Bioevolution vol 1 (3) september 2014

Editorial Board BioEvolution Chief Editor

Dr Kanika Sharma Professor

Mohanlal Sukhadia University, Udaipur, Rajasthan

Managing Editor

Dr Arti Goel Assistant Professor

Amity University, Noida, UP

Dr Nidhee Chaudhary Professor Amity Institute of Biotechnology, Amity University, Sector- 125, Noida (U.P.)

Dr Rajshree Saxena Lecturer Amity Institute of Microbial Biotechnology, Amity University, Sector- 125, Noida (U.P.)

Dr Arpita Bhattacharya Associate Professor Amity Institute of Nanotechnology, Amity University, Sector- 125, Noida (U.P.)

Dr U K Tomar Scientist F Arid Forest Research Institure Jodhpur, Rajasthan

Dr Era Upadhyay Assistant Professor Ansal Institute of Technology & Management, Sector C, Pocket 9, Sushant Golf City, Lucknow (U.P.)

Dr Pranita Roy Assistant Professor Amity Institute of Biotechnology, Amity University, Sector- 125, Noida (U.P.)

Dr Prachi Bhargava Assistant Professor Department of Biotechnology, Sri RamSwaroop Memorial University, Deva Road, Lucknow (U.P.)

Dr Ritu Mawar Senior Scientist (Plant Pathology) PCFC Unit, AICRP (FC),

IGFRI, Jhansi

Dr. Jitendra K. Saini Scientific Officer DBT-IOC Centre for Advanced Bio-Energy Research Haridwar, Uttarakhand,

India

Vikas Mishra, Ph.D. Postdoctoral Research Associate, Department of Pediatrics, Penn State Milton S. Hershey Medical Center, Hershey, PA, USA.

Dr. Muraleedharan Damodaran Emertus Professor and Senior Researcher DARIS Centre for Scientific Research and Technology Development, University of Nizwa,

Sultanate of Oman

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Vol 1 (3) September 2014 ISBN 987-93-83006-02-1

BIOSENSORE AND MEDICINE

Dr. Namita Bedi* and Manish Kumar

*Assistant Professor, Amity Institute of Biotech,

Amity University, Noida (U.P.).

DIOXIN CONTAINING DIAPERS: IS IT SAFE FOR BABIES?

Dr.Rishav Raj

Department of Pediatrics, Hi Tech Medical College,

Utkal Univesity Bhubaneshwar, Odisha

POLYETHYLENE OXIDE-POLYPROPYLENE OXIDE BASED BLOCK COPOLYMERS AS NANOVEHICLES FOR DRUG FORMULATIONS

Dr. Rakesh K. Sharma and Maheshwari Sharma

Applied Chemistry Department, Faculty of Technology & Engineering,

The M. S. University of Baroda, Gujarat, India

EVOLUTION OF NANOTECHNOLOGY

Sneha Bhatnagar and Arti Goel

*Assistant Professor, Amity Institute of Microbial Biotechnology,

Amity University, Noida (U.P.).

RAPID DIAGNOSTIC TEST FOR PREDICTION OF PRETERM DELIVERY BY ACTIM PARTUS KIT

Dr Mitali Mahapatra,

Department of Obstetrics and Gynecology, Hi Tech Medical College,

Utkal University, Bhubaneshwar, India

ROLE OF SUPEROXIDE DISMUTASE IN THERAPEUTICS: AN OVERVIEW

Nameet Kaur and Nidhee Chaudhary

Amity Institute of Biotechnology,

Amity University, Noida, UP, India

Published by Gyandhara International Academic Publications, (GIAP) Journals, Flat 303, Building 3, Sai Saraswati Dham CHS, Shantivan, Opposite Silver Park, Mira Road (E), Thane, India

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BioEvolution Vol 1 (03), September 2014, ISBN 978-93-83006-02-1, pg 55-65

55 www.giapjournals.org/bioevolution.html

BIOSENSORE AND MEDICINE Dr. Namita Bedi and Manish Kumar

Amity Institute of Biotech, Amity University, Noida [email protected]

Abstract

Biosensors are sensor frameworks that are consolidated with natural frameworks. They comprise of two primary parts; one of them is organic distinguishment component and the other one is physicochemical transducer. Organic distinguishment component might be biocatalysts (protein, microorganism, tissue materials) or bioligands (antibodies, nucleic acids, lectins) that associate specifically with the target analyte. Transducer changes over the physical reaction coming about because of the association between the target analyte and natural distinguishment component into a measurable indicator. Biosensors which give observing organic and manufactured methods could be utilized other than therapeutic measuring and examining, for natural perceptions and controls, barrier industry, agribusiness, sustenance and pharmaceutical industry. In this way, they have a paramount part in day by day life. In this audit, qualities of biosensors that are utilized as a part of restorative measuring and dissecting have been dead set and classifications of biosensors

concurring diverse gimmicks that are found in writing are displaye.

Keywords: Biosensors, Transducers, DNA, Therapeutic

INTRODUCTION

Biosensors: are expository devices for the investigation of bio-material examples to addition an understanding of their bio-creation, structure and capacity by changing over a living reaction into an electrical indicator. The expository gadgets made out of an organic distinguishment component straightforwardly interfaced to an indicator transducer which together relate the centralization of an analyte (or gathering of related analytes) to a measurable reaction.

Individuals, alongside different vertebrates, intentionally connect with the encompassing scene by method for seven sensing components. Notwithstanding the five faculties, the capabilities to recognize temperature and Variations in height are just about as paramount. One really want to be awed by the evolutionary process that realized the improvement of such faculties and by their combination into a mind equipped for data transforming and stockpiling.

In the event that a finer consciousness of our surroundings, nourishment supplies, and predators was the principle driver for this Evolutionary methodology, a parallel may be drawn with our human venture of making sensors that help us comprehend the world with which we communicate. A sensor could be seen as the "essential component of an estimation chain, which changes over the data variable into a sign suitable for estimation.

Sensing plan is typically focused around a transduction standard or system; an info variable is changed into a yield variable through a transduction instrument. Transduction standards are known physical or concoction impacts that associate perceptions in distinctive spaces. Case in point, the photoelectric impact is utilized to associate number of photons with electric current.

BIOSENSORS: CLASSIFICATION

Two crucial components of a biosensor are"biological distinguishment" and "sensing." Therefore, a biosensor could be for the most part characterized as a gadget that comprises of two essential parts associated in arrangement:

(1) An organic distinguishment framework, regularly called a bioreceptor, and

(2) A Transducer

The essential rule of a biosensor is to recognize this atomic distinguishment and to change it into an alternate sort of sign utilizing a transducer. The primary reason for the distinguishment framework is to give the sensor a high level of selectivity for the analyte to be measured. The collaboration of the analyte with the bioreceptor is intended to deliver an impact measured by the transducer, which changes over the data into a measurable impact, for example, an electrical sign. Delineates the reasonable rule of the biosensing methodology. Biosensors could be arranged by bioreceptor or transducer sort. A bioreceptor is a living atomic animal varieties (e.g., a neutralizer, a protein, a protein, or a nucleic corrosive) or a living natural framework (e.g., cells, tissue, or entire life forms) that uses a biochemical component for distinguishment. The inspecting segment of a biosensor holds a biosensitive layer that can hold bioreceptors or be made of bioreceptors covalently appended to the transducer.




FIGURE 1: Conceptual Diagram of the Biosensing Principle

Measurement

Data Recording and Display

Analyte

Bioreceptor

Molecular Recognition

Transducer




FIGURE 2 Biosensor Classification Schemes

The most common forms of bioreceptors used in biosensing are based on

1. Antibody/antigen interactions,

2. Nucleic acid interactions,

3. Enzymatic interactions,

4. Cellular interactions (i.e., microorganisms, proteins), and

5. Interactions using biomimetic materials (i.e., synthetic bioreceptors).

For transducer classification, the previously mentioned techniques (optical, electrochemical, and mass-sensitive) are used. Bioreceptors are the key to specificity for biosensor technologies. They are responsible for binding the analyte of interest to the sensor for the measurement. These bioreceptors can take many forms; the different bioreceptors that have been used are as numerous as the different analytes that have been monitored using biosensors.

However, bioreceptors can generally be classified into five different major categories:

1. Antibody/antigen,

2. Enzymes,

3. Nucleic acids/DNA,

4. Cellular structures cells, and

5. Biomimetic.

Figure 2 shows a schematic diagram of two types of bioreceptors.the structure of an immunoglobulin G (IgG) antibody molecule and DNA and the principle of base pairing in hybridization . Since the first biosensors were reported in the early 1960s growth of research activities in this area has accelerated.3–28 Biosensors have seen a wide variety of applications, primarily in three major areas: biological monitoring, biomedical diagnostics and environmental sensing applications.

BIOSENSORS

Biomimetic Cell DNA Enzyme Antibody Other Mass Based Electrochemical

Optical

BIORECEPTOR TRANSDUCER

Cellular Systems Non-Enzymatic Proteins




BIORECEPTORS AND BIOSENSOR SYSTEMS

A. ANTIBODY

Antibody Bioreceptors

The basis for the specificity of immunoassays is the antigen-antibody (Ag-Ab) binding reaction, which is a key mechanism by which the immune system detects and eliminates foreign matter.The enormous range of potential applications of immunosensors is due, at least in part, to the astonishing diversity possible in one of their key components, antibody molecules. Antibodies are complex biomolecules made up of hundreds of individual amino acids arranged in a highly ordered sequence. The structure of an IgG antibody molecule is schematically illustrated in. The antibodies are actually produced by immune system cells (B cells) when such cells are exposed to substances or molecules called antigens. The antibodies called forth following antigen exposure have recognition/ binding sites for specific molecular structures (or substructures) of the antigen. The way in which an antigen and an antigen-specific antibody interact may perhaps be understood as analogous to a lock-and-key fit, in which the specific configurations of a unique key enable it to open a lock. In the same way, an antigen-specific antibody fits its unique antigen in a highly specific manner, so that hollows, protrusions, planes, and ridges on the antigen and the antibody molecules. (in a word, the total three-dimensional structure) are complementary. This unique property of antibodies is the key to their usefulness in immunosensors; the ability to recognize molecular structures allows one to develop antibodies that bind specifically to chemicals, biomolecules, microorganism components, etc. Nanometer-scale fiberoptic biosensors were used for monitoring biomarkers related to human health effects associated with exposure to polycyclic aromatic hydrocarbons (PAHs). These sensors use a monoclonal antibody for benzo pyrene tetrol (BPT), a metabolite of the carcinogen benzo pyrene, as the bioreceptor. Excitation light is launched into the fiber and the resulting evanescent field at the tip of the fiber is used to excite any of the BPT molecules that have bound to the antibody. The fluorescent light is then collected via a microscope. Using these antibody-based nanosensors, absolute detection limits for BPT of approximately 300 zeptomol (10–21 mol) have been reported. These nanosensors allow probing of cellular and subcellular environments in single cells.

Immunoassay Formats

Biomolecular interactions can be classified into two categories according to the test format performed (direct or indirect). In a direct format the immobilized target molecule interacts with a ligand molecule or the immobilized ligand interacts with a target molecule directly. For immunosensors, the simplest situation involves in situ incubation followed by direct measurement of a naturally fluorescent analyte. Nonfluorescent analyte systems, in situ incubation is followed by development of a fluorophor-labeled second antibody.

Biomolecular interactions can be classified into two categories according to the test format performed (direct or indirect). In a direct format the immobilized target molecule interacts with a ligand molecule or the immobilized ligand interacts with a target molecule directly. For immunosensors, the simplest situation involves in situ incubation followed by direct measurement of a naturally fluorescent analyte. Nonfluorescent analyte systems, in situ incubation is followed by development of a fluorophor-labeled second antibody.

Classical avidin–biotin and IgG–anti-IgG interactions. No difference was observed in the binding of fluorescently labeled goat IgG by rabbit anti-goat IgG, regardless of whether the capture antibody was bound to the probe surface via protein A or covalently attached. However, in a sandwich immunoassay for the F1 antigen of Yersinia pestis, probes with rabbit anti-plague IgG bound to the surface via protein A generated twice the signal generated by the probes with the antibody covalently attached. Assay regeneration was also examined with protein A probes because Ab–Ag complexesm have been successfully eluted from protein A under low pH conditions.

Antibody Probe Regeneration

Removal of antigens bound to antibodies covalently attached to optical fiber surfaces is one of the limiting factors in the development of reusable, inexpensive, and reliable optical fiber immunosensors for environmental and clinical analysis. Chemical reagents were supposed to cleave the binding between antibody and antigen, thus regenerating the biosensor80 — a chemical procedure that is simple but ineffective after multiple regeneration operations (less than five cycles), due to possible denaturation of the antibody.




FIGURE 3. Immunoassay formats: (A) competitive assay; (B) Direct assay; (C) Sandwich assay.

The mechanisms of megasonic cleaning likely involve nonlinear effects such as acoustic Streaming and radiation pressure as well as the oscillatory linear forces. Stable cavitations (a less Intense process than inertial cavitations) may also play an important role, particularly in enhancing Streaming effects. The success of the gentle but effective megasonic cleaning process suggests that MHz-range ultrasound may have some utility in regeneration of biosensor surfaces.

B. ENZYME

Enzymes are often used as bioreceptors because of their specific binding capabilities as well as their catalytic activity. In biocatalytic recognition mechanisms, the detection is amplified by a catalytic reaction. This is the basis for the now commonplace enzyme-linked immunosorbent assay (ELISA) technique. With the exception of a small group of catalytic ribonucleic acid molecules, all enzymes are proteins. Some enzymes require no chemical groups other than their amino acid residues for activity. Others require an additional component called a cofactor, which may be either one or more inorganic ions, such as Fe2+, Mg2+, Mn2+, or Zn2+, or a more complex organic or organometallic molecule called a coenzyme. The catalytic activity provided by enzymes allows for much lower




limits of detection than would be obtained with common binding techniques. As expected, the catalytic activity of enzymes depends upon the integrity of their native protein conformation. If an enzyme is denatured, dissociated into its subunits, or broken down into its component amino acids, its catalytic activity is destroyed. Enzyme-coupled receptors can also be used to modify the recognition mechanisms.

The response of this evanescent wave immunosensor was then compared to a commercially available SPR-based biosensor for lactate dehydrogenase detection using similar assay techniques, and similar results were

C. NUCLEIC ACID

Nucleic Acid Bioreceptors

Another biorecognition mechanism involves hybridization of DNA or RNA. In the last decade, interest in nucleic acids as bioreceptors for biosensor and biochip technologies has increased.96,98–102 The complementarity of the pairing of the nucleotides adenine:thymine (A:T) and cytosine:guanine(C:G) in a DNA ladder forms the basis for the specificity of biorecognition in DNA biosensors, often referred to as genosensors. If the sequence of bases composing a certain part of the DNA molecule is known, then the complementary sequence, often called a probe, can be synthesized and labeled with an optically detectable compound (e.g., a fluorescent label).

The effectiveness of the new detection scheme is demonstrated using the gag gene sequence of the human immunodefficiency (HIV) virus. A SERSbased DNA assay for the breast cancer susceptibility gene (BRCA1) has also been developed. The assay is based on the immobilization of oligonucleotides on a thin silver surface and hybridization with Rhodamine-labeled probes. The silver surface serves as the hybridization support and the means for Raman signal enhancement. The development of a biosensor for DNA diagnostics using visible and NIR dyes has been reported. The system employed a two-dimensional charge-coupled device and was used to detect the cancer suppressor p53 gene.

DNA Biosensors

DNA sensors are usually based on hybridization assays and may incorporate simultaneous analytical capability to detect a large number of oligonucleotide fragments. A fiber-optic DNA biosensor microarray for the analysis of gene expression has been reported for the simultaneous analysis of multiple DNA sequences using fluorescent probes. Fluorescent intercalating and groove-binding dyes that can associate with double-stranded DNA (dsDNA) are used for detection of hybridization in some sensor and biochip designs.

The experimental results suggested that the thermodynamic stability of duplexes immobilized on a surface is dependent on the density of immobilized DNA.Additionally, the deviation in melt temperature, arising as a result of the presence of a centrallylocated single base-pair mismatch, was significantly larger for thermal denaturation occurring at the surface of the optical fibers relative to that observed in bulk solution These results suggest that hybridization at an interface occurs in a significantly different physical environment from hybridization in bulk solution, and that surface density can be tuned to design analytical figures of merit. Increased immobilization density resulted in significantly higher sensitivity but reduced dynamic range in all hybridization assays conducted. Sensitivity and selectivitywere functions of temperature; however, the selectivity of hybridization assays done using the sensors could not be predicted by consideration of thermal denaturation temperatures alone.

D. CELL-BASED SYSTEMS

Cellular structures and cells comprise a broad category of bioreceptors that have been used in the development of biosensors and biochips. These bioreceptors are based on biorecognition by an entire cell or microorganism or by a specific cellular component capable of specific binding to certain species. There are presently three major subclasses of this category: cellular systems, enzymes, and nonenzymatic proteins. Due to the importance and large number of biosensors based on enzymes, these have been given their own classification and were previously discussed in This section deals with cellular systems and nonenzymatic proteins.

Cellular Bioreceptors

Microorganisms offer a form of bioreceptor that often allows a whole class of compounds to be monitored. Generally these microorganism biosensors rely on the uptake of certain chemicals into the microorganism for digestion. Often, a class of chemicals is ingested by a microorganism, therefore allowing a class-specific biosensor to be created. Microorganisms such as bacteria and fungi have been used as indicators of toxicity or for the measurement of specific substances. For example, cell metabolism (e.g., growth inhibition, cell viability, substrate uptake), cell respiration, and bacterial bioluminescence have been used to evaluate the effects of toxic heavy metals.




Many cell organelles can be isolated and used as bioreceptors. Cell organelles are essentially closed systems, so they can be used over long periods of time. Whole mammalian tissue slices or in vitro cultured mammalian cells are used as biosensing elements in bioreceptors. Plant tissues are also used in plant-based biosensors; they are effective catalysts because of the enzymatic pathways they possess. Bilitewski and co-workers have developed a microbial biosensor for monitoring short-chain fatty acids in milk. Arthrobacter nicotianae microorganisms were immobilized in a calcium-alginate gel on an electrode surface. To this gel was added 0.5 mM CaCl2 to help stabilize it. Monitoring the oxygen consumption of the A. nicotianae electrochemically allowed its respiratory activity to be monitored, thereby providing an indirect means of monitoring fatty acid consumption. Detection of short-chain fatty acids in milk, ranging from 4 to 12 carbons in length, was accomplished with butyric acid as the major substrate. A linear dynamic range from 9.5 to 165.5 M was reported, with a response time of 3 min.

Non enzymatic Proteins

Many proteins found within cells often serve the purpose of bioreception for intracellular reactions that will take place later or in another part of the cell. These proteins could simply be used for transport of a chemical from one place to another, e.g., a carrier protein or channel protein on a cellular surface. In any case, these proteins provide a means of molecular recognition through one or another type of mechanism (i.e., active site or potential sensitive site). By attaching these proteins to various types of transducers, many researchers have constructed biosensors based on nonenzymatic protein biorecognition. In one recent application, Cusanovich and co-workers developed micro- and nanobiosensors for nitric oxide that are free from most potential interferents.96 These sensors are based on bioreception of nitric oxide by cytochrome

Two different techniques of immobilization of the cytochrome to fibers were tested: polymerization in an acrylamide gel and reversible binding using a gold colloid-based attachment. The cytochrome used in this work was labeled with a fluorescent dye that is excited via an energy transfer from the hemoprotein. Response times of faster than 1 s were reported along with a detection limit of 20 M. Cytochrome samples from three different species of bacteria were evaluated. Vogel and co-workers have reported on the use of lipopeptides as bioreceptors for biosensors. A lipopeptide containing an antigenic peptide segment of VP1, a capsid protein of the picornavirus that causes foot-and-mouth diseases in cattle, was evaluated as a technique for monitoring antigen– antibody interactions. The protein was characterized via circular dichroism and infrared spectroscopy to verify that upon self-assembly onto a solid surface it retained the same structure as inits free form. Based on surface plasmon resonance measurements, it was found that the protein was still fully accessible for antibody binding. This technique could provide an effective means of developing biomimetic ligands for binding to cell surfaces.

Biomimetic Receptors

A receptor fabricated and designed to mimic a bioreceptor is often termed a “biomimetic receptor.”Several different

methods have been developed over the years for the construction of biomimetic receptors. These methods include genetically engineered molecules, artificial membrane fabrication, and molecular imprinting. The molecular imprinting technique, which has recently received great interest, consists of mixing analyte molecules with monomers and a large number of cross linkers. Following polymerization, the hard polymer is ground into a powder, and the analyte molecules are extracted with organic solvents to remove them from the polymer network. As a result, the polymer has molecular holes or binding sites that are complementary to the selected analyte. Recombinant techniques, which allow for the synthesis or modification of a wide variety of binding sites using chemical means, have provided powerful tools for designing synthetic bioreceptors with desired properties.

Hellinga and co-workers reported the development of a genetically engineered single-chain antibody fragment for monitoring phosphorylcholine.129 In this work, protein engineering techniques are used to fuse a peptide sequence that mimics the binding properties of biotin to the carboxyterminus of the phosphorylcholine-binding fragment of IgA. This genetically engineered molecule can be attached to a streptavidin monolayer, and total internal reflection fluorescence was used to monitor the binding of a fluorescently labeled phosphorylcholine analog. Artificial membranes have been developed for many different bioreception applications. Stevens and co-workers developed one by incorporating gangliosides into a matrix of diacetylenic lipids (5 to 10% of which were derivatized with sialic acid). The lipids were allowed to self-assemble into Langmuir-Blodgett layers and were then photopolymerized via ultraviolet irradiation into polydiacetylenemembranes.

BIOMEDICAL APPLICATIONS

This section presents an overview of various medical applications of biosensors, especially in the diagnosis of diseases. A large body of work was accomplished using SPR commercial instruments and involves the study of




protein or DNA interactions relevant for medical applications. Other widely used methods include fluorescence spectroscopy, NIR, and circular dichroism.

Cellular Processes

Staining cellular organelles is a classic laboratory method that utilizes visible or fluorescent dyes that have high affinity for specific organelles. After staining, visible or UV illumination is used for microscopy identification. Newer techniques explore the same principles to obtain more information on cellular processes as well. For example, G protein-coupled receptors (GPCRs) represent one of the most important drug targets for medical therapy, and information from genome sequencing and genomic databases has substantially accelerated their discovery.

Taken together, these data suggest that JIM19 interacts with a functional PM complex involved in ABA signaling. Another receptor binding study done with optical biosensor technology investigated the affinity and specificity of the putative proximal tubular scavenging receptor for protein reabsorption and the specificity of AGE-modified protein interactions with primary human mesangial cells.

The main antigenic site (site A) of the foot-andmouth disease virus (FMDV, strain C-S8c1) may be adequately reproduced by a 15-peptide with the amino acid sequence H-YTASARGDLAHLTTT-NH2 (A15), corresponding to the residues 136 to 150 of the viral protein VPI.158 The SPR sensor surface was modified with monoclonal antibodies raised against antigenic site A. Although these antigenicities have previously been determined from ELISA methods, the SPR-based technique is superior in that it allows a fast and straightforward screening of antigens while simultaneously providing kinetic data for the Ag–Ab interaction.

Human Immunodeficiency Virus (HIV)

The human immunodeficiency virus (HIV) has been the target of intense research in the past two decades. Some of the research efforts involving optical biosensors are outlined below. The two main proteins on the HIV envelope are glycoproteins gp120 and gp41 (named for their approximate size in kilodaltons). Glycoproteins gp120 and gp41 are associated together noncovalently. Binding ofHIV-1 gp120 to T-cell receptor CD4 initiates conformational changes in the viral envelope that trigger viral entry into the host cells.

Studies showed that cv-n is also capable of binding to the glycosylated ectodomain of the HIV-envelope protein gp41 (sgp41), albeit with considerably lower affinity than the sgp120/cv-n interaction. These optical techniques shed light on the binding of cv-n with sgp120 and sgp41, providing direct evidence that conformational changes are a consequence of cv-n interactions with both HIV-1 envelope glycoproteins.

Bacterial Pathogens

Several physicochemical instrumental techniques for direct and indirect identification of bacteria such as IR and fluorescence spectroscopy, flow cytometry, chromatography, and chemiluminescence have been reviewed as feasible biosensor technologies.

S. aureus (Cowan-1) cells were detected in spiked milk samples at cell concentrations from 4103 to 1.6106 cells/ml using the sandwich assay. The same resonant mirror optical biosensor technology was used to characterize Helicobacter pylori strains according to their sialic acid binding. In that work, intact bacteria were used in real-time measurements of competition and displacement assays using different glycoconjugates. The authors found that that several, but not all, H. pylori strains express sialic acid-binding adhesin specific for alpha-2,3-sialyllactose. The adhesin, removable from the bacterial surface by water extraction, is not related to other reported H. pylori cell surface proteins with binding ability to sialylated compounds such as sialylglycoceramides.

The magnetic microbeads coated with anti-Salmonella were used to separate Salmonella from sample solutions at room temperature for 30 min. A sandwich complex with alkaline phosphatase and the Salmonella immobilized on the magnetic beads was formed, separated from the solution by a magnetic filtration, and incubated with a p-nitrophenyl phosphate substrate at 37C for 30 min to produce p-nitrophenol by the enzymatic hydrolysis. Salmonella was detected by measuring the absorbance of p-nitrophenol at 404nm, with a linear response between 2.2104 and 2.2106 CFU/ml.




Cancer

Optical biosensors have been utilized as tools to aid in the direct diagnosis of carcinogenesis, the identification of genetic markers associated with it, and the quantification of known carcinogens. Fluorescent detection has become a technique of choice for oligonucleotide hybridization detection.

Pairs of high density oligonucleotide arrays (DNA chips) consisting of more than 96,000 oligonucleotides were designed to screen the entire 5.53-kb coding region of the hereditary breast and ovarian cancer BRCA1 gene for all possible sequence changes in the homozygous and heterozygous states. Fluorescent hybridization signals from targets, containing the 4 natural bases to more than 5592 different, fully complementary 25mer oligonucleotide probes on the chip, varied over two orders of magnitude. To examine the thermodynamic contribution of rU.dA and rA.dT target probe base pairs to this variability, modified uridine [5-methyluridine and 5-(1-propynyl)-uridine)] and modified adenosine (2,6- diaminopurine riboside) 5-triphosphates were incorporated into BRCA1 targets. Hybridization specificity was assessed based upon hybridization signals from >33,200 probes containing centrally localized single base pair mismatches relative to target sequence.

Targets containing 5-methyluridine displayed promising localized enhancements in the hybridization signal, especially in pyrimidine-rich target tracts, while maintaining single nucleotide mismatch hybridization specificities comparable with those of unmodified targets. In another study, the breast cancer susceptibility gene BRCA1 was also detected by a relatively new technique based on SERS. A single 24-mer sequence was used as the capture probe and was immobilized on a silver-coated microarray platform for hybridization. Breast cancer was also the target of a biosensor design to measure the interaction of S100A4 and potential binding partners. Elevated levels of S100A4 induce a metastatic phenotype in benign mammary tumor cells in vivo. In humans, the presence of S100A4 in breast cancer cells correlates strongly with reduced patient survival. There was significant interaction of S100A4 with nonmuscle myosin and p53 but not with actin, tropomyosin, or tubulin.

The carcinogen benzo pyrene (BAP) was the target of an antibody-based fiberoptics biosensor.In that biosensor, BAP was the analyte and fluorophore because it has a large fluorescence cross section. An antibody with high specificity for BAP was immobilized on the tip of an optical fiber.Upon exposure to contaminated samples, the optical fiber was irradiated with a laser and the resulting fluorescence correlated with the BAP concentration.

Parasites

Detection of antibodies specific for the parasite L. donovani in human serum samples has been reported. The method is based on an evanescent wave fluorescence collected by optical fibers that have the purified cell surface protein of L. donovani immobilized on their surface. The sensing fibers are incubated with the patient serum for 10 min and then incubated with goat anti-human IgG. Fluorescence was proportional to L. donovani–specific antibodies present in the test sera.

Toxins

Ricin, a potently toxic protein, has been detected with an evanescent-wave fiber-optic biosensor witha detection limit of 100 pg/ml and 1 ng/ml for buffer solutions and river water, respectively. This detection was based on a sandwich immunoassay scheme, using an immobilized anti-ricin IgG on the surface of the optical fiber. Two coupling methods were used. In the first, the antibody was directly coated to the silanized fiber using a cross linker; the second method utilized avidin-coated fibers incubated with biotinylated antiricin IgG to immobilize the antibody using an avidin-biotin bridge. The assay using the avidin–biotin-linked antibody demonstrated higher sensitivity and a wider linear dynamic range than the assay using the antibody directly conjugated to the surface. The linear dynamic range of detection for ricin in buffer using the avidin–biotin chemistry is 100 pg/ml to 250 ng/ml. The lipopolysaccharide (LPS) endotoxin is the most powerful immune stimulant known and a causative agent in the clinical syndrome known as sepsis. Sepsis is responsible for more than 100,000 deaths annually, in large part due to the lack of a rapid, reliable, and sensitive diagnostic technique. An evanescent wave fiberoptic biosensor was developed for the detection of LPS from E. coli at concentrations as low as 10 ng/ml in 30 s.18 Polymyxin B covalently immobilized onto the surface of the fiber-optic probe was able to bind fluorescently labeled LPS selectively.

Unlabeled LPS present in the biological samples was detected in a competitive assay format, by displacing the labeled LPS. The competitive assay format worked in buffer and in plasma with similar sensitivities. This method might also be used with other LPS capture molecules, such as antibodies, lectins, or antibiotics, to simultaneously detect LPS and determine the LPS serotype.18 An immunoaffinity fluorometric biosensor was developed for the




detection of aflatoxins, a family of potent fungi-produced carcinogens commonly found in a variety of agriculture products. Developed into a fully automated instrument based on immunoassays with fluorescent tags, the detection system was able to detect aflatoxins from 0.1 to 50 ppb in 2 min with a 1-ml sample volume.

Parathion was detected with a biosensor based on total internal reflection using a competitivedisplacement immunoassay. This biosensor utilizes casein–parathion conjugates, immobilized by adsorption on quartz fibers, and selectively adsorbed antiparathion rabbit antibodies raised against bovine serum albumin(BSA)–parathion conjugates from polyclonal immune sera. The presence of free parathion inhibited the binding of the rabbit’s anti-(BSA)-parathion. Fluorescein isothiocyanate (FITC) goat antirabbit IgG was used to generate the optical signal. It could detect 0.3 ppb of parathion and had a detection limit 100-fold higher for the detection of its oxygen analog, paraoxon. Other biosensor approaches for the detection of parathion include another competition assay that inhibits the alkaline phosphatase generation of a chemiluminescent substance and a direct detection method based onanother enzymatic sensor.

Congenital Diseases

SPR and biospecific interaction analysis (BIA) have been used to detect the Delta F508 mutation of the cystic fibrosis transmembrane regulator (CFTR) gene in homozygous as well as heterozygous human subjects.189 The detection method involved the immobilization on an SA5 sensor chip of two biotinylated oligonucleotide probes (one normal, N-508, and the other mutant, Delta F508) that are able to hybridize to the CFTR gene region involved in F508del mutation. A hybridization step between the oligonucleotide probes immobilized on the sensor chips and (1) wild-type or mutant oligonucleotides, as well as (2) ssDNA.

These nucleic acid samples were obtained using asymmetric polymerase chain reaction (PCR), performed using genomic DNA from normal individuals and from F508del heterozygous and Delta 508del homozygous patients. The different stabilities of DNA/DNA molecular complexes generated after hybridization of normal and Delta F508 probes immobilized on the sensor chips were then evaluated. The results strongly suggest that the SPR technology enables a onestep, nonradioactive protocol for the molecular diagnosis of F508del mutation of the CFTR gene. This approach could be of interest in clinical genetics, because the hybridization step is often required to detect microdeletions present within PCR products.

CONCLUSIONS

The past decade has witnessed the rapid development of a wide variety of biosensors for many different analyses and has even seen them begin to advance to clinical and, in some cases, commercially available technologies, such as SPR biosensors. The increasing interest in the field of optical biosensors has provided a great deal of information about the biochemistry of clinically important ailments and has provided drug discovery research with faster analytical tools to investigate drug–receptor interactions.

Optical methods have also been extensively applied to DNA fingerprinting and genotyping, techniques that might find enormous applications in clinical diagnosis in the near future. As a very positive sign, optical biosensors are coming of age as a bioanalytical tool. A larger number of researchers in different areas now have access to a more user-friendly technology, and are able to develop custom applications specific to their research needs, expanding the range of applications for the technology.

For practical medical diagnostic applications, there is a strong need for a truly integrated biosensor system that can be easily operated by relatively unskilled personnel. Some of the current commercially available technologies have dramatically simplified the data collection operation. Although these systems have demonstrated their usefulness in genomic detection, protein interaction analysis, carcinogen monitoring, etc., they are laboratory oriented and involve relatively expensive equipment and trained, supervised operation.

The outlook is promising for optical biosensor systems; the near future should bring a larger number of multichannel applications for the simultaneous detection of multiple biotargets; improvements in size and performance; and lower production costs due to a more integrated package. Highly integrated systems lead to reduction in noise and increase in signal due to the improved efficiency of sample collection and the reduction of interfaces. The capability of large-scale production using low-cost integrated circuit (IC) technology is an important advantage that cannot be overlooked. For medical applications, the development of low-cost, disposable biosensor surfaces that can be used for clinical diagnostics at the point of care can be a major driving force for the expansion of optical biosensor technologies.




REFERENCES

1. Nauta, J.M., vanLeengoed, H., Star, W.M., Roodenburg, J.L.N., Witjes, M.J.H., and Vermey, A.,Photodynamic therapy of oral cancer — a review of basic mechanisms and clinical applications, Oral Sci., 1996

2. Clark, L.C., Jr. and Lions, C., Electrode systems for continuous monitoring in cardiovascular surgery, Ann. N.Y. Acad. Sci. 1962.

3. Weetall, H.H., Chemical sensors and biosensors, update, what, where, when and how, Biosensors Bioelectron. 1999.

4. Tess, M.E. and Cox, J.A., Chemical and biochemical sensors based on advances in materials chemistry, J. Pharm. Biomed. Anal. 1999.

5. Vo-Dinh, T., Tromberg, B.J., Griffin, G.D., Ambrose, K.R., Sepaniak, M.J., and Gardenhire, E.M., Antibody-based fiberoptics biosensor for the carcinogen benzo(a)pyrene, Appl. Spectrosc., 1987.

6. Braguglia, C.M., Biosensors: an outline of general principles and application, Chem. Biochem. Eng. 1998. 7. Huber, A., Demartis, S., and Neri, D., The use of biosensor technology for the engineering of antibodies and

enzymes, J. Mol. Recognition, 1999.

8. Arya SK, Datta M, Malhotra BD. 2008. Recent advances in cholesterol biosensor.

9. Aptamers in Bioanalysis. M. Mascini Ed., John Wiley & Sons, Inc., Hoboken, New Jersey, 2008.




DIOXIN CONTAINING DIAPERS: IS IT SAFE FOR BABIES? Dr.Rishav Raj, MBBS, MD

Department of Pediatrics, Hi Tech Medical College, Utkal Univesity Bhubaneshwar, Odisha

[email protected]

INTRODUCTION

From time immemorial there has been veritable revolution in cloth diapering. If we look back and at present age we see cloth diapers don’t stand in the market world in comparison to Dioxin Sodium Polyacrylate, Polyster, Synthetic rubber and Polyolefin Diapers. The Dioxin is a Carcinogenic chemical listed by EPA (Environment Protecting Agency). During the past decades the mother were fond of using cloth diaper containing 100% cotton. In fact during those day hardly anybody can have Pampers, Huggies for their babies. Kudos! to our grandmother for not using these synthetic rubber and dioxin containing diaper. As a matter of fact this advertising agencies promote use of disposable diaper by poking grandmothers by saying” hamare jamane me diaper nahi tha aaj acha hai diaper hai”

Modern mother imagine cloth diaper as thin sheet of cloth material not containing absorbent for soaking the urine they opt for Dioxin containing diaper during journey of poop a pee for easy and convenient method but they will be shocked to know how hazardous is these dioxin containing diaper for babies and environment too.

The World Health Organisation (WHO) hypothesizes that Dioxin exposure has most serious consequences on developing features of new born babies. The disposable diaper not only affect our new born babies but also our environment by adding pollution to our mother earth. Dioxin is no doubt toxic if persistent use of disposable diaper allowed to babies can be fatal and can accumulate in the body. It is like “An eye for an eye makes everyone blind”.

WHY CLOTH DIAPERS AND NOT DISPOSABLE DIAPERS?

They are Healthier Indeed, cloth diapers are comfortable, give baby more space and lightness. Cloth diapering these days has nothing to do with diaper pins or rubber pants. On the other hand disposable diapers contain Dioxin, which in various form has shown to cause cancer, birth defects, liver damage, skin disease and genetic damage. Another compound known as TBT (tributyl tin) also seen to be used by manufacturers of disposable diaper which is a toxic pollutant known to cause hormonal problems in human and animals. This sodium polyacrylate is a type of super absorbent polymer (the gel like substance that can be seen in disposable diaper when extremely wet .The tampons use to contains sodium polyacrylate which has been removed from tampons as it links to bacterial illness toxic shock syndrome. They are more Convenient It is convenient to reuse cloth diaper which we can’t do with disposable diapers. Money Saving These disposable diapers are costly and in purchase of each diaper we are throwing away money with each diaper we throw away. But modern mother don’t hesitate in purchasing these diapers. When we use cloth diaper that allow us to reuse the product we are investing it making it economical. You will be shocked to know 7 billion gallons of oil are used to make these disposable diapers. Another point to be noted is that if a baby wears 5 diaper a day and if the average child wear diaper for 34 months a single child will use 5000 diaper from birth to toilet training. On the other hand if a baby use cloth diaper for 150 times the baby will need only 34 diapers of cloth those 34 diaper wearing month. So,cloth diaper is economical and rash free diaper. Disposable Diapers can Overheat the genitals of baby boys Recent studies have shown that higher temperature of the scrotum can decrease the sperm count and increase the risk of testicular cancer. Disposabe Diaper can lead to diaper rash As it contains dioxin, synthetic rubber,polyster,polyolefin,absorbant gel ,sodium polyacrylate. Cause Asthma If exposed to disposable diaper containing harmful chemical called acrylate powder may land you asthma like symptoms. Environmental Factors Pollution in the environment is increasing by leaps and bounds. The use of disposable diaper have added poison to our mother earth. As the plastic diapers leak noxious gas and pollute our rivers, streams and oceans.





CONCLUSION

Indeed, cloth diaper like g -diaper containing cotton is superior to these dioxin containing diaper. In these era of cosmetic world our mother earth has suffered a lot by these human made disposable diaper causing environmental hazard. Let us join together and save our babies life and our mother earth. Enough is enough. I exhort an appeal to all the doctors of the world and WHO to take initiative for banning the disposable diapers. I know it will take a hard nut to crack for WHO to take stern action against these companies manufacturing these disposable diapers. So act fast because every hour every minute an every seconds matter as people purchasing it every now and then are adding noxious gas to atmosphere.

REFERENCES 1. MADHUKAR B.V., BREWSTER D.W., MATSUMURAF. Effects of in vivo-administered 2,3,7,8-tetrachlorodibenzo-p-dioxin on receptor

binding of epidermal growth factor in the hepatic plasma membrane of rat, guinea pig,mouse, and hamster. Proc. Natl. Acad. Sci. USA, 81, 7407,1984.

2. MADHUKAR B.V., EBNER K., MATSUMURA F.,BOMBICK D.W., BREWSTER D.W., KAWAMOTO T. 3. 2,3,7,8-tetrachlorodibenzo-p-dioxin causes an increase inprotein kinases associated with epidermal growth factor receptor in the hepatic

plasma membrane. J. BiochemToxicol., 3, 261, 1988. 4. Ahlborg, UG; Becking, G; Birnbaum, L; et al. (1994) Toxic equivalency factors for dioxin-like PCBs. Report on a WHO-ECEH and

IPCS consultation, December, 1993. Chemosphere 28(6):1049−1067. 5. 4, Barnes, D; Alford-Stevens, A; Birnbaum, L; Kutz, F; Wood, W; Patton, D. (1991) Toxicity equivalency factors for PCBs? Qual Assur

1(1):70−81. 6. Birnbaum, LS. (1994) The mechanism of dioxin toxicity: relationship to risk assessment. Environ Health Perspect 102(Suppl. 9):157−167




POLYETHYLENE OXIDE-POLYPROPYLENE OXIDE BASED BLOCK COPOLYMERS AS NANOVEHICLES FOR DRUG FORMULATIONS

Dr. Rakesh K. Sharma* and Maheshwari Sharma Applied Chemistry Department, Faculty of Technology & Engineering,

The M. S. University of Baroda, Gujarat, India [email protected]

Abstract

Concepts utilizing polymers as therapeutic agents have been widely investigated for a number of decades. Nanosized drug delivery systems formulated from biocompatible and biodegradable polymers constitute an evolving approach to drug delivery and tumor targeting. The increasing interest in Polyethylene oxide (PEO)-Polypropylene oxide (PPO) block copolymers(known as Pluronics®) arises mainly due to their surface-active and self-associative characteristics leading to micellar formation. Pluronic polymers are approved by FDA for pharmaceutical and medical applications and listed in the US and British pharmacopoeia as pharmaceutical excipients. The present article will be explained the basics of these smart polymers, its nanomicelles and applications of such nanomicelles in drug formulations specifically for an anticarcinogenic drug, Curcumin for improved solubility, stability, release, and bioavailability.

Keywords: Block copolymers, Pluronic® , Micelles, Nanoparticles, Drug delivery, Curcumin drug

Polymers have been used in a range of pharmaceutical and biotechnology products for more than five decades. Since the 1940s polymers have been explored as therapeutics with a rapidly increase in research publications year on year (Fig.1a). The number of papers in 2011 interestingly reached to ~9000, still some 10-fold higher than is seen for the descriptor “nanomedicine,” a wider discipline that is growing well.

The term “Polymer therapeutics” was coined to describe polymeric drugs, polymer conjugates of proteins, drugs and aptamers, together with those block copolymer micelles and multicomponent non-viral vectors which contain covalent linkages (Fig.1b). These often complex, multicomponent constructs are actually “drugs” and

“macromolecular prodrugs” in contrast to drug delivery systems that simply entrap (non-covalently) drug molecules(Fig.1b). They have also been called as nanomedicines.

Nanomedicine can be defined as the design of diagnostics and/or therapeutics on the nanoscale, which provides advantages due to the high degree of coincident transport and delivery of the active species with mediation of their navigation within the biological systems for the treatment, prevention and diagnosis of diseases [1,2].

Fig.1: (a) Literature against “Polymer Therapeutic” in search panel (b) Various Polymer Therapeutics

As drug carriers, the Polymeric micelles have manifested a number of attractive features and advantages over the other types of carriers. Over the years, various synthetic, structural, physicochemical, and biomedical aspects of polymeric micelles have been discussed in a number of excellent reviews[3,4]. In this article, we mainly discussed the Polyethylene oxide(PEO)-Polypropylene oxide(PPO) based block copolymeric micelles as the smart materials for the drug formulations.

Block copolymers are a special type of macromolecules in which each molecule consists of two or more segments of simple polymers (blocks) covalently joined together with and/or radial arrangement. In the




simplest case, a diblock copolymer (A-B) is composed of two different homopolymers linked end to end. An extension of this concept leads to (A-B-A) or (B-A-B) triblocks and to (A-B)n linear multiblocks, A and B can be made into a copolymer in many different ways[5].Where, n block copolymer chains are linked by one of their ends to a multifunctional moiety. Some arrangements are linear, in which the blocks are connected end-to-end, and star, in which all of the blocks are connected via one of their ends at a single junction. The number of monomer types in a block copolymer may be less than or equal to the number of blocks. Even more complex architectures in block copolymers have also been reported, such as cyclic, heteroarm, ‘‘palm tree’’, H-shaped, dumb-bell structures, etc[6].

Pluronic® Polymers- The Smart Materials

A number of symmetric tri-block copolymers of propylene oxide (PO) and ethylene oxide (EO) have been commercially available since 1950’s under the generic name Poloxamers and the trade marks Pluronic® (BASF, Mount Olive, NJ, USA) and Synperonic® (ICI, Cleveland, UK). It is a under the well class of typical nonionic surfactant[5,7,8]. The polypropylene oxide block, PPO, is sandwiched between two polyethylene oxide (PEO) blocks. PEO blocks are hydrophilic while PPO is a hydrophobic block in the Pluronic molecule(Fig.2a). Depending on the number of EO and PO units, various types of Pluronics are available commercially with molecular weights ranging from 1,100 to 14,600 and the weight fraction of the hydrophilic PEO block ranging between 0.1 and 0.8. BASF has generated a Pluronic grid (Fig.2b), which can be utilized to select the Pluronic® that is appropriate for the need of applications [7].

Fig.2: (a) Chemical structure of Pluronic®polymers (b) Pluronic grid. Liquids in blue, pastes in green and solids in the pink region.(from Ref.9)

In the Pluronic grid[9], the alphabetical designation explains the physical form of the product: ‘P’ for pastes, ‘F’

for solid and ‘L’ for liquid form. The first digit (two digits in a three-digit number) in the numerical designation, multiplied by 300, indicates the approximate molecular weight of the hydrophobe part. The last digit, when multiplied by 10, indicates the approximate ethylene oxide content in percentage in the molecules.

Micelles of Pluronic® Polymers

The micellization of PEO–PPO–PEO triblock copolymers in water has been a subject of much debate in the past. These are highly surface active compounds and form core-shell micelles with core consisting of hydrophobic middle PPO block surrounded by an outer shell of the hydrated hydrophilic PEO end blocks above the critical micelle concentration(CMC) or critical micelle temperature(CMT) in water(Fig.3)[7,8]. In contrast to the conventional nonionic surfactants of the type PEO condensates, Pluronics show some unique behavior in water which mostly arises around room temperature when the PPO no longer remains hydrophobic. The shape or morphology of micelles prepared from amphiphilic Pluronic polymers includes spherical (‘core-corona’ or

‘crew-cut’), network-like, cylindrical, lamellar and so on(Fig.3). The shape varies with the block length ratio and the composition of block copolymers.

Pluronic micelles can be prepared by several techniques, but most of them are focused on the variations for drug loading. The representative method is a direct dissolution of Pluronic polymer around CMC and followed by the elevation of temperature or the addition of hydrophobic drugs. The other one might be solvent (e.g., DMF, DMSO, acetone, THF) exchange. In this method, drugs are dissolved in a solvent with the block copolymers (rather water insoluble), and then water can be added into this solution or vice versa, viz., the solution can be poured into the water.




Fig.3: Formation and types of Pluronic Micelles

Pluronic micelles as Nanovehicles for Drug Delivery Systems

The Pluronic®polymers are approved by Food and Drug Administration (FDA ) also listed in the US and British pharmacopoeia as pharmaceutical excipients and extensively used as emulsification, solubilization, dispersion, thickening, coating, and wetting agents[10].

Pluronic® self-assemble into micelles (5-50 nm) in the aqueous solution with core–shell structure above their CMC and the hydrophobic PPO core of micelles serves as a cargo space for the incorporation of drug molecules, while the hydrophilic PEO shell contributes greatly by maintaining the micelles in a dispersed state as well as by decreasing undesirable drug interactions with cells and proteins through steric-stabilization effects[10-13]. Fig.4 represented the complete mechanism of an encapsulation of drugs into Pluronic micelles. The incorporation of drugs into the core of the micelle results in increased solubility, metabolic stability and circulation time for the drug. By attaching a peptide or other biospecific molecule that can promote site-specific drug delivery to surface of the EO corona, a micelle can be targeted to a specific site in the body. Not only that, Pluronic® unimers exhibit biological response modifying activities in certain drug formulations. For example, the interactions of the unimers with multidrug-resistant cancer cells result in sensitization of these cells with respect to various anticancer agents. Further, Pluronic®unimers have been shown to inhibit drug efflux transporters in both the blood-brain barrier and in the small intestine. This can provide for enhanced transport of select drugs to the brain and enhanced oral bioavailability [12].

Fig.4: Encapsulation of drugs into Pluronic micelles: Work mechanism

There have been many important studies on the solubilization of hydrophobic drugs having different pharmacological activity in Pluronic®micelles were reported. Increased solubility and longer half-life of naproxen and rofecoxib was shown by Suh et al.[13] and Ahuja et al.[14]. Solubility of tropicamide a mydriatic/cycloplegic drug was studied with a series of Pluronic® with different molecular characteristics and found that ocular solubility increased linearly with increasing surfactant concentration and enhanced solubility




for higher EO content[15]. Neuroleptic drug haloperidol showed a 5-fold increase in solubility in P85[16]. A novel doxorubicin formulation (SP1049C) has been developed using a combination of two Pluronics (L61 and F127) by Alakhov et al.[17]. The cytotoxic activity of this product has shown that it is highly effective against MDR cells. Further mechanistic studies have revealed that SP1049C has higher activity than doxorubicin due to increase in the drug uptake, inhibition of the energy-dependent drug efflux and changes in intracellular drug trafficking. The experiments on in vivo tumour models have confirmed high efficacy of SP1049C against drug-resistant tumours, as well as suggested that this product has considerably broader efficacy than doxorubicin. The analysis of pharmacokinetics and biodistribution of SP1049C has shown that it accumulates in tumour tissue more effectively than doxorubicin, while distribution of the formulation in normal tissues is similar to that of doxorubicin. Micelles formed by Pluronic® polymers(PBC) have been studied in multiple applications as drug delivery systems by Kabanov et al[18]. Hydrophobic PBC forms lamellar aggregates with a higher solubilization capacity than spherical micelles formed by hydrophilic PBC. However, they also have a larger size and low stability. To overcome these limitations, binary mixtures of hydrophobic (L121, L101, L81, and L61) and hydrophilic (F127, P105, F87, P85, and F68) Pluronics were also studied. PBC mixtures were not stable, revealing formation of large aggregates and phase separation within 1–2 day(s). However, stable aqueous dispersions of the particles were obtained upon sonication for 1 or 2 min and/or heating at 70˚C for 30 min. In many combinations, L121/F127 mixtures (1:1% weight ratio) formed stable dispersions with a small particle size. The solubilizing capacity of this system was examined using a dye, Sudan(III). Mixed L121/F127 aggregates exhibited approximately 10-fold higher solubilization capacity compared to that of F127 micelles. It concluded that the mixed PBC aggregates can efficiently incorporate hydrophobic compounds.

Oh et al.[19] developed a binary mixing system with two Pluronics, L121/P123, as a nano-sized drug delivery carrier. The lamellar-forming Pluronic L121 (0.1 wt%) was incorporated with Pluronic P123 to produce nano-sized dispersions (in case of 0.1 and 0.5 wt% P123) with high stability due to Pluronic P123 and high solubilization capacity due to Pluronic L121. The binary systems were spherical and less than 200-nm diameter, with high thermodynamic stability (at least 2 weeks) in aqueous solution. The CMC of the binary system was located in the middle of the CMC of each polymer. In particular, the solubilization capacity of the binary system (0.1/0.1 wt%) was higher than mono-systems of P123. The main advantage of binary systems is overcoming limitations of mono systems to allow tailored mixing of block copolymers with different physicochemical characteristics. These nano-sized systems may have potential as anticancer drug delivery systems with simple preparation method, high stability, and high loading capacity.

Nanomicelles of Pluronic® Polymers as nanovehicles for Curcumin drug

Curcumin is a natural polyphenol molecule derived from the Curcuma longa plant which exhibits anticancer, chemo-preventive, chemo- and radio-sensitization properties. Curcumin’s widespread availability, safety, low

cost and multiple cancer fighting functions justify its development as a drug for cancer treatment[20]. However, various basic and clinical studies elucidate curcumin’s limited efficacy due to its low solubility, high rate of

metabolism, poor bioavailability and pharmacokinetics[21]. To increase its solubility and bioavailability, attempts have been made through encapsulation in liposomes, polymeric and lipo-NPs, biodegradable microspheres, cyclodextrin, and hydrogels. Nano-sized drug delivery systems formulated from Pluronic polymers constitute an evolving approach to drug delivery and tumor targeting. Fig.5 shows the solubilization of curcumin in Pluronic as the nanocarriers.

Fig.5: Solubilization/encapsulation of Curcumin drug into Pluronic micelles




Pluronic® F127 and F68 were used for the encapsulation of curcumin for drug delivery application by Sahu et al[22]. The encapsulation of drug in pluronic micelle was highly dependent on drug-to-polymer ratio. Copolymer F127 showed better encapsulation efficiency than F68. Tonnessen et al[23] have been studied that the concentration of solubilized curcumin is related to the number of PO units of Pluronics. All Pluronics showed a maximum solubilizing capacity at a certain drug-to-polymer molar ratio and exceeding this ratio resulted in precipitation of curcumin, when keeping it the samples for 15 days. Addition of PEG-400 could to a certain extent stabilize the supersaturated samples, while ethanol physically destabilized the samples. Ionic strength did not influence the solubilization of curcumin in the Pluronic micelles.

Curcumin-loaded mixed micelles using P123 and F68, was prepared through thin-film hydration method and evaluated in vitro by Zhai and group[24]. The preparation method was optimized with a central composite design (CCD). The average size of the mixed micelles was 68.2 nm, and the encapsulating efficiency for curcumin was 86.93%, and 6.996% drug-loading. Compared with the curcumin in propylene glycol (PPG) solutions, the in vitro release of curcumin from loaded mixed P123/F68 micelles showed the sustained-release property. The in vitro cytotoxicity assay showed that the IC50 values on MCF-7 cells for Cur-PF and free Cur in DMSO solution were 5.04μg/mL and 8.35 μg/mL, while 2.52 μg/mL and 8.27 μg/mL on MCF-7/ADR cells. Results concluded that mixed P123/F68 micelles might serve as a potential nanovehicles to improve the solubility and biological activity of curcumin.

Bora et al[25] have also been reported the nanoformulation of curcumin with a tripolymeric composite for delivery to cancer cells. The composite nanoparticles (NPs) were prepared by using three polymers; alginate(Alg), chitosan(CS), and F127 by ionotropic pregelation followed by polycationic cross-linking. F127 was used to enhance the solubility of curcumin in the Alg-CS NPs. The AFM and SEM analysis showed that the particles were spherical in shape with an average size of 100 ± 20 nm. FTIR analysis revealed potential interactions among the constituents in the composite NPs. Encapsulation efficiency(%) of curcumin in composite NPs showed considerable increase over Alg-CS NPs without Pluronics. The in vitro drug release profile alongwith release kinetics and mechanism from the composite NPs were studied under simulated physiological conditions for different incubation periods. A cytotoxicity assay showed that composite NPs at a concentration of 500μg/mL were nontoxic to He-La cells. Cellular internalization of curcumin-loaded composite NPs was confirmed from green fluorescence inside the He-La cells. The half-maximal inhibitory concentrations for free curcumin and encapsulated curcumin were found to be 13.28 and 14.34 μM, respectively.

Pluronic®F127 (EO99-PO65-EO99) has a good solubilizing capacity and is approved by FDA for pharmaceutical and medical applications, including parenteral administration and extensively used as emulsification, solubilization, dispersion and wetting agents. Therefore, it was chosen as parent copolymer for our study too.

Curcumin has negligible solubility (0.11 ng/mL) in water, but a linear increase in solubility with F127 concentrations can simply be attributed to interaction between drug and polymer molecules and increased no. of micelles(shown in Fig.6a). The intensity of yellow colour of drug was clearly increased with the F127 concentrations suggested the high amount of drug in aqueous media. Solubility is almost 1000 folds more in presence of Pluronic micelles encourages to use of such smart materials for encapsulation of curcumin to it.

Fig.6: a) Phase solubility curve of curcumin in Pluronic solutions (b) Synthesis of Curcumin loaded Pluronic nanoparticles by

Thin film hydration method.




Curcumin-loaded nanoparticles (CUR-NPs) were prepared by thin-film hydration method and represented in Fig.6b. CURNP1(using hydrophobic F127 polymer) clearly shows the more intense orange colour in compare to CURNP2(using hydrophilic F68 polymer). The drug loading and nanoparticles yield of the optimized NPs were 47.72% and 34.51% for CURNP1 and CURNP2, respectively.

Fig.7: Characterization of curcumin loaded Pluronic nanoparticles by various techniques.

The Characterization of the synthesized CURNPs was performed by variety of techniques presented in Fig.7. It can be observed that micelles for both the formulations has the particle sizes were about 99 nm (for CURNP1) and 107 nm (for CURNP2), which are practically useful for drug delivery applications. The SEM pattern of pure curcumin shows its crystalline nature. The Pluronic itself was semi-crystalline, but the SEM of CURNP1 showed absence of major curcumin crystals. It indicates the masking of crystallity of drug after encapsulation into Pluronic micelles. Results of UV-Visible spectra (1% aqueous solution of NPs) clearly show that drug has weak/negligible absorption (no intense peak) in the wavelength range of 300 to 500 nm as hydrophobic, but CURNPs gave sharp intense peaks of curcumin. Results suggested the enacapsulation of drug in Pluronic® micelles. In all the three samples, a characteristic intensity of the O-H stretch is observed around 3500 cm-1. The chemical structure curcumin shows the functional groups of phenolic OH, C=O and aromatic C=C peaks at 1623 cm-1 and 1520 cm-1. The F127 showed two major peaks at 1101 cm-1of –C–O stretching and 2882 cm-1 of –C–H stretching. Whereas, CURNP1 gave three major peaks at 1104 cm-1 of –C–O stretching, 2882 cm-1 of –C–

H stretching and 1350 cm-1 of C=C aromatic. The FTIR spectra also confirmed the interaction of curcumin with F127 micelles and identified the formation of NPs between them. The PXRD pattern of pure curcumin shows several characteristic peaks indicative of its crystalline nature. The Pluronic itself was also crystalline in nature. The two sharp peaks at 2θ of 20.00C and 23.90C were due to the presence of PEO groups present in F127. But the CUR-NPs showed absence, broadening and reduction of major curcumin diffraction peak. And it indicated the masking of crystalline peaks of curcumin after encapsulation into Pluronic micelles. DSC curves obtained for pure curcumin, F127, and CURNP1 are displayed in Fig.7. A broaden melting peak was observed at about 1760C for free curcumin and in the thermogram of F127, a sharp peak (60.05°C) was observed, which was associated with the endothermic melting of F127. In DSC spectra of CURNP1 was showed peak at 59.65 °C for F127, but no peak at around 1760C.

The in vitro release of both the formulations, a typical two-phase release profile was observed(Fig.8). A relatively rapid release was in the first stage followed by a sustained and slow release over a prolonged time up to 11 days. Relase of drug was faster in case of F68 based NPs and almost 80% was released in 10 days while in the CURNP1, 63% drug released. The stronger the interaction between the drug and the core-forming block, the slower the release of drug from micelle.




Fig.8: In Vitro release of curcumin from CURNPs.

Conclusions

Overall, Pluronic®

of biological response modifying activity, and an ability to self-assemble into micelles, incorporate drug molecules and transport them within the body. These properties warrant further investigation regarding the use

Pluronic micelles as nanovehicles for curcumin drug has not only become a frontier movement but is also future prospects. Nanomicelles of Pluronic resulted in increased solubility, metabolic stability, and improved circulation time for curcumin drug.

Acknowledgement

Head, Applied Chemistry Department, FTE, The M. S. University of Baroda, Vadodara and the University Grants commission(UGC), New Delhi Project no. F.41-1327/2012(SR) for financial assistance are gratefully acknowledged.

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A.V., Morozov, G.V., Severin, E.S. and Kabanov, V.A. 1989, The neuroleptic activity of haloperidol increases after its solubilisation in surfactant micelles : micelles as macrocontainer for drug targeting, Febs Lett., 258 : 343.

17. Alakhov, V., Klinski, E., Li, S., Pietrzynski, G., Venne, A., Batrakova, A., Bronitch, T. and Kabanov, A.V. 1999, Block copolymer-based formulation of doxorubicin. From cell screen to clinical trials Colloids and Surfaces B: Biointerfaces, 16 : 113–134.

18. Kabanov, A. V., Bronich, T. K.and Oh, K.T. 2004, Micellar formulations for drug delivery based on mixtures of hydrophobic and hydrophilic Pluronic® block copolymers, J. Control.Release, 94 : 411-422.

19. Lee, E. S., Oh, Y. T., Seo, Y., Youn, Y. S., Nam, M., Park, B., Yun, J., Kim, J. H., Song, H.T. and Oh, K. T. 2011, Colloids and Surfaces B: Biointerfaces , 82 : 190–195.




20. Anand, P., Sundaram, C., Jurani, S., Kunnumakkara, A.B. and Aggarwal, B.B. 2008, Curcumin and cancer: An “old-age” disease with

an “age-old” solution, Cancer letters, 267 : 133-164. 21. Anand, P., Kunnumakkara, A.B., Newman, R.A. and Aggarwal, B.B. 2007, Bioavailibility of curcumin: problems and promises,

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About the Authors

Dr. Rakesh K. Sharma, M.Sc., M.Phil., Ph.D., is the Assistant Professor in the Applied Chemistry Department, Faculty of Technology & Engineering, The M. S. University of Baroda, Vadodara, Gujarat, India. He has almost 11 years of teaching and 14 years of research experience. His research interests are in aggregation and phase behavior of surfactants and EO-PO based block copolymers and its application in drug delivery and detergency. He has published 17 research papers in reputed peer-reviewed international journals, presented 14 papers and almost attended 25 conferences and symposia of international and national repute. He has reviewed papers of Tenside Surfactants Detergents (Hanser Verlag, Germany), J. Dispersion Science and Technology (Taylor and

Francis), J. of Colloid and Interface Science(Elsevier),etc reputed journals. He is recognized Ph.D. guide and successfully guided 16 dissertations of master students in Applied Chemistry.

Maheshwari Sharma, M.Sc., is a project student in the Applied Chemistry Department, Faculty of Technology & Engineering, The M. S. University of Baroda, Vadodara, Gujarat, India. She is working in the area of applications of surfactants in pharmaceutical sciences.




EVOLUTION OF NANOTECHNOLOGY Sneha Bhatnagar and Arti Goel*

*Assistant Professor, Amity Institute of Microbial Biotechnology, Amity University, Noida (U.P.).

[email protected], [email protected]

Abstract

Nanotechnology is basically the technology which allows the atoms or molecule to get manipulated and form small sized products. The national nanotechnology initiative, which defines nanotechnology as the manipulation of matter with at least one dimension sized from 1 to 100 nanometers. Hence, constructing or the engineering of functional systems at micro level is considered under nanotechnology. The field of nanotechnology is growing with an exponential phase as academic and commercial interest grows in the field. New researches are published and work is being constantly done in the field. The basic development in the field occurred in the eighteenth and the early nineteenth century an understanding of development and the criticism of this vision is integral for comprehending the realities and potential of nanotechnology today. Nanotechnology is relatively a new field of scientific research. Nanotechnology emerged out of experimental advancements like the discovery of scanning tunneling microscope, the discovery of fullerenes, atomic field microscopy etc. Around 2000s, the field took attention of people and started blooming with new scientific researches and commercial applications.

PREHISTORIC NANOTECHNOLOGY

In the early decades examples of nanostructured materials are supposed as craftsmen’s empirical understanding and manipulation of materials. For example In the 4th century “The Lycurgus Cup” (Rome) which is considered as a type of dichroic glass; colloidal gold and silver in the glass allow this cup to look opaque green when lit from outside but translucent red when light shines through the inside. In Addition to the examples, the lustrous ceramic glazes that were used in the Islamic world during 9th to 17th century for decoration contained silver or copper or metallic nanoparticles.

Also gold nanoparticles were used to make stained glass windows that also were composed of gold chloride and the oxides of other metals. In the 13th to 18th century "Damascus saber blades" were developed which contained carbon nanotube (scale bar, 5 nm) and cementite nanowires that provided them with a strength and ability to hold edge.

EARLY DISCOVERIES AND DEVELOPMENTS

Certain discoveries that were done in the early era lead to the development of nanotechnological sciences and formed a base to the nascent field. The experimentations and discoveries helped nanotechnology to develop and further grow. These developments were based on the basic understanding of scientific technologies and instrumentation.

In 1857, Michael Faraday discovered that if nanostructured gold was exposed to certain lighting conditions it produced colored solutions and thus developed colloidal "ruby" gold. Then in 1936, Ervin Muller developed the field electron microscopy which provided near atomic resolution images of materials. 1947 was then a remarkable year when semiconductor transit were developed by John Bardeen, William Shockley, and Walter Brattain at Bell Labs which was the foundation to electronic devices.

1950 was the year when Victor La Mer and Robert Dinegar developed the process for development of monodisperse colloidal materials which lead to the development of paints, paper, and thin films etc. the next year, 1951 was when field ion microscope was developed by Erwin Müller.

In 1956, Arthur von Hippel at MIT introduced many concept and coined the term “molecular engineering” as applied to dielectrics, ferroelectrics, and piezoelectrics. Jack Kilby designed the first integrated circuit in 1958.

All these developments lead to a strong foundation to the field of nanotechnology. The first spark to the field was given by Richard Feynman which led to the development of an all new magical world of nanotechnology.Richard Feynman was an American physicist who delivered a lecture “there’s plenty of room at the bottom” on December

29, 1959 in the American physical society meeting. He delivered the lecture on the field of nanotechnology that inspired and gave a spark to new researchers and scientists to work for the new potentially filled field. He described




http://en.wikipedia.org/wiki/Nanometers

http://www.britishmuseum.org/explore/highlights/highlight_objects/pe_mla/t/the_lycurgus_cup.aspx



the nascent field with endless potential where atoms and molecules can be manipulated and turned into new particles that could be used for different applications. Feynman talked majorly about the possibilities to manipulate matter at the atomic level. His major concern of the talk was the possibility of creating computer circuits and also microscopes that are able to see things that are smaller than what is possible with a scanning electron microscopy. He said that if nanoscale machineries are developed, it can result into manipulation of atoms the way we want. He also suggested a weird though of the ability to “swallow a doctor” where a tiny surgical robot can be swallowed and

manipulate faults in the body.

Norio Taniguchi was a Japanese scientist who was the first to introduce the term “nanotechnology” in a conference

held in the year 1974. He described the semiconductor processes which included ion bead milling and thin film deposition. He defined nanotechnology as the process that that includes separation, consolidation, and deformation of materials by one atom or one molecule.

Drexler in the year 1959 encountered Feynman’s talk “There's Plenty of Room at the Bottom" when he was working

on the field of molecular engineering and manipulations. And as by the time the term “nanotechnology” was coined

the idea of molecular nanotechnology or molecular manufacturing stroked Drexler. In his first book “Engines of Creation: The Coming Era of Nanotechnology” he gave the idea of nanoscale “assembler” that would be able to

build a similar copy of itself and also of other things. In his PhD work his thesis was on "Molecular Machinery and Manufacturing with Applications to Computation” Drexler was the founder of the Foresight institute in 1986 with an

aim of preparing for nanotechnology.

OTHER DISCOVERIES

As years flew by many new inventions came in the field of nanotechnology, now making it more advanced and developed. The discoveries and developments continue to grow with time and promise a bright future for nanotechnology.

The co-founder of Intel Gordon Moore described the density of transistors on an integrated chip (IC) doubling every 12 months (later amended to every 2 years). He said that as the size of the chips shrink, its cost will also go down with the coming time and the functionalities will go up subsequently.

Gerd Binnig and Heinrich Rohrer at the IBM’s Zurich lab invented the scanning tunneling microscope in the year 1981. This discovery proved to be of utmost importance and allowed to create images of atoms individually. This allowed a great outflow of provisions in the field of nanotechnology.

In the year 1985, Harold Kroto, Sean O’Brien, Robert Curl, and Richard Smalley discovered

the Buckminsterfullerene (C60), more commonly known as the buckyball. This was a molecule which resembled the shape of a soccer ball and the whole molecule was composed of only carbon atoms. This was the third molecule after diamond and graphite that was only composed of carbon atoms.

Bell Labs’s Louis Brus discovered the colloidal semiconductor nanocrystals (quantum dots) in the year 1985. Gerd Binnig, Calvin Quate, and Christoph Gerber in the year 1986 invented the atomic force microscope. This microscope was capable of viewing, measuring and manipulating materials to a fraction of nanometers in the size. It could also measure various intrinsic forces in the nanomaterials.

In 1989, IBM's Almaden Research Center Don Eigler and Erhard Schweizer manipulated 35 individual xenon atoms to spell out the IBM logo; this showed that atomic manipulations can be applied to nanotechnology.

In the early 1990s nanotechnology companies started like Nanophase technologies in the year 1989, Nano- Tex 1998, Helix Energy Solutions Group in 1990 etc.

In 1991, Sumio Iijima of NEC discovered carbon nanotube (CNT) , these carbon nanotube like bulky balls are made of carbon and are basically tubular in their shape, also these tubes exhibit enormous strength, thermal and electrical conductivity.

Then 1992 was the year when C.T. Kresge and colleagues developed nanostructured catalytic materials MCM-41 and MCM-48 which is now used for crude oil refining. Moungi Bawendi of MIT then in 1993 invented a method for controlled synthesis of nanocrystals also called as quantum dots.


http://en.wikipedia.org/wiki/Engines_of_creation

http://en.wikipedia.org/wiki/Engines_of_creation



Few years later in 1998, the Interagency Working Group on Nanotechnology (IWGN) was formed under the National Science and Technology Council. This was formed so as to forecast the future developments and possibilities of growth of nanosciences.

The next year, 1999 was when the chemical bonding secrets for assembly of molecule [iron carbonyl Fe (CO) 2] from constituent components [iron (Fe) and carbon monoxide (CO)] was done with the help of a scanning tunneling microscope. Also, dip-pen nanolithography® which lead to the production of reproducible and manufacturable electronic circuits with a wide range of applications.

Naomi Halas, Jennifer West, Rebekah Drezek, and Renata Pasqualin at Rice University developed gold nanoshells in 2003. These nanoshells were able to discover, diagnose and treat breast cancer.

Then in the year 2005, Erik Winfree and Paul Rothemund from the California Institute of Technology discovered the DNA based computation and algorithmic self assembly. In these, nanocrystal growth is done by computations.

A nanoscale car made of Nanoscale carmade of phenyl ethynylene along with alkynyl axles and four spherical C60 fullerene wheels was build by James Tour and colleagues at Rice University in the year 2006.

In the year 2009-2010 Nadrian Seeman and colleagues at New York University were able to create DNA like robotic nanoscale assembly device which led to creating 3D DNA structures using DNA crystals that were synthetically developed. These were able to assemble and generate sticky ends as they are programmed for the functions.

Many new discoveries and processes are developed in a remarkable way in the field of nanotechnology and more growth is expected in the coming years.

CONCLUSION

In the past years many products that are nanotechnology based have been produced and provided for consumer’s benefit that are available in the markets. These products include golf balls that are able to fly straight, tennis rackets which are stiff, better baseballs, nanotechnology enabled automobile bumpers, scratch resistant glass coats, improved television displays, digital cameras, cell phones, nano-silver antibacterial socks and other endless variety of applications.

The field opens provisions and endless dimensions for discoveries that can serve well for science and technology. As commercial and public interest grows in the field, it promises a constant growth in the future. With Richard Feynman being the one who enlightened the idea of the potential of the nano things, the growth in the field took the input of many brilliant scientists and researchers. Brilliant brains have taken this field along a new way with years and as more and more are adding on there is a definite sparkling future for nanotechnology- “The next big thing

which is really small”.

REFERENCES-

1. Feynman, R. P. Engineering and Science 22–36 (February 1960).

2. Feynman, R. P., Leighton, R. B. & Sands, M. Feynman Lectures on Physics (Vols 1–3) (Addison Wesley, 1963).

3. Feynman, R. P. 'Surely You're Joking, Mr Feynman!' Adventures of a Curious Character (W. W. Norton, 1985). Toumey, C. Engineering and Science 16–23 (June 2005)

4. M. Reibold, P. Paufler, A. A. Levin, W. Kochmann, N. Pätzke & D. C. Meyer, Nature 444, 286, 2006

5. Yong-Hoon Kim, Tae-Hyoung Kim, Yeji Lee, Jong-Woong Kim, Jaekyun Kim, and Sung Kyu Park J. Nanosci. Nanotechnol. 14, 8158-8162 (2014).

6. Gold Bulletin 2007 40,4, p. 267

7. Enhanced Transparent Silver Nanowire Network for All Solution-Based Top-Contact Metal-Oxide Thin-Film Transistors , Yong-Hoon Kim, Tae-Hyoung Kim, Yeji Lee, Jong-Woong Kim, Jaekyun Kim, and Sung Kyu Park J. Nanosci. Nanotechnol. 14, 8158-8162 (2014)

8. http://www.nano.gov/timeline

9. http://nanotechweb.org/cws/article/multimedia/57535

10. Hommel D, Leonardi K, Heinke H, Selke H, Ohkawa K, Gindele F, Woggon U. CdSe/ZnSe Quantum Dot Structures: Structural and Optical Investigations. Phys. Stat. Sol. (b) 1997; 202,835-43


http://www.nano.gov/timeline

http://nanotechweb.org/cws/article/multimedia/57535



11. Binder Free SnO2-CNT Composite as Anode Material for Li-Ion Battery, Dionne Hernandez, Frank Mendoza, Emmanuel Febus, Brad R. Weiner, and Gerardo Morell Volume 2014 (2014), Article ID 381273, 9 pages

12. Catalytic Chemical Vapor Deposition of Methane to Carbon Nanotubes: Copper Promoted Effect of Ni/MgO Catalysts, Wen Yang, Yanyan Feng, and Wei Chu Volume 2014 (2014), Article ID 547030, 5 pages

13. Controlled Synthesis of Gold Nanoparticles Using Aspergillus terreus IF0 and Its Antibacterial Potential against Gram Negative Pathogenic Bacteria, Eepsita Priyadarshini, Nilotpala Pradhan, Lala B. Sukla, and Prasanna K. Panda Volume 2014 (2014), Article ID 653198, 9 pages


http://www.hindawi.com/journals/jnt/2014/381273/






RAPID DIAGNOSTIC TEST FOR PREDICTION OF PRETERM DELIVERY BY ACTIM PARTUS KIT

Dr Mitali Mahapatra, MBBS, MD Department of Obstetrics and Gynecology, Hi Tech Medical College,

Utkal University, Bhubaneshwar, India [email protected]

INTRODUCTION

In this era of modern world preterm delivery is the leading cause of neonatal morbidity and mortality. In India the incidence of perinatal deaths has been increased by leaps and bounds so to predict who is most likely to deliver preterm is important. The Actim partus kit is the tool to know about the preterm labour. This actim partus kit test is an immune –enzymatic test which is the best tool to know preterm labor at bedside. The test is most useful for symptomatic women with gestational age between 22 and 34 wks presenting with intact membrane and it is not reasonable to use in gestational age above 36 wks. It is a simple test and there is no risk of fetus and the mother.

PROCEDURE

1. Do speculum examination. 2. Hold the polyester swab provided in the kit. 3. Collect the cervical secretion /fluid from the endocervix with polyester swab. 4. Quantity to be collected 150micro L of sample for proper function of dipstick.

Note: The sample collection should be done before digital examination. 5. After the swab has absorbed the sample it should be inserted and swirled vigorously for 10 sec in an extraction

solution in which the dipstick can be dipped after the swab has been discarded. 6. Now keep the dipstick in the extraction solution until the liquid front reaches the result window . 7. Then the dipstick is removed from the extraction solution (as soon as the liquid front becomes visible in the

result window) and let to develop for 5 min in horizontal position. 8. Total time from sample collection to the availability of results is no more than 10 mins. 9. Interpret the result. 10. Count the no. of lines in result window. 11. Interpret the positive result if 2 blue lines (a control line and a test line) become visible in the result window.

The motto of the study is to know effectiveness of actim partus kit in prediction of preterm delivery.

MATERIALS AND METHODS

The study was conducted in the department of obstetrics and gynecology at Hitech Hospital, Bhubaneshwar. A Total of 108 patient were selected who attended OPD/LABOUR ROOM during the period of Feb 2013 to Feb 2014.

INCLUSION & EXCLUSION CRITERIA

All pregnant women were included who were presented with symptoms of preterm labor between 24 wks and 34 wks a singleton gestation and intact membrane. But women whose pregnancies were complicated by multiple gestation, PROM, Cervical cerclage, cervical dilation of 3 cm or greater, placenta previa , chorioamnioitis ,intra uterine growth restriction of fetus, preeclampsia, suspected fetal asphyxia or a major fetal anomaly excluded.

OBSERVATION

108 Patient were recruited for the study. 14 patient were excluded from the final analysis due to incomplete data. 94 patient had complete data for analysis.

Table: 1.1 Demographic and Clinical Characteristics of Study Population

MATERNAL AGE (T=94) GA (wks) Parity Gravidity Os

Positive n=28 (27) 30.5 0.5 2 1

Negative n=66 (27) 32.2 1 2 0





Table 1.2 Gestational Age at Delivery and Admission to Delivery Interval

Gestational age at delivery (wks) Admission to delivery interval (wks)

Positive n= 28 32.9 +_4.0 2.3

Negative n=60

P

37.4 +_1.8

<0.001

5.1

<0.001

Table 1.3 The Sensitivity, Specificity, PPV and NPV for IGFBP1 Test

PIGFBF-1 Test Sensitivity Specificity PPV NPV

48 hrs 1 0.74 0.18 1

7 Days 0.69 0.78 0.39 0.92

14 Days 0.72 0.80 0.46 0.92

NPV: Negative Predictive Value, PPV: Positive Predictive Value.

CONCLUSION

Actim partus test is lucid, faster, convenient method of predicting preterm labour. The test is not affected by semen .it is less expensive. With symptomatic patients a negative actim partus test result is a clear indication that the delivery will not start within the next 7 days .Pigfb1 has higher NPV of 1 in predicting risk of delivery within 48 hrs.

REFERENCES

1. Honest H, Bachmann LM, Gupta JK, Kleijnen J, Khan KS. Accuracy of cervicovaginal fetal fibronectin test in predicting risk of spontaneouspreterm birth: systematic review. BMJ 2002;325:301.

2. Kwek K, Khi C, Ting HS, Yeo GS. Evaluation of a bedside test for phosphorylated insulin-like growth factor binding protein-1 in preterm labour. Ann Acad Med Singapore 2004;33:780-3.

3. Lockwood CJ, Senyei AE, Dische MR, Casal D, Shah KD, Thung SN, et al. Fetal fi bronectin in cervical and vaginal secretions as a predictor of preterm delivery. N Engl J Med 1991;325:669-74.

4. Iams JD, Casal D, McGregor JA, Goodwin TM, Kreaden US, Lowensohn R, et al. Fetal fibronectin improves the accuracy of diagnosis of preterm labor. Am J Obstet Gynecol 1995;173:141-5.

5. Peaceman AM, Andres WW, Thorp JM, Cliver SP, Lukes A, Iams JD, etal. Fetal fibronectin as a predictor of preterm birth in patients with symptoms: a multicenter trial. Am J Obstet Gynecol 1997;177:13-8.

6. Joffe GM, Jacques D, Bemis-Heys R, Burton R, Skram B, Shelburne P. Impact of the fetal fibronectin assay on admissions for preterm labor. Am J Obstet Gynecol 1999;180:581-6.



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www.giapjournals.org/bioevolution.html

ROLE OF SUPEROXIDE DISMUTASE IN THERAPEUTICS: AN OVERVIEW Nameet Kaur and Nidhee Chaudhary*

Amity Institute of Biotechnology, Amity University, Noida, UP, India

*[email protected]; [email protected]

The greatest excitement in recent years in the area of free radical biology research has been focused on the pathological production of oxygen- derived free radicals, and on the possibilities of anti-radical and anti-oxidant approaches to therapy and prevention of diseases. There are large numbers of disease caused due to over production of free radicals or Reactive Oxygen species (ROS). Superoxide dismutase (SOD) has played an invaluable role in exploring the mechanisms of disease by virtue of its absolute specificity for its substrate; due to the fact that it is an enzyme, whereas the non-enzymatic radicals interfere in any other number of processes. SODs are used for preparation of many pharmaceutical compositions for treatment of many diseases including myocardial ischemia (Emerit et al., 2006; Downey et al., 1991), multiple sclerosis (Namaki et al., 2009), colitis and in improving the clinical irradiation treatment of malignant diseases such as breast cancer (Pajovic et al., 2003). Superoxide dismutase mimetics (Warner et al., 2004) such as tempol has also beneficial effects in several experimental models of hypertension and acute kidney injury (Quiroz et al., 2009). SOD mimetics have also been reported to extend the mean life-span of the wild-type worm Caenorhabditis elegans by a mean of 67 percent increase (Melov et al., 2000). SOD in its pharmaceutical form "Orgotein" is a potent anti-inflammatory agent approved in many countries, and because of their effectiveness in the general treatment of inflammatory and degenerative diseases, researchers are constantly looking at SODs and related agents in experimental animal models of disease (Marberger et al., 1974). Numerous studies have proved the safety of superoxide dismutase drugs in animals and man and studies prove SOD as a strong anti-inflammatory agent (Yi et al., 2002). Apart from invaluable health benefits, significant contributions towards various diseases are discussed below.

CARDIOVASCULAR DISORDER

Atherosclerosis is the root cause of heart attacks, strokes, and peripheral vascular disease all together known as "cardiovascular diseases". Atherosclerosis development depends on the balance between proinflammatory; antiinflammatory and antioxidative defense mechanisms. The significant role of oxygen-derived free radicals in myocardial reperfusion injury is very well documented (Bolli et al., 1988). The involvement of SOD in lipid peroxidation was analyzed using reperfused heart. The toxicity of high-dose superoxide dismutase suggests that superoxide can both initiate and terminate lipid peroxidation (Nelson et al., 1994). Role of SOD, oxidants and free radicals in reperfusion injury (a unique form of myocardial damage) has studied (Zweier and Talukder et al., 2006).

NEUROLOGICAL DISORDERS

As we are aware of the fact that Superoxide dismutase enzyme, plays an important role in protecting tissues from superoxide anions, which are normally produced in all aerobic cells. In brain tissue, aerobic metabolism increases with maturation and an increased level of SOD for anti-oxidant defense is therefore required. Animal experiments demonstrate that the activity of SOD in brain remains at a constant low level during fetal life and increases in the postnatal period (Mavelli et al., 1982). Various strategies have been carried out in order to understand the mechanisms involved in central nervous system. (Shukla,1984). Free radical injury plays a significant role in traumatic brain injury and SOD activity has been visualized in rat brain by histochemical localization (Okabe et al., 1998).

DIABETES

Oxidative stress plays an important role in the development of diabetes complications, both microvascular and cardiovascular. The outcome and progression of diabetic retinopathy is highly influenced by SOD and its overexpression in transgenic diabetic mice prevents diabetic retinopathy, nephropathy and cardiomyopathy (Ferdinando and Michael, 2010).

AGING

All cells are hypothesized to originate from stem cells. These stem cells mature as they divide and eventually reach a fully differentiated cell, which cannot divide. Aging is caused by the loss of stem cells, either due to cell death or terminal differentiation, or by eventual death of fully differentiated cells; both brought about by oxygen radicals. Young people naturally produce the antioxidant enzymes superoxide dismutase (SOD) and catalase as a defence


mailto:*[email protected]


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against destructive free radicals. Unfortunately, the levels of catalase and SOD continuously decline with age. Recent research suggests that SOD boosts immunity, quenches the free radicals and promotes longevity. Over the past 60 years, numerous studies have been performed to demonstrate the role of SOD and its contribution to aging. SOD serves as an important antioxidant defense mechanism in organisms, and any deletion or alteration of these genes shortens the life span as proved in experiments on budding yeast Saccharomyces cerevisiae (Muid et al., 2014).

HEPATOPATHOLOLICAL DISEASES

Many hepatic disorders are related to free radical formation and Superoxide dismutase plays an important role in curing various hepatopathological disorders. Liver damage due to alcohol-induced liver injury is associated with an increase in oxidants from a variety of possible sources; SOD is used as a potential anti-fibrotic drug for hepatitis C related fibrosis (Emerit et al., 2006). Superoxide dismutase extracted from fruits and vegetables has been potentially used for curing various diseases. It has been found that an SOD rich melon extract “Extramel” prevents aortic lipids and liver steatosis in diet-induced model of atherosclerosis. The findings support the view that good quantity intake of melon juice extract rich in SOD has potential beneficial effects for the treatment of atherosclerosis and liver steatosis, emphasizing its use as potential dietary therapy (Intes et al., 2012).

RENAL DISORDERS

Kidney is the only organ in human body which excretes toxins and harmful wastes and plays an important role in filtration of body. Superoxide dismutase activity was investigated in Human Glomerulonephritis. The study showed that renal function deterioration is directly proportional to SOD activity in patients. Lower level of SOD activity, a decreases the scavenger reaction of superoxide, thus making the tissue more prone to oxidative stress (Kashem et al., 1996). Novel approaches to therapy have been explored including SOD gene therapy and SOD targeting to the lung. In the future, new drugs interacting with superoxide may provide significant advances in the treatment of lung diseases (Demiryurek et al., 1999). Radiation-mediated lung injury is one of the limiting factors for radiation therapy. SOD-TAT is a fusion protein of hCuZn-superoxide dismutase (SOD) and HIV-1 Tat protein transduction domain; it is useful in the prevention and treatment of skin damage of guinea pigs by UVB radiation (Pan et al., 2012).

ARTHRITIS

Arthritis can be cured and its potential is analyzed by using various animal models. Study was performed by a rat model of arthritis. Evaluation of the therapeutic efficacy was performed by SOD transdermal delivery using Tfs. The amelioration of disease symptoms on animal treated with SOD-Tfs showed that epicutaneous application of SOD in especially developed mixed lipid vesicles play a significant role in the reduction of inflammation, in the adjuvant arthritis model (Simoes et al., 2005).

INFLAMMATORY DISEASES

SOD potentially decreases ROS and oxidative stress related diseases by interrupting the inflammatory cascades and scavenging free radicals (Riedl et al., 2005). It is highly effective in treatment of colonic inflammation and to generate new therapies for the treatment of inflammatory bowel diseases (Seguil et al., 2004). It possesses steroid-like anti-inflammatory activity; repeatedly injecting low doses of SOD maintained the suppressed state of edema (Yoshihiko et al., 1981). Liposomal-encapsulated SOD injections were used to treat systemic inflammatory diseases and skin ulcer lesions (Niwa et al., 1989). Catalase and superoxide dismutase mimics (Conjugation of a metal ion chelator to aromatic amino acids generates a series of novel metal-binding anti-oxidant enzyme) have been used for the treatment of inflammatory diseases (Fisher et al., 2003).

CANCER

Since SOD is the key enzyme in the first metabolic step of superoxide elimination, deficiency in SOD or inhibition of the enzyme activity may cause severe accumulation of O2 • - in cells thus, leading to cell death. Studies have demonstrated that SOD treatments of rodents before and after irradiation result in increased survival and cellularity of the bone marrow and reduced skin reactions (erythema, hair loss, scarring), pneumonitis and pulmonary fibrosis (Schwartz et al., 1996). A study was performed as an in vitro investigation using rat glioma cell line in culture to understand the change of superoxide dismutase (SOD) in tumor cells by hypoxia and hypoxia-normoxia exposure. The results obtained suggest that the SOD in glioma cells can be activated to compensate the damage from free


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radicals during hypoxic stress (Chang et al., 1997). Targeting SOD is a most reliable approach towards the selective killing of cancer cells.

SOD AS A FUTURE GENE THERAPY

Gene therapy is a novel therapeutic branch of modern medicine. Its emergence is a direct consequence of the revolution heralded by the introduction of recombinant DNA methodology in the 1970s. Many reports showed that SOD gene therapy has been shown to attenuate tissue damages and improve the recovery of the tissue injuries (Laurila et al., 2009); to provide the heart with protection against myocardial stunning (Li et al., 1998); to ameliorate delayed diabetic wound healing (Luo et al., 2004); to stent-induced vascular injury (Brasen et al., 2007); to attenuate ischemia-reperfusion injury of testes in rats (Palffy et al., 2006); to attenuates ischemia-reperfusion injury in the rat kidney if delivered to the kidney by intravenous injection (Yin et al., 2001). Reports also demonstrated that MnSOD gene therapy could be applied in combination with other therapies, for example with chemotherapy as a dual therapy achieving synergetic action in suppressing the tumour growth of colorectal cancer (Zhang et al., 2006); another example is in the rabbit model of vein graft disease, combination extracellular SOD gene therapy, anti-inflammatory, and anti-proliferative genes was found to be effective in decreasing neointimal formation (Turunen et al., 2006).

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Bioevolution vol 1 (3) september 2014

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