**1. Introduction**

150 Energy Efficiency – The Innovative Ways for Smart Energy, the Future Towards Modern Utilities

algorithm presented above without major implications.

**Author details**

Bjørn R. Sørensen

**7. References**

*Narvik University College, Norway*

Most commercial controllers allow for programming and are thus able to incorporate the

**Figure 12.** Fan static pressure difference control system (supply side only). The CO2 concentrations of the zones (or exhaust) are used to determine the degree of set point variation (represented by Kpath).

Daly, B. B. (1988). *Woods Practical Guide to Fan Engineering.* Woods of Colchester Limited. Eck, B. (1973). *Fans: Design and Operation of Centrifugal, Axial-flow and Cross-flow Fans*. 1st

Lorenzetti D. (1993). Modelling Adjustable Speed Drive Fans to Predict Energy savings in VAV Systems. *Proceedings of the 14th AIVC Conference*, Copenhagen, Denmark. Sørensen, B. R. (2011). An Energy Efficient Control Strategy for Fan Static Pressure Difference, *Proceedings of the International Conference on Information Engineering and*

Sørensen, B. R. (2010). A Model Library for VAV Systems, *Proceedings of the 10th REHVA*

Sørensen, B. R., Riise, R. (2010). Rotary heat exchanger model for control and energy

Sørensen, B. R. (2008). Rotary heat exchangers and VAV systems. *Proceedings of the 11th international conference on indoor air quality - Indoor Air 2008*, Copenhagen, Denmark. Sørensen, B. R. (2006) Demand Controlled Ventilation - A detailed study of energy usage by

Sørensen, B. R. (2006) Modelling and simulation of solar and atmospheric irradiation of

*World Congress – Clima 2010*, Antalya, Turkey. ISBN<PHONE_NUMBER>-14-6.

simulation. *Proceedings of the 27th annual AIVC conference*, Lyon, France.

Buildings. *Proceedings of the Cold Climate HVAC 2006*, Moscow, Russia.

calculations. *Proceedings of AIVC 2010 conference*, Seoul, Korea.

English Edition, Pergamon Press, ISBN 0 08 015872 2.

*Mechanical Engineering (IEME)*, Xianning, China.

The increase of energy efficiency is a general trend in a worldwide relation. According to the Kyoto Protocol from 1997, the EU has to reduce greenhouse gas emission by 8% below the level from 1990 by the 2008 - 2012 period. To achieve these reductions, substantial efforts have to be undertaken in all branches of human enterprise.

One of the important industry utilities that has to be encompassed by this energy policy are compressed air systems (CASs). The application of compressed air has had a growing trend due to its easy and safe generation, manipulation, and usage. In previous years, the research efforts in this domain were concentrated on the CASs development and application aimed at boosting the productivity regardless of the energy consumption. With increased awareness of the energy costs as well as the effects of greenhouse gas emission, the attention has been recently placed on the energy efficient use of compressed air.

The experience gained in numerous CAS optimisation projects, as well as the opinions of the experts in the field, indicated that many industrial systems are missing the chance to improve energy savings with the relatively low costs of projects for increasing energy efficiency. Energy saving measures in CASs that have been identified in the course of energy audits in the small and medium industrial enterprises may yield an average energy saving of nearly 15%, with a payback of two years, the energy saving potential in some of them amounting from 30% up to even 60% (USDOE, 2001). The basis for all decisions concerning energy efficiency of the existing CASs is the understanding of the way of their functioning and existence of appropriate data. In that sense, it would be necessary to make measurements of consumed electricity of compressors, airflow, system leakage and pressure drop in the system.

Besides energy savings, increasing energy efficiency of CASs may ensure other significant benefits for the enterprise. Energy saving measures imply a high monitoring level of CASs and

© 2012 Šešlija et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

appropriate maintenance. That leads to decreased breakdowns of production equipment, avoiding the loss of raw materials or other inputs, longer life cycle of pneumatic devices and higher reliability of CASs. Reduction in energy consumption will also lower the emissions of dangerous and polluting substances, which will lessen the influence on the environment. Often, these benefits are more valuable than the energy savings.

Increasing the Energy Efficiency in Compressed Air Systems 153

2000, of the overall 2,574 billion kWh, 951 billion kWh were used in the industry. Of this, 614 billion kWh, or 65%, was consumed by motor-driven systems. It was estimated that the potential saving could be 181 billion kWh, (29%), or seven percent of the overall electricity

According to the study "Compressed Air Systems in European Union" (Radgen and Blaustein, 2001), the EU-15 was spending 10% of the total electricity consumed in the industry for the production of compressed air. The most important potential energy savings are related to the system installation and renewal (the overall system design, improvement of drives, use of sophisticated control systems, recovering heat waste, improved cooling, drying and filtering, reducing frictional pressure losses, etc.) and system operation and maintenance (reducing air leaks, more frequent filter replacement, etc.). The percentages of potential saving varied from country to country. For instance, Germany spent seven percent, United Kingdom 10%, Italy, France and the rest of EU 11% (Radgen and Blaustein, 2001). Details on potential energy savings can be found in the corresponding references: for Germany in (Radgen, 2003; Radgen, 2004), for Switzerland in (Gloor, 2000), for Sweden in

The electricity consumption of CASs in Chinese enterprises goes from 10% up to 40% (Li et al., 2008) of the total industrial electricity consumed. According to (Li et al., 2008), the most widely used compressors in China are reciprocating compressors, often several decades old. To meet increased compressed air demands, many enterprises have undertaken retrofits of their CASs, yielding increased compressor capacity, improved system piping, etc. The most frequently implemented energy saving measures are: purchasing rotary screw compressors, application of variable speed drives and changes to the piping system to allow centralized

Based on data of the Electric Power Industry of Serbia (EPS, 2009a), the amount of electricity consumed in Serbia in 2008 was 27,639 GWh. Industrial CASs installed in Serbia consume about 8% of the electricity used by industry (Šešlija et al., 2011). Although this percentage is low compared to the values reported for some other countries (Radgen and Blaustein, 2001; Radgen, 2003; Radgen, 2004), this does not mean that the CASs in Serbia are more efficient. This low consumption percentage is a consequence of the inefficient electricity utilization in the industry, the value of energy intensity being three times higher than in the developed European countries (USEIA, 2006). Besides, the price of electricity in Serbia is relatively low,

There is a high potential for increasing energy efficiency of CASs in Serbia. One of possible ways of increasing the energy efficiency of CASs is the replacement of the reciprocating single-acting compressors with rotary screw compressors, which would reduce the CAS

(Henning, 2005), and for Austria in (Kulterer and Weberstorfer, 2007).

and it does not exist appropriate attention to its economic utilization.

consumption (De Keulenaer et al., 2004).

**2.3. State of energy efficiency in China**

production of compressed air, etc.

**2.4. State of energy efficiency in Serbia**

This paper is concerned with the identification of the current state of energy efficiency in the production and usage of compressed air and possibilities for improvements that would yield the corresponding energy saving.


*Edited by Md. Zahurul Haq*

Measurement is a multidisciplinary experimental science. Measurement systems synergistically blend science, engineering and statistical methods to provide fundamental data for research, design and development, control of processes and operations, and facilitate safe and economic performance of systems. In recent years, measuring techniques have expanded rapidly and gained maturity, through extensive research activities and hardware advancements. With individual chapters authored by eminent professionals in their respective topics, Advanced Topics in Measurements attempts to provide a comprehensive presentation and in-depth guidance on some of the key applied and advanced topics in measurements for scientists, engineers and educators.

Photo by Rost-9D / iStock

Advanced Topics in Measurements

Advanced Topics in

Measurements

*Edited by Md. Zahurul Haq*


**7. Companion electronic circuitry**

A block diagram of this circuitry is depicted in Fig. 7. All photodetector p-n diodes in the array of FPPD under the channel produce a current whose magnitude contains information related to light intensity. Furthermore this light intensity, which is absorbed by the photodiodes, depends on the content of the chemicals present in the micro-channel fluid. The main purpose of this electronic circuitry is to collect, condition, and interface these current signals to the post processing circuit. Since the information signals are in the form of diode currents, it is preferred to work with current mode (CM) electronic circuits.

Fig. 7. Companion Electronic circuit connects array of FPPD to the microprocessor


*3.2. Antioxidant Activity (DPPH and ABTS Method)*

Figures 3 and 4 report the results of antioxidant activity, obtained with DPPH and ABTS antiradical activity methods, of differently obtained carrot and apple juices, respectively.

**Figure 3.** Antioxidant activity (DPPH and ABTS method) of carrot juices obtained from pretreated mash. Different letters indicate significant differences (*p* < 0.05) between samples.

**Figure 4.** Antioxidant activity (DPPH and ABTS method) of apple juices obtained from pretreated mash. Different letters indicate significant differences (*p* < 0.05) between samples.

For carrot juice (Figure 3), a significantly higher antioxidant activity was obtained for carrot juices preheated to 80 ◦C with or without additional PEF or OH treatment with the ABTS method. Higher retention of bioactive compounds with DPPH method was observed for carrot mash pretreated at 80 ◦C coupled with PEF treatment. However, detected with DPPH method, the application of OH treatment reaching 40 and 60 ◦C reduced the antioxidant activity, in comparison with that of juice from the untreated control carrot mash. With ABTS method, no significant difference was found (Figure 3).

Significantly higher antioxidant activity, detected with both DPPH and ABTS methods, was obtained for apple juices preheated to 80 ◦C with or without additional PEF or OH treatment (Figure 4). Instead, OH reaching 40 ◦C and 60 ◦C reduced the antioxidant activity, with both used method, compared with that of the apple control juices, which might be due to the activation of degradative enzymes, such as peroxidase and polyphenoloxidase that lead to bioactive compounds' oxidative degradation.

Fruits and vegetables are good sources of natural antioxidants, containing carotenoids, vitamins, phenolic compounds, flavonoids, dietary glutathione and endogenous metabolites. However, the majority of the antioxidant activity of fruits and vegetables is derived from phenolic compounds (hydroxycinnamic acids, flavan-3-ols, anthocyanidins, flavonols and dihydrochalcones) rather than vitamin C and E, or β-carotene, due to their stronger activity against peroxil [27]. The peroxidase and polyphenoloxidase enzymes lead to the degradation of phenolic compounds and a subsequent loss of nutritional and sensorial values such as browning and off-flavour [28]. Moreover, heat treatment may influence the binding properties of bioactive compounds causing their higher release, but at the same time increase enzymatic or nonenzymatic degradation processes that can cause subsequent negative effects on quality of processed products [15].

In fact, higher temperature leads to the inactivation of the oxidative enzymes, thus reducing degradation effects and resulting in higher antioxidant activity in the juice. Moreover, additional effects other than those from thermal treatment need to be taken into account, since the electropermeabilization, induced by PEF and OH treatment, may contribute to the increased release of antioxidant compounds.

The detected difference between the two different methods used could be due to the fact that DPPH method is more sensitive to detect flavanones, while ABTS for the radical scavengers such as vitamin C [29].

For carrot and apple juice, PEF treatment without preheating did also not affect the extractability of bioactive compounds, which is in accordance with Shilling et al. [15], who reported no significant differences on total antioxidant activity between control and PEF-treated apple mash for different electric fields (1, 3 and 5 kV/cm).

#### *3.3. Enzyme Activity*

#### 3.3.1. POD Activity

Process pretreatment for the juice production is an important operation in order to improve the quality of the vegetable and fruit raw materials as well as to avoid the activation of degradative enzymes such as POD and PPO that consequently provoke pigments and nutrients loss [30].

yy j g

Peroxidase (POD) activity for differently obtained carrot juice is shown in Figure 5.

**Figure 5.** Peroxidase (POD) activity in carrot juice obtained from pretreated mash. Different letters indicate significant differences (*p* < 0.05) between samples.

This study revealed that the effect of only PEF treatment at 20 ◦C and 40 ◦C preheating could not reduce the activity of POD in both carrot (Figure 5) and apple juice [26], while for the samples preheated at 60◦C, the reduction of POD activity was observed.

The highest POD inactivation could be reached by preheating to 80 ◦C with and without additional PEF or OH treatment. All pretreatment conditions with temperature at or up to 60 ◦C and 40–80 ◦C by OH treatment led to a decrease in POD activity in the carrot juice, while for apple juices, a greater reduction of the activity (from 50% to 90%) was achieved by 60 and 80 ◦C preheating temperatures with and without additional PEF or OH application. Enzyme inactivation in the juice, after higher PEF treatment intensities, for microbial inactivation and preservation purposes, is mainly related to secondary effects such as local temperature distributions, electrochemical reactions or formation of free radicals, instead of primary effects of electric field. For the treatment of mash, the PEF treatment intensity can be considered 10-fold lower and having no direct effect on fruit and vegetable mash ingredients.

POD activity decreased with increasing temperature, and almost no POD activity was detected in juice extracted when the treatment temperature reached 80 ◦C, especially in apple juice samples. High temperature leads to an increase in the internal energy of the enzymes, thus consequently causes the break of bonds that determine the three-dimensional structure of enzymes [18].

Moreover, with the increasing of temperature, the enzyme activity decreased and required a particular temperature–time combination for complete inactivation. Inadequate temperature led to a decrease in the enzyme activity time rather than complete inactivation, which may cause browning effect. In fact, Bhat et al. [18] reported similar results for OH-treated bottle gourd juice, where the temperature of 60 and 70 ◦C seemed to be not enough for complete enzyme inactivation, which instead was observed at 80 ◦C for 4 min.

Icier et al. [31] showed that OH treatment could be used for POD inactivation on pea puree at the range of 30–50 V/cm combined with the water blanching. Elez-Martínez et al. [32] reported a completely POD deactivation in orange juice after the application of PEF treatment at 35 kV/cm for 1500 μs.

Moreover, the variations of colour for carrot and apple juice pretreated with 80 ◦C with both PEF and OH applications could be explained by the decrease of enzyme activity, in fact, a correlation between colour and POD activity was found to be 0.8902 and 0.5166, respectively, for carrot and apple juice.

#### 3.3.2. PPO Activity

Polyphenoloxidase (PPO) activity of differently obtained apple juice samples is shown in Figure 6.

**Figure 6.** Polyphenoloxidase (PPO) activity of apple juice obtained from pretreated mash. Different letters indicate significant differences (*p* < 0.05) between samples.

PPO is an oxidoreductase enzyme which catalyses the oxidation of phenolic compounds in o-quinones, which are subsequently polymerized into brown pigments [33]. Heating treatment seems to be the most effective applied treatment for the stabilization of food products against microbial and enzyme activity. Nevertheless, thermal treatment has been shown to cause negative effects on quality and related nutritional compounds [9]. The mechanism of enzyme inactivation is not completely clear. Current results show empirical proof of protein modification by electrical fields [34] that may provoke a deformation or structural change of a protein, due to the interaction between the external electric field and the functional groups of the protein that allow its unfolding [32].

PPO activity was significantly decreased by PEF application at room temperature compared with that of the untreated control sample. In addition, a greater inactivation was achieved when the treatment temperature reached 80 ◦C, as well as with OH treatment and just preheating. Moreover, PPO inactivation was even more effective when a combination of temperatures and PEF or OH applications were used.

Similar results were reported by Turk et al. [35]. PPO activity was reduced in apple cider mash pretreated with PEF at 1 kV/cm for 100 μs. The loss of PPO activity could be explained also by the inhibition of the enzyme with oxidised phenolic compounds, in particular procyanidins [36].

Previous work reported similar results for PPO deactivation; the residual PPO activity was 35% after 14 min with OH treatment at 70 ◦C, by applying 35 V/cm in grape juice [30].

Liang et al. [37] found a significant decrease (33%) in PPO activity in freshly squeezed apple juice when preheated at 50 ◦C and treated with PEF at 27 kV/cm for 58.7 μs.

Saxena et al. [20] found a reduction of PPO activity up to 97.8 % by applying 32 V/cm with OH treatment at 90 ◦C for 5 min in sugarcane juice. Moreover, a greater increase in residual PPO activity was visible at 90 ◦C by increasing the holding times of OH treatment (5, 10, 15 and 20 min). The increase of the enzyme activity with the holding time at constant temperature was attributed to the pulsating OH treatment that may cause biochemical reactions by changing the molecular spacing and may result in a better interaction between substrate and enzyme [38]. A recent review on the impact of electric fields on enzymes is provided by [39].

#### **4. Conclusions**

Obtained results emphasize the role of thermal treatment for the inactivation of enzymes, reflected by improved colour values for juices exposed to 80 ◦C, independent of the PEF or OH application. The inactivation of POD and PPO was more pronounced when a temperature of 80 ◦C was achieved for both carrot and apple mash (around 90%).

However, a better retention of plant secondary metabolites from carrot and apple mashes could be achieved by additional PEF or OH application. PEF treatment was found to improve the release of such compounds, whereas OH contributed to a very fast volumetric heating that reduced the overall thermal load that the sample was exposed to. Based on the results, a combination of thermal and electric field pretreatments is required for the controlled release, inactivation and retention of ingredients. Thermal effects contributing to the colour, bioactive compounds retention and enzyme inactivation were found to be still important when applying nonthermal cell disintegration techniques such as PEF. However, both electrotechnologies, PEF and OH were found to positively contribute to improved juice quality by enhanced ingredient release and retention.

**Author Contributions:** C.M., T.F. and H.J. conceived and designed the experiments; C.M. and K.R. performed the experiments; C.M., K.R. and U.T. analysed the data; H.J. contributed reagents/ materials/ analysis tools; S.R. as Supervisor; C.M. contributed Writing-Original Draft Preparation; C.M., T.F., U.T., S.R., M.D.R. and H.J. contributed Writing-Review and Editing.

**Funding:** This work was partly supported by the Austrian Research Promotion Agency (FFG Project No. 859077).

**Acknowledgments:** Part of the equipment used in this study was financed by EQ-BOKU VIBT GmbH and belongs to the Center for Preservation and Aseptic Processing. Cinzia Mannozzi acknowledges the Marco Polo Programme (University of Bologna) for the travel grant.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*


**NANOWIRES - SYNTHESIS, PROPERTIES AND APPLICATIONS**

#### **Nanowires - Synthesis, Properties and Applications**

http://dx.doi.org/10.5772/intechopen.75337 Edited by Simas Rackauskas

#### **Contributors**

Michael Koblischka, Uttam Kumar Das, Tarun K. Bhattacharyya, Kien Wen Sun, Muthaiah Shellaiah, Natasha Ross, Shrok Allami, Dereje Seifu, Aiyeshah Alhodaib, Anthony Krier, Andrew R.J. Marshall, Yasir Noori

#### **© The Editor(s) and the Author(s) 2019**

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**3. Delivery systems**

Over the past 20 years, a variety of techniques have been developed for encapsulating both conventional drugs (such as anticancer drugs and antibiotics) and new genetic drugs (plasmid DNA containing therapeutic genes, antisense oligonucleotides and small interfering RNA) within nanoparticles. Nanoparticle delivery systems for gene delivery possess useful inherent attributes, including a diameter of approximately 100 nm or less, a high drug-to-lipid ratio (in the case of lipid based systems), excellent retention of the encapsulated drug, and a long (> 6 h) circulation lifetime. These properties permit nanoparticles to protect their contents during circulation, prevent contact with healthy normal tissues, and accumulate at sites of disease.

### **3.1 Viral and non-viral delivery systems**

A major impediment to the successful application of gene therapy for the treatment of a range of diseases is not a paucity of therapeutic genes, but the lack of an efficient non-toxic gene delivery system. Gene delivery is generally accomplished using one of two categories of delivery vector: 1) Viral delivery vectors derived from engineered retrovirus, adenovirus, herpesvirus, lentivirus or hybrid retro/adeno virus), and 2) Non-viral (synthetic) vectors comprised of polymers and lipids, naked DNA, plasmid-protein conjugates. Other (physical as opposed to chemical) methods include microneedle coating, gene gun, and ultrasound/microbubble-mediated gene delivery.

Viral vectors account for nearly 75% of all clinical trials conducted thus far (http://www.wiley.co.uk/genetherapy/ clinical). These vectors are essentially viruses that have been genetically modified to remove any replication/pathogenic genes, and instead encode a gene of interest (Hesdorffer et al. 1998). This strategy preserves the viruses highly efficient ability to infect cells, while eliminating/attenuating toxicity. The most commonly used viral vectors are retroviral, adenoviral, adeno-associated viral and herpes simplex viral-based (Young et al. 2006). Although Vitravene, an antisense oligonucleotide-based product, is the only gene delivery product approved so far by US-FDA, there are several other products in late stages of clinical trials. Gendicine, an adenovirus encoding the *p53* gene, developed by SiBiono GeneTech Co., Ltd., was recently approved by China's state Food and Drug Administration, for the treatment of head and neck squamous cell carcinoma. Virus-based treatments tend to maximize efficiency of gene transfer, often at the cost of safety, while non-viral options generally capitalize on a higher safety profile, but usually at the expense of Gene of Interest (GOI) expression efficiency. Many viruses including retroviruses, adenoviruses, herpes simplex viruses, adeno-associated viruses (AAV) and pox viruses have been modified to eliminate their toxicity, maintain their high gene transfer capability and long term gene expression.

nanoparticles. Third, to avoid P-glycoprotein-mediated drug efflux, they further designed another delivery vehicle, LPD-II, which showed much higher entrapment efficiency of Dox than LPD. Finally, the authors delivered a therapeutic siRNA to inhibit MDR transporter. Three daily intravenous injections of therapeutic siRNA and Dox (1.2 mg/kg) co-formulated in either LPD or LPD-II nanoparticles showed a significant improvement in tumor growth inhibition (Chen et al. 2010a). Numerous other similar studies highlights a potential clinical use for these multifunctional nanoparticles with an effective delivery property and a

Over the past 20 years, a variety of techniques have been developed for encapsulating both conventional drugs (such as anticancer drugs and antibiotics) and new genetic drugs (plasmid DNA containing therapeutic genes, antisense oligonucleotides and small interfering RNA) within nanoparticles. Nanoparticle delivery systems for gene delivery possess useful inherent attributes, including a diameter of approximately 100 nm or less, a high drug-to-lipid ratio (in the case of lipid based systems), excellent retention of the encapsulated drug, and a long (> 6 h) circulation lifetime. These properties permit nanoparticles to protect their contents during circulation, prevent contact with healthy

A major impediment to the successful application of gene therapy for the treatment of a range of diseases is not a paucity of therapeutic genes, but the lack of an efficient non-toxic gene delivery system. Gene delivery is generally accomplished using one of two categories of delivery vector: 1) Viral delivery vectors derived from engineered retrovirus, adenovirus, herpesvirus, lentivirus or hybrid retro/adeno virus), and 2) Non-viral (synthetic) vectors comprised of polymers and lipids, naked DNA, plasmid-protein conjugates. Other (physical as opposed to chemical) methods include microneedle coating, gene gun, and

Viral vectors account for nearly 75% of all clinical trials conducted thus far (http://www.wiley.co.uk/genetherapy/ clinical). These vectors are essentially viruses that have been genetically modified to remove any replication/pathogenic genes, and instead encode a gene of interest (Hesdorffer et al. 1998). This strategy preserves the viruses highly efficient ability to infect cells, while eliminating/attenuating toxicity. The most commonly used viral vectors are retroviral, adenoviral, adeno-associated viral and herpes simplex viral-based (Young et al. 2006). Although Vitravene, an antisense oligonucleotide-based product, is the only gene delivery product approved so far by US-FDA, there are several other products in late stages of clinical trials. Gendicine, an adenovirus encoding the *p53* gene, developed by SiBiono GeneTech Co., Ltd., was recently approved by China's state Food and Drug Administration, for the treatment of head and neck squamous cell carcinoma. Virus-based treatments tend to maximize efficiency of gene transfer, often at the cost of safety, while non-viral options generally capitalize on a higher safety profile, but usually at the expense of Gene of Interest (GOI) expression efficiency. Many viruses including retroviruses, adenoviruses, herpes simplex viruses, adeno-associated viruses (AAV) and pox viruses have been modified to eliminate their toxicity, maintain their high gene transfer capability and long term gene expression.

function to overcome drug resistance in cancer.

normal tissues, and accumulate at sites of disease.

ultrasound/microbubble-mediated gene delivery.

**3.1 Viral and non-viral delivery systems**

**3. Delivery systems**

However, despite these advantages, there are currently major limitations to the use of viruses as gene delivery vectors. These include the limited carrying capacity of transgenic materials (i.e. size of plasmid) since the packaging capacity of viral vectors (usually up to 5 kb) is constrained; the potential for oncogenesis due to chromosomal integration or activation of oncogenes/inactivation of oncogene regulators; the generation of infectious viruses due to recombination; and concerns regarding instability that challenge production and storage.

The potentially hazardous nature of viral vectors has been observed in a number of gene therapy-related patient mortalities in various clinical trials. Patient complications to date have included the rejection of DNA carriers, resulting in immune response that have led to already one death, Jesse Gelsinger, who died in 1999 from a rare metabolic disorder during a gene-therapy clinical trial at the University of Pennsylvania (Branca 2005;Ledford 2007;Stolberg 1999;Wilson 2010). DNA constructs need to be optimized to carry minimal immunogenic components without compromising both efficient and longterm transgene expression. In this context, technology employing minimal immunological defined gene expression technology (MIDGE) represents a promising future alternative to conventional plasmids in terms of biosafety, improved gene transfer, potential bioavailability, minimal size and low immunogenicity associated with these chemically engineered miniDNA vectors. MIDGEs are non-viral, lcc (linear covalently closed) miniplasmids synthesized *in vitro* via a patented chemical modification of linear open (lo). These plasmids confer the advantage of a minimal coding sequence that relieves complications from expression of additional unwanted genes and CpG motifs that have a 20-fold higher occurrence in bacterial cells. MIDGE lcc plasmid transfection expression was reported to increase luciferase transgene expression from 2.5 to 17 fold *ex vivo* compared to circular covalently closed (ccc), isogenic forms in a tissue-dependent manner (Schakowski et al. 2007) . In addition, the mean numbers of MIDGE vector molecules per cell was also found to be significantly higher, suggesting that linear lcc plasmids transfect cells more efficiently. MIDGE technology has already been applied, with promising results, to the development of a Leishmania DNA vaccine and a colon carcinoma treatment. Here, therapy was based on IL-2 delivery to specific tumor cell lines *ex vivo*, and revealed 2 to 4 fold higher relative transgene expression compared to the ccc control (Schakowski et al. 2001). In combination, the existing studies employing lcc DNA systems suggest that lcc DNA is superior to lo DNA in transfection efficiency, and to ccc DNA in transgene expression. Purified TelN (closely related to Tel) was previously reported to generate lcc plasmids *in vitro* in (*pal*-related) *telRL*-dependent manner and was shown to successfully deliver EGFP to human embryonal kidney cells *in vitro,* as well as IL-12 in an untargeted manner to inhibit metastasis formation in B16F10C57BL/6 melanoma model mice *in vivo* (Heinrich et al. 2002).

At present, our laboratory is involved in design, and construction of bacteriophageencoded recombination systems to generate linear covalently closed (lcc) DNA minivectors. Conditional expression recombinase systems *in vivo* in *E. coli* provide a one step production system for step minivectors from specialized parental plasmids. The exploitation of this recombination system allows us to generate lcc miniplasmids that should have a preferential safety profile, preventing viable vector-chromosome, single recombination products in mammalian cells. In theory, the integration of covalently closed linear exogenous DNA into double-stranded (ds) DNA breaks is unlikely due to the unavailability of terminal ends, and a vector single recombination event into a target

Nanomedicine Based Approaches to Cancer Diagonsis and Therapy 527

Cationic liposomes are one of the most efficient, and among the most widely used non-viral vector systems. They are composed of positively charged lipid bilayers that can be complexed to DNA through electrostatic interactions resulting in complexes, termed lipoplexes. The application of cationic liposomes to gene therapy was first described in 1987 by Felgner (Felgner et al. 1987). The lipoplexes are generally composed of a positively charged lipidic component (see Figure 2), such as dioleylpropyltrimethylammonium chloride, dioleoyl triethylammonium propane (DOTAP), or dimethylaminoethane carbamoyl cholesterol (DC-Chol), that is capable of complexing and condensing the DNA (Giatrellis et al. 2009). Most lipoplex formulations include a ''helper'' lipid, such as dioleoylphosphatidyl-ethanolamine (DOPE, as seen in Figure 2), or cholesterol that provides added stability to the lipoplexes and enhances DNA release from endosomal compartments. While transfection activity of lipoplexes is shown to vary depending on their DNA/cationic lipid ratio, it is necessary for them to be positively charged to interact with the negatively charge cell membranes and exhibit transfection activity. Lipoplex uptake occurs through endosome formation (via a number of mechnisms including caveosomes, clathrin dependent, etc.), followed by disruption of the endosomal membrane by fusion with, or incorporation of the lipoplex lipids resulting in

The advantages of liposomes in delivery system designs include their simplicity in preparation, ability to complex relatively large amounts of DNA, versatility for use with any size or type of DNA/RNA, ability to transfect non-dividing cells and overall stability (Dutta et al. 2010). In addition, lipids are non-immunogenic allowing for repeated administration without adverse immunologic reaction(Barron et al. 1999). The primary disadvantages of lipoplex delivery vectors include low tumor transfection efficiency and lack of tumor

In order to enhance gene transfection, based upon structure-activity relationship, various gemini surfactants (Gemini surfactants consist of two hydrophobic chains and two polar headgroups linked chemically by a spacer group) have been designed. In order to enhance gene transfection based upon structure-activity relationship, various gemini surfactants have been designed, synthesized and tested for gene delivery in our laboratory (Donkuru et al. 2010;Wang and Wettig 2011;Wettig et al. 2007a;Wettig et al. 2007b;Wettig and Verrall 2001). Recent reports have shown that obstacles can be overcome by exploiting receptormediated endocytosis for highly efficient internalization of ligands naturally employed by eukaryotic cells. Advances along this line include the conjugation of mAbs ("immunoliposomes"), ligands such as growth factors, or hormones to liposomes to confer targeting capability. Nanoparticle based anti-cancer gene/drug delivery first reached clinical trial in mid 1980s and the first nanomedicine Doxil® (liposomal encapsulated doxorubicin) was marketed in 1995. Other example of liposome-mediated drug delivery include daunorubicin (Daunoxome), which is also currently being marketed as liposome delivery systems. Numerous new nanoparticle based cancer gene therapy systems are under

Polyplexes differ from lipoplexes in that they are comprised of charged complexes of plasmid DNA and a cationic polymer, such as poly-L-lysine (PLL), polyethylenimine, polyamidoamine (starburst) dendrimers, and chitosan with a net positive charge (See Figure 2). Cationic polymers differ from cationic lipids primarily in that they do not contain

**3.1.2 Cationic liposomes**

DNA release.

specificity.

development.

**3.1.3 Polyplexes**

chromosome should theoretically result in the disruption of the chromosome, killing the cell (see Figure 1).

Fig. 1. Plasmid Integration Events: A miniplasmid that undergoes a single recombination event with the host chromosome should be rare due to the removal of all elements except the gene of interest. Any integration events by: A. A lcc miniplasmid should result in chromosomal disruption that is likely lethal and cannot be replicated or segregated; B. A ccc miniplasmid can integrate without disrupting the host chromosome.

Non-viral gene delivery is emerging as a realistic alternative to the use of viral vectors with the potential to have a significant impact on clinical therapies. Synthetic vectors provide flexibility in formulation design and can be tailored to the size and topology of the DNA cargo and the specific route of vector administration, and can be delivered selectively to a specific tissue type through the incorporation of a targeting ligand. Compared to viral vectors, synthetic vectors are potentially less immunogenic, relatively easy to produce in clinically relevant quantities, and are associated with fewer safety concerns (Liu and Huang 2002). As a result of these advantages, their use has rapidly moved from transfection of cell cultures to clinical cancer gene therapy applications.

Synthetic vectors designed for parenteral administration encompass a wide range of formulations. These include unmodified (naked) DNA, which is designed for direct intratissue injection, cationic polymer–DNA complexes, cationic lipid–DNA complexes, and cationic polymer–lipid–DNA ternary complexes (lipopolyplexes), which are mostly aimed at systemic administration.

#### **3.1.1 Naked DNA**

The simplest employed method of transgene delivery is the injection of naked DNA. It shows very little dissemination and transfection at distant sites following delivery and can be re-administered multiple times into mammals (including primates) without inducing an antibody response against itself (i.e., no anti-DNA antibodies generated) (Wolff and Budker, 2005). The simplicity of this approach is offset by serious limitations such as inefficient uptake of the therapeutic gene into the target cells and rapid clearance of the DNA from the circulation. Direct *in vivo* gene transfer with naked DNA was first demonstrated by efficient transfection of myofibers following injection of mRNA or pDNA into skeletal muscle (Wolff JA, 1990). This novel approach was heralded as a superior method of *in vivo* transfection and its application was later advanced to confer high level expression in hepatocytes in mice by the rapid injection of naked DNA in large volumes into the tail vein (Zhang et al., 1999). This hydrodynamic tail vein (HTV) procedure has proven very useful not only to gene expression studies, but also more recently for the delivery of siRNA (Lewis et al., 2002; McCaffrey et al., 2002). Ultrasoundmediated eruption of polyethyleneglycol (PEG)-modified liposomes loaded with naked plasmid-DNA is also a feasible and efficient technique for gene delivery (Negishi et al. 2010).