilitate to accumulate more viruses like particles and helping endoplasmic reticulum retention signals to improve the uptake ( Chorny et al., 2006 ).Adjuvant responsible for enhancing the immunogenic response along with the vaccine DNA can all be allowed to interact with the nanoparticles and a joint vaccine-adjuvant nanoparticle combination can be created ( Shahiwala et al., 2007 ).

Nanoparticle protein or DNA interaction can convert this complex into biosensors, molecule scale fluorescent tags, imaging agents, targeted molecular delivery vehicles and other useful biological tools ( Fortina et al. 2006 ). The unprecedented freedom to design and modify the nanomaterials to target cells, chaperone drugs, image biomolecular processes, sense and signal molecular responses to therapeutic agents and guide surgical procedures is the fundamental capability offered by nanotechnology, which promises to impact drug development, medical diagnostics and clinical applications profoundly ( Garnett and Kallinteri, 2006 ). An obvious advantage of nano biotechnology as it relates to the biological systems is the ability to control the size of the resulting particles and devices. Nanoscale constructs are smaller than cells/organelles and similar in size such as enzymes, receptors, hemoglobin, and lipid bilayer. Nanoparticles smaller than 20 nm, can transit through blood vessel walls.

The size of nanoscale devices also allows them to interact readily with the biomolecules on the cell surface and within the cells, often in that they do not alter the behavior and biochemical properties of those molecules ( Jin and Ye, 2007 ). Such ready access to the interior of a living cell affords the opportunity for unprecedented gains on the clinical and basic research frontiers ( Cuenca et al., 2006 ). The ability to interact with receptors, nucleic acids, transcription factors and other signaling proteins at their own molecular scales provide the data needed to better understand the complex regulatory and signaling networks and transport processes that govern the behavior of cells in their normal state and as they undergo the changes that transform them during the disease process.

Application of nanoparticles in DNA, RNA and siRNA delivery

Genetic materials such as DNA, plasmids, RNA and siRNA can be either encapsulated inside or conjugated to the NPs ( Jin and Ye 2007, Tan et al., 2004 ). One of the easiest ways to link DNA to a NP is to modify the surface of NPs to a positive charge so that the NP-DNA complexes can be formed simply through electrostatic binding between the positive charges of NPs and the negative charges of DNA ( Jin and Ye 2007 ). This mechanism has been widely used in liposome and other polymer mediated gene transfer. The electrostatic bound DNA can be released the NP-DNA complexes at alkaline pH or by presence of high salt concentration. The enzymatic digestion of DNA can be inhibited by these NP-DNA complexes ( He et al., 2003 ). The smallness of NPs may force the DNA to become bound in such a way that cleavage is either impossible or at least greatly slowed on the NP surface. It has been observed that silica –NPs released the DNA inside the cytoplasm and migrated into the nucleus for gene delivery. In addition, DNA or RNA can be encapsulated inside biodegradable polymeric NPs for controlled the gene release when polymeric NPs are degraded or digested by the enzymes. The encapsulation of nucleic acids into NPs provides the nucleic acids protection from enzymatic digestion during their transit in systematic circulation and allows targeting to tissues or cells in the body through the surface functionalization of the NPs ( Jin and Ye, 2007 ).The encapsulation also avoids uptake of the nucleic acids, and plasmid DNA, by the mononuclear phagocytic system, which happens all the time when naked plasmid DNA used in the systematic administration ( Jin and Ye 2007, Luo et al., 1999, Kaul and Amiji 2005 ). To improve the efficiency of targeted gene delivery further efforts have been made by ligands conjugate antibodies to biodegradable NPs in which DNA are encapsulated. Gold nanoparticle based method that allows the DNA detection and quantification and is capable of single nucleotide polymorphism (SNP) discrimination ( Qin and Yung 2007 ).

Gene silencing mediated by the double stranded small interfering RNA (siRNA) has been widely investigated as a potential therapeutic approach for disease of the genetic defects ( Jin and Ye, 2007, Galun 2005, Sato, 2005 ). However, siRNA therapy is hindered as result of the poor stability of SiRNA in physiological fluids and hence their limitations in intracellular uptake of NPs have been exploited to maintain a sufficient local cone of siRNAs. These nanoparticles act as the positively charged vehicles carrying negatively charged siRNAs. Further, most of the nanoplexes are biodegradable in nature and are degraded once the siRNAs is delivered to the target site. This method ensures the enhanced stability, solubility, absorption and affinity to the specific targets ( Toub et al., 2006, Belhke 2006 ).

The particle size of NPs can significantly impact the gene transfer efficiency in vivo. It has been reported that the optimization of particle size can dramatically improve the clearance behavior and the tissue distribution of intravenously injected NPs and hence ameliorate the efficiency of drug or gene delivery( Kong et al., 2000, Prabha et al., 2002). .

Nano particles for the drug delivery

Nanoparticles of the size less than 20 nm can transit blood vessel walls and have the ability to penetrate blood-brain barrier or gastro intestinal epithelium( Garnett and Kallinteri, 2006 ). Because of the small size, nanoparticles avoid rapid filtration by spleen whose filamental meshwork, spaced roughly at 200 nm serves for phagocytic cells. Nanoparticles can pass through organs like liver with 150-200 nm sized fenestrate and avoids Kupffer cell lined sieve plates. Nanoparticles can accommodate tens and thousands of atoms or small molecules such as magnetic resonance imaging (MRI) contrast agent gadolinium for the improved detection sensitivity.

Since our body is a set of complex mechanism and our immune system recognizes any foreign particle in the body and generates a response that efficiently phagocytes the particle. Nano complexes can avoid such phagocyte capture and non specific immune stimulation and thus increases the half life by providing the better stability to the complex and extended blood circulation with the low toxicity. Conjugation of PEG (polyethylene glycol ) to nano-DNA complex helps to a successful transportation across the cell membrane and gives protection against proteolysis. This complex has increased pH and thermal stability. This results in the increased effective potency, improved response to drugs and diminished side effects.

Mucosal vaccines currently been investigated using a broad spectrum of nanocarrier systems are liposome, water in oil emulsions, multiple emulsions, polymeric nanoparticle, alginate coated chitosan nanoparticle, nanoparticulate vesicular formulations, micro and nano particles, cationic nanoparticle and dendrimers ( Shahiwala et al., 2007 ).

Liposome is an organized phospholipids vesicle that has been used to encapsulate protein and DNA based vaccines( Chikh and Schutze Redelmeier, 2002 ). Considerable evidence suggests that liposome or suspensions of lipids and/or phospholipids can exert the immunomodulatory effects when introduced into the body as a vaccine adjuvant. Polymerized liposome have been modified with a targeting molecule selected from a group consisting of antibodies and antibody fragments, antigens and other molecules capable of binding to the specific cell surface receptors found in the mucosal tissue. Nanoparticles carriers for use as vaccines have also been from lipids or other fatty acids and further it has been observed that the nanoparticle vaccines having multivalent surface antigen or encapsulated antigen elicit significantly increased immune responses.

Emulsions have also been explored for the vaccine delivery systems ( Shahiwala et al., 2007 ). Enveloped virus or a recombinant protein antigen is simply mixed with nano emulsions and used. The mechanism depends on the small size and high potential energy of the nano emulsion droplets and the efficiency by which this material is endocytosed by cells. Nanoemulsion based mucosal vaccines appear to require fewer doses to achieve the effective immune responses than traditional vaccines. The nano emulsion even appears to stabilize the antigen and because it is antimicrobial it may alleviate the need for other preservatives or refrigeration and can present the accidental cross contamination between vaccines. Polymeric nanoparticles comprise adjuvant and an immunogenic antigen together encapsulated in a solid hydrophobic nano spheres in moisture sensitive or pH sensitive polymeric micro spheres ( des Rieux et al., 2006 ). Polymeric nano particles are also made up of a cocktail of different plasmid DNAs encoding corresponding antigens. Micelles have been well investigated as potential antigen carriers. The structure consists of PEG that prevents protein adsorption, cellular adhesion and removal from endothelial system. The core is hydrophobic that consists of poorly water soluble and less biocompatible toxic drugs. The side chains of the micelles can easily be adjusted which facilitate drug incorporation and their controlled release.

Source:Role of Nanoparticles in Plant Molecular Farming

V. A. Bapat1 . G.B. Sunil Kumar2, J.P. Jadhav1. S.P. Govindwar3 and T.R. Ganapathi2 * Plant genetic transformations and molecular markers (Ashwani Kumar eds) 2009  pp 288 Pointer Publishers, Jaipur   email: Price Rs 1800   or $ 90