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=Nanotechologies in DNA, Drug delivery, and Tissue regenerations=

Abstract
Nanotechnologies have been used in numerous areas such as in medical fields; they give some remarkable applications in terms of DNA robots, which tailors specific piece of DNA for specific treatments. The robots are able to work at nano scales which make surgery process a lot easier. To increase the concentrations of drugs and to have better effects, nanoparticles are used to enclose the drugs in a capsulate and they are send to the body to target the specific cancer cells. multiple studies have shown the great effects of this capsule. Lastly, tissue regeneration also benefits from nanofiber. The nanofibers resemble the large surface of extracellular matrix; thus they are able to give support and helps cells to proliferate.

Introduction
Nanotechnology is study of matter on a molecular or atomic level; usually matters are defined from sizes 1-100 nanometers. To picture this, it is about 3-5 atoms wide, that why in ancient Greek, “nano” is referred to as “dwarf” (Stylios, Giannoudis & Wan 2005). Matters on such small scales have been expanding remarkably fast; it has potential usages from electronic to construction to energy production and to medication. In medical field, it promises revolutionary applications, such as manipulation on DNA, drug delivery, tissue regenerations, and much more (Zhang et al. 2005). The uses of nanotechnology have offered so many possibilities that many investments and techniques are still being imagined while others are at their testing stages (Stylios, Giannoudis & Wan 2005).

Nanotechology and DNA
Deoxyribonucleic acid or DNA is a double helix that encodes the genetic materials that used in the development of all living things. The fundamental base pairs influenced the genetic expressions, and missing pairs may result in many serious diseases. Nowadays, therapies have been involved to manipulate the individual genes or any other possibilities that might have an influence on the DNA expressions. Since DNA is on a very small scale, a small tool is needed for scientists to make such treatments on diseases. Many different researches have been done to invest the DNA nanotechnologies, and one area that has been focused on is DNA robots or DNA walkers. The DNA robots are used to tailor the specific DNA pieces to give specific treatments. Milan and his research team have named such robots “ molecular spiders.” The spiders’ different legs have different functions such as the fourth leg is used s a ‘tether-release’ system. They are also able to perform functions such as ‘start’, ‘stop’, ‘turn’, and ‘follow’(Sealy 2010). The spiders are able to tailor treatments based on the genetic makeups and able to “cut” a piece of DNA bases for examination and perform “surgery” based on the needs. The convenient of those robots can repair inside the nucleus and inside the cells to target the individual parts. Those DNA based nano-robots are wisely invested because of its high potentials in the medical worlds. Another use of DNA robots is to target the cancer cells in the body (Sealy 2010).

Drug Delivery
According to World Health Organization, cancer is one of the leading deaths and each year over 7.6 million deaths are reported. Chemotherapy is the beginning of cancer treatment, and it is often incorporates with anti-cancer drugs and cytotoxic agent. However, cancer chemotherapies often have many side effects. To limit the side effects, nanotechnology come in to rescue again. One of the remarkable applications of nanotechnologies in medicine is drug deliver. The specific drugs are being engineered in an enclosed nanoparticle, so it can attack the diseased cells; this device allows the drugs to have direct contact with diseases. The technique not only increases the effective of the drugs, but it also reduces some damage to healthy cells in the body (Ma, Kohli & Smith 2013). Multidrug resistance (MDR) is one of the major obstacles in treating the cancer cells, because many cancers have developed resistance to chemotherapy due to many drug exposures (Emilienne Soma et al. 2000). Also, due to lack of selectivity, chemotherapy drugs always have some side effects, which end up damaging normal organ cells in the body. To overcome this problem, drug delivery system is invested. Drug delivery system, DDS, is a nanoparticulate system that has been used to target cancer cells in the body to avoid the damage and to increase the effectiveness of the drugs to specific cancers (Kibria 2013). For instance, DDS deliver the specific inhibitor to the cancer sites by encapsulating the inhibitors in nanoparticles, which are modified with specific ligands to direct them to specific cells. This specificity function can down-regulate the expression of the MDR proteins. Sometimes, cytotoxic drugs, which are used in chemotherapy to inhibit the proliferation of cancerous cells, are delivered con-currently or separately with the inhibitors to the cancer site. One study has shown the resistant cell culture experiment due to the combination of both cyclosporin A and doxorubicin (chemotherapeutic agent) within a single nanoparticles. The formulation has the most effective growth rate of inhibition as compared to the free cyclosporin A or doxorubicin alone in the leukemia cells (Emilienne Soma et al. 2000). Below figures showed the structures of cyclosporin A or doxorubicin (Fliri et al. 1993) (Mahmud, Xiong & Lavasanifar 2008).

[[image:structure of cyclosporin A.jpg]], [[image:structure of doxorubicin.jpg]]
Figure 1: chemical structure of cyclosporin A and doxorubicin (Fliri et al. 1993) (Mahmud, Xiong & Lavasanifar 2008)

Another study was done to study the K562 leukemia cells. The design of this study was aim at selective targeting of cancer cells and at same time to reduce the cardiotoxicity drugs, as well as over the multidrug resistance. They used conjugated liposomal which contained both doxorubicin and verapamil that were selectively targeting the drug resistance in K 562 cells. The outcome resulted in a higher accumulation of doxorubicin in a doxorubicin resistant K562 cells (Wu et al. 2007). Based on those two studies, it is fair to conclude that selective delivery of encapsulated inhibitors in a nanoparticles give some promises for cancer treatments. Besides inhibitors, gene delivery in another powerful tool in cancer therapy; gene delivery controls the activity of specific gene that interfere with RNA interference (RNAi) (Panyam, Labhasetwar 2012). RNAi is also known as post-transcriptional gene silencing (PTGS) because RNA molecules inhibit the gene expression and cause the destruction of specific mRNA (translates genetic information from DNA to ribosome that directs amino acid sequences to protein) molecules. In tumors, the transporters from membrane play the role of distributing and excreting chemotherapeutic drugs. Recently, attempts were made to control the expression of transporters by delivering nucleic acids that are loaded with nanoparticles to tumor cells. In one of MDR cancer studies, it showed that siRNA could silence the MDR-1 by reducing the MDR-related proteins. Si-RNA are short interfering RNA that interfere with specific gene expression (Liu et al. 2009). To make deliveries toward more specific cancer cells, nanoparticles come in play again. siRNA and doxorubicin are encapsulated in a nanoparticle and was delivered to the tumor cells; the delivery had silence the MDR-related proteins, specifically the P-glycoprotein (P-gp), and this resulted an increase of intracellular concentration of doxorubicin (Meng et al. 2010). Figure 2 displayed the fluorescent intensity of doxorubicin in the nuclear cells. Free doxorubicin was used as control and it is obvious from the chart that siRNA-PEI-Dox-MSNP (mesoporous silica nanoparticles) has the strongest intensity. The data illustrated that the enclosed nanoparticles has successfully delivered doxorubicin and Pgp siRNA to a drug resistant cancer cells. The dual delivery was capable of increasing the intracellular and intranuclear drug concentration levels (Meng et al. 2010). Some other study had also compared the freed siRNA verses encapsulated siRNA in a nanoparticles in the cellular uptake, and the result shown that the free siRNA’s stability was way shorter than the enclosed siRNA in the cell. The free siRNA was unstable in serums and the stability of siRNA could be improved by delivering the material in a nanoparticles (Gao, et al. 2009).

[[image:figure 1 copy.jpg]]
Figure 2: Quantitative analysis of the fluorescence signals in the nuclei (Meng et al. 2010). Further more, co-delivery has also been studied, and results were significantly remarkable. In this study, the nanoparticles were made of a biodegradable amphiphilic copolymer, and the co-delivery of paclitaxel with an interleukin-12-encoded plasmid in a nanoparticle have overpowered cancer growth more efficiently than the delivery of either the plasmid or paclitaxel alone in a 4T1 mouse breast cancer model. Moreover, the co-delivery of paclitaxel with siRNA has decreased the MDR activities (Wang et al. 2006). As mentioned above, there are numerous remarkably advantages of nanoparticles in drug delivery path in the body. But one has to notice that there are different properties in the free drugs and the drugs that are enclosed in a nanoparticles. Generally, free drugs are diffused across the cellular membranes, and the efflux pumps on the cell membrane surface can detect the free molecules as they make their ways cross the membrane. Some of the free molecules are pumped out of the cell, as little of them actually go to the targeting cells (figure 3) (Kibria 2013).

[[image:figure 2.jpg]]
Figure 3 shows the route of free drugs vs. drugs that are encapsulated in a nanoparticle (Kibria 2013).

To over this problem, nanoparticles come in place to increase the efficiency of the drugs. The nanoparticles enclosed the drugs and deliver them to the specific cellular internal organelles; they cross the membrane and were able to hide themselves to avoid being pumped out of the cells. This type of activity is known as “stealth endocytosis,” and it results in higher concentration of drugs in the target cells (Kibria 2013). As mentioned above, in chemotherapy repairs, drugs are sending in the combination of chemotherapeutic agent and other drugs. If the individual drug is sent to body alone, then it requires some kind of toxic solvent, which will be harmful to the body. In the right ratio of combination of drugs in a nanoparticles, the drugs can be delivered harmlessly and has a higher concentration amount after injection compared to the free drugs, and figure 4 proved this point too (Ma, Kohli & Smith 2013). That is why from the above experiments, the drugs are delivered together in a nanoparticle to achieve a higher efficiency rather than deliver the drug freely.

[[image:figure 3 copy.jpg]]
Figure 4: combination drug therapy exhibits higher concentration in the body compared to the free drugs ((Ma, Kohli & Smith 2013).

Tissue regenerations
Besides drug delivery, there are vast amount of applications in nanotechnologies. Another essential usage of nanotechnology in medical industry is polymer nanofibers. Nanofibers have diameter less than 100nm, and they are usually manufactured by a technique called electrospinning, and a picture of nanofibers can be shown in figure 5 (Zhang et al. 2005).

[[image:figure 4.jpg]]
Figure 5: SEM photograph of electrospinning nanofiber (Zhang et al. 2005).

According to American Burn Associate, more than 500, 000 people receive burn wounds in the United States every year. Among these patients, numerous require alternatives to fix the wounds. To heal from those wounds in a timely manner, patients would benefit greatly from advanced tissue regeneration technologies, such as nanofiber. Nanofibers are very similar to human’s natural biological tissues, thus this area is being researched intensively. Nanofibers are very useful because of their surface areas and energy is a lot higher compared to bulk materials, which allows for “enhanced adhesion with cells, proteins, and drugs” (Leung, Ko 2011). An excellent application of nanofiber is tissue regeneration. In surgeries, common obstacles are autografts and allografts (Mata et al. 2010). Autografts occur when treatments are depended on the identical genetic origin; this can create problems because it depends on the donor sites and availability. Allografts are when the recipient bodies reject the donor organs due to immune responses of foreign unrecognized materials. To overcome these obstacles, new tissue generation technologies are developed to obtain a “biocompatible platform” that can be used as a temporary host which are able to “attach, proliferate, and differentiate into the specific tissues that needs to be repaired” (Leung, Ko 2011). The availability of large surface areas of nanofibers also resembles the extracellular matrix (ECM) that provides structural and biochemical support to the surrounding cells (Tuzlakoglu et al. 2005). The ability to mimic the ECM and form scaffolds is the beginning of tissue regenerations. As mentioned above, nanofibers can be made from electrospinning; it can also be made from self-assembly and phase separation. Electrospinning generates larger nanofibers in diameters than self-assembly. The larger diameter nanofibers are on the upper end of ECM collagen, while the smaller diameters are on the lowest end of ECM collagen. The diameters of nanofibers that are made from phase separation are similar to the natural ECM collagen. Phase separation allows the three dimensional network into the scaffolds (Smith, Ma 2004). The nanofibers scaffolds then act as a host cell to perform the necessary activities, such as “to help cells attach, proliferate, or differentiate” (Leung, Ko 2011). When tissues are damaged, the structural support of ECM is loss. New ECM can be regenerated at the site, but lack of connectivity of tissues can slow the healing process. Thus the temporary nanofiber scaffolds come in play; it regenerates the tissue cells at a greater speed, thus increase the healing process. The scaffolds must have two requirements: 1) surface compatibility and 2) structural compatibility. Surface compatibility as in terms of surface chemistry in the cell life, such as signaling, regulating and adhesion. The structural compatibility is in terms of able to support cells’ migration and supporting the surrounding cells. Also, the scaffolds must be biodegradable, so that during the sealing process, the cells are able to synthesis new ECM (Leung, Ko 2011). Nanofibers for tissue regenerations are highly available due to their flexibilities and resources. Studies have shown that chitosan can help to aid blood clothing (Jayakumar et al. 2010), and different techniques for electrospinning can enhance the nanofiber scaffold performances (Leung, Ko 2011). Proteins can also be added to the scaffolds to enhance their properties and performances. Koh and his team has invested adding laminin onto nanofibers using covalent binding, and cells and neurite outgrowth assays proved that the nanofibers were able to enhance axonal extensions (Koh et al. 2008).As in skin tissue regeneration, Leung and his team has found that silk, collagen, chitosan, and alginate can be used as potential skin tissue scaffold. They also electrospun sodium alginate nanofiber with ethylene oxide to aid the fiber formations; the results was that the new nanofibers could crosslink with calcium nitrate to enrich the structural properties in some aqueous environments. They also added poly(l-lysine) to their scaffolds to increase the cell attachments (Leung, Ko 2011). The products can be used to treat some several wounds or burns. His team has also demonstrated that to enhance the properties with tissue cells, nanofiber scaffolds can be modified. This allows the tissue cells to attach and proliferate to fill in the defect injury site. Not only that, but it is possible to pre-seed the tissues onto the scaffolds and then apply the scaffolds to the injury sites which can be a great benefit to the severe injuries. ((Leung, Ko 2011). Besides skin regenerations, nanofiber scaffolds can also be used in stem cells. By optimizing the materials and electrospinning process, the growth factors can be carefully chosen to treat the specific range of tissues with the stem cells. Janjanin and her group demonstrated the mesenchymal stem cell (MSC)-based as an alternative to cartilage grafting. They basically let MSCs seeded into the biodegradable nanofibrous scaffolds and engineered it into cartilages with defined dimensions and shapes for easier use than grafts. The research result was that the molding system of nanofibrous scaffold has a higher functional matrix, and it supports efficient cell differentiation (Janjanin et al. 2008). Another research group had a similar experiment as Janjanin’s; Li and his teammates compared chondrogenic activities in cell pellet and bone marrow-derived MSC in nanofiber scaffolds. They found that cell pellet culture system does not give the mechanical properties that they needed for cartilage repairs; the properties were weak and fragile. However, when they seeded the bone marrow-derived MSC into nanofiber scaffolds, the results were stunning. With nanofibers scaffold, the level of MSC chondrogenesis is enhanced and they provided better mechanical stability that can be used for cartilage repairs (Li et al. 2005). Polymer nanofibers are also great use in bone regenerations, and they are often mixed with calcium phosphate to enhance the scaffold matrix properties by giving a better compressive strength (Ramay, Zhang 2004). Bone morphoenetic protein-2 (BMP-2) is known to be able to help with bone healing process, and hydroxyapatitie is known to enhance attachment to bone cells (Leung, Ko 2011). The presence of hydroxyapatite can help BMP-2 to be released in the bone cells. In one study, the researchers implanted the scaffold with hydroxyapatitie-composed nanofiber with BMP-2 in mouse cells, and the injuries in the bone cells healed faster (Nie et al. 2009). Because of BMP-2’s bone healing effects, it can also be used in orthopedic implants. If a nanofiber scaffold can control the release of BMP-2, then it can be processed into a formulation that can be delivered to the injury implant site (Nair, Laurencin 2008). Another research group has used poly-L-lactic acid (PLLA) in their nanofibers because PLLA is biodegradable, biocompatible, and FDA approved. The group has incorporated PLLA nanofiber with BMP-2 in the implants, and this scaffold was able to heal some seriously injured calvarial defects within eight weeks (Schofer et al. 2011). In figure 6, PLLA/BMP-2 showed faster results than any other bone regenerations. The nanofibers in orthopedic implants have to be made of carbon nanofibers in order to reduce the weakness in the bone implant (Price et al. 2003). Study has reported that decrease in carbon fiber size on the nanofibers can increase the osteoblast cell adhesion and at same time increasing the fiber surface energy. Basically, the osteoblast adhesion on carbon nanofiber seemed to have a higher concentration than the other polymer nanofibers (Price et al. 2003).

[[image:figure 5.jpg]]
Figure 6: Formation of new bones in relation of defected areas (Schofer et al. 2011).

Conclusion
In conclusion, tremendous efforts have been made recently on the nanotechnologies in medical fields. There are so many potentials that many investments are still on going or in process. Nanotechology has made DNA robots possible; the robots are able to “walk” or “work” with specific pieces and able to give treatment to the defected pieces. Nanoparticles are made to give incredible results in cancer treatments. Not only can they increase the effective of drugs in the body, they are also able to deliver the drugs to specific areas of body and safely transport the drugs to their desired destinations. Nanoparticles improved drug solubility, reduced toxicity in the body, and reduce many side effects of cancer treatments. Lastly, nanofibers can made wounds heal faster and they are excellent in terms of tissue regenerations. The large surface areas of nanofibers resemble the extracellular matrix (ECM) that provides structural supports and helps cells to proliferate. Studies have also concluded that carbon nanofibers are stronger than the other nanofibers in terms of support and mechanical properties, and carbon nano sizes are related to cell adhesions and energies. With specific size and dimensions of nanofibers, they can enhance the properties in terms of mechanical supports in maintaining and supporting different organ tissue functions.

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