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Rakesh Sharma,Ph.D Certified Grant Writer
Innovations And Applied Solutions, Inc. 3945 West Pensacola Street, Tallahassee, FL 32304
technical or scientific development in both Public information and Technical abstract. readable. What is research proposal? Research proposal is a study plan to be followed in the timely manner.PUBLIC INFORMATION 2. What are key factors? Creative writing in effective writing style is the key of successful proposals.Date e.purpose.TECHNICAL ABSTRACT 3. its feasibility and direction of project writing process. contents and steps of a proposal.I like to introduce you first with the purpose. Use subheadings as guide indicators 4. public interests. Type of document b.Place or location or institution 2 . Strictly follow specific guidelines to complete proposal outline In general. specific aim. implications. It gives a continuity to various stages in steps of the work done. It serves as document to convince funding sources.PROPOSAL BODY 4. It serves as a guide in the research process. Tips of Research Outline Explanation: Mention clear.BUGET JUSTIFICATION Next important issue is Format of each component.Author d. The federal agencies have specific styles of proposal body such as: problem statement. Proposal body: 1. sponsors and federal research bodies.Title c. Face page: a. the proposal has the following contents: 1.Name of sponsor(s) f. What are the components of a proposal? 1. First use headings as note taking device 3. The proposal needs the purpose. Organization of the proposal outline 2. major and minor research questions.
2. Table of Contents a. tools of measuring variables and softwares Validity and reliability Pretesting the data collection instruments Definition and concepts Data analysis procedures Special techniques Human subjects Footnotes and appendices Bibliography 3 . LITERATURE REVIEW Historical background Theory relevant to research Current literature c. METHODOLOGY Major or general hypothesis Research Design and schematics Study subjects or population and characteristics Location and place of research Calender table of study to carry out Sample design and procedures Data collection and Data processing procedures Instruments. INTRODUCTION Brief description of the area of concern Problem to study Purpose of the study Major research question Minor research questions Major hypothesis and minor hypotheses Significance of the problem Justification to investigate the problem Feasibility of doing proposed study b.
 To develop a data base. …………………. Technical Objectives: Specific aims  To optimize the parameters  To optimize the technique  To characterize the data and analyze  To develop technique in order to design a new ―criterion‖.EXAMPLE OF STEP-BY STEP OUTLINE OF PLUGGING COMPONENTS IN PROPOSAL BODY PROPOSAL BODY Description of research problem: May resolve the problem of noninvasive skin imaging and measurement of structures still unsolved: May develop better theoretical models: May influence public interest or government policy: May assist in quick screening: Contribution in non-invasive skin evaluation: Literature review: Description of proposed research: Hypotheses: Example: A causes B to make effect C 1.  To image process to develop ―methodology‖. Specific Aim 1: To measure structures. Aim: Molecular and structural analysis with its main focus. 2. Specific Aim 2: To develop assessment. Preliminary studies done: Sampling: Healthy human volunteers For preliminary data 4 . ………………….
Sample size and sample power: analysis. 2. Data analysis Image J Statistics: SPSS and Prism Analysis and Statistical Evaluation: a. 6th month-16th month: Accquisition of imaging data and image processing. Assessment d. % difference. 16th month to 24th month: Analysis of data. Student ‗t‘ test and ANOVA Time Frame: First 6 month: standardization and calibrations of methods. 3. reporting and publication Implications of research findings: Work Cited 1. Assessment of c. ---------------------------------------------------------------------------------------------------------------- TASKS AFTER FIRST DRAFT OF RESEARCH PROPOSAL IS FINISHED To do list: Consultation with experienced knowledgeable person Repeated thoughts and incorporate them Not to do things: Document is not another paper or duplicate 5 . Association between b.Research methodology: Clinical evaluation Image processing and data analysis Imaging instruments: Data collection and measurement: Data collection and analysis by Office Access and microcomputing techniques .
REVIEW OF LITERATURE.SPECIAL TECHNIQUES BUDGET Review Process Which are Good proposals? Concise.METHODOLOGY. 2. processing phase is over Research Report writing SHAPE THE FIRST THREE CHAPTERS OF RESEARCH REPORT Seperate out different sections for use and examine them Reread them and edit them. 4. deadlines. adding material and removing inappropriate parts to keep the limit Update literature Methodology chapter as reflecting actual facts of research experience Special techniques Check 4 chapters in accomplished report: 1. one-time only grantsand matching grants Knowledge of funding agencies: financial range. Unimportant proposal Good material Enough money to use Professional formating by professional person To do things after research is completed Data collection. 3.INTRODUCTION. changing tenses. limitations Contacts and reputation of grant writer/researcher Read carefully an example of research funded. specific proposals. Proposal Biotechnology Research Grant 6 . patterns.
Greensboro. Scientist Center for Research Excellence in Nanobiosciences University of North Carolina at Greensboro 203 Eberhart 1000 Spring Garden St. NC 27403 7 .D.Tumor-Targeting Nanoparticles Carrier for Imaging and Therapy Co-PI: Rakesh Sharma. Ph.
Following the heart diseases..Tumor-Targeting Nanoparticles As Carrier for Imaging and Therapeutics Abstract: Superparamagnetic nanoparticles have emerged as potential dual-purpose localized hyperthermia therapeutic agents and imaging contrast agents. However. reduced septic shock potential (deletion of msbB gene) and antibiotic susceptibility. In this process magnetic material is introduced at the tumor site then subjected to an oscillating magnetic field that will cause the material to heat . A novel delivery technique is proposed that makes use of impregnating modified strains(name and subtype) bacteria species with characteristics of genetically stable. Magnetic Resonance Imaging (MRI) technology has grown over the recent years as most accepted imaging modality to answer tissue characteristics and non invasive intracellular events. Genetically modified strains of salmonella typhimurium bacteria after delivery systematically accumulate at tumor sites when injected in tumorbearing mice while these were rapidly cleared away from the blood stream in normal mice [6-8]. attenuated virulence. Utilizing the attenuated bacteria as contrast agent carrier will further improve the needed superior specificity. The levels of magnetizations at different gradient levels create MRI image with different contrasts . magnetic particles were synthesized and these particles displayed the 8 . However. Different types of superparamagnetic particles can be used as contrast agents to enhance the MRI signal. malignant tumors remain as the second largest leading cause of death in the United States . Achievement of high and steady magnetic fields has enabled the accurate MRI tissue characterization of pathological areas at high resolution. Heating of organs and tissues as a treatment of cancer has been known for a long time . At our lab. It generates MRI signal based on the difference of magnetization of the tissue protons in high magnetic fields subjected to a specific radiofrequency pulse sequence. The current proposed work is continuation of our previous research effects to develop novel magnetic nanoparticles as contrast agents for MRI microimaging with self-controlled tumor heating characteristics when subjected to alternating magnetic fields. Magnetic hyperthermia is the method of heating body tissue using magnetic materials. reduced septic shock potential and antibiotic susceptibility. The enforced delivery and residence of the magnetic carriers at localized tumor sites for longer times still remains a challenge. Development of superparamagnetic particles as novel imaging contrast agent and therapeutic agent carriers is a novel technique. The major objective of the present proposal is to upload magnetic nanoparticles into genetically modified strains of bacteria bearing attenuated virulence (depletion of purl gene). most commercially available contrast agents display poor specificity. Background and Significance Superparamagnetic nanoparticles as imaging contrast agents have emerged in MRI diagnosis and as heating therapeutic agents at malignant tumors sites [1-4]. magnetic particles selective for cancer cells with limited toxicity to adjoining normal tissues is a major challenge to accomplish.
The specific aim of the present proposal is to provide a smart delivery vehicle of the multifunctional nanoparticles at tumor sites to enable tissue characterization and hyperthermia therapy. 6132607). Mn-Zn-ferrite and Ni-Cu has been synthesized by the principal investigator . Self controlled hyperthermia controls the spot overheating otherwise that may cause necrosis. There is paucity in the literature on possibility of loading bacteria with imaging contrast agents or magnetic hyperthermia agents. Attenuated bacterial strains were reported as potential carriers for gene vectors . Rationale of the proposal The proposed research plan includes genetically-modified. Magnetic nanoparticles were used in separation of biological components from various samples such as blood and food samples [15-17]. Properties of these synthesized magnetic nanoparticles may be controlled by managing the parameters of their synthesis process [19-21]. as hyperthermia agent [1-3. Our approach will address the existing challenge of a site specific carrier without any change in the environment. Magnetic iron oxide nanoparticles (MION) are routinely used as imaging contrast agents for magnetic resonance imaging . to the knowledge of the PIs. These particles were functionalized for applications such as red cell separation from whole blood (US patents 6036857.. non-pathogenic bacteria as magnetic nanoparticles carrier. these bacteria would be ideal carriers for developing imaging contrast agents and therapeutic agents. Aims and Objectives The major objective of proposed research is to develop enabling technology to address the current strategy of nanoparticles as imaging contrast agent and selective hyperthermia agent carriers for cancer tissue without any toxicity to normal tissues. Utilizing the genetically modified bacteria as hyperthermia therapeutic agent carrier will enable the controlled delivery and release of the therapeutic agent at the tumor site. impregnating wild bacteria or attenuated bacteria strains with nanoparticles (naked or encapsulated) have not been available. vaccines  and chemotherapeutic agents . Nonpathogenic attenuated Salmonella. Clostridium and Bifidobacterium bacterial strains are capable of residing selectively in tumors at specific locations. To impregnate the magnetic nanoparticles in attenuated strain of bacteria Research Plan Synthesis of Magnetic Nanoparticles: Magnetic particles (6-30nm) made of iron oxide (Fe2O3). self-controlled magnetic 9 . 6129848.overheating self-control properties [1-3]. However. The specific aims are: 1. Neodymium Iron Boron (NdFe-B). 18]. Because of their selectivity for tumor tissue. To synthesize magnetic nanoparticles with size distribution less than 25 nm as dual-purpose potential imaging contrast agent and hyperthermia therapeutic agents 2. The present study anticipates a novel approach of loading the attenuated strains of bacteria with entrapped superparamagnetic nanoparticles that would function as imaging contrast agents and/or hyperthermia agents.
Cellular diffusion: Small size particles (less than 10 nm) can infuse into the cellular membrane. These particles will need to maintain a narrow size distribution with mean diameter in the order of 25 nm. Compared to wild-type Salmonella. Due to the requirement of larger particle size (25-50nm) to effectively function as hyperthermia agent and imaging contrast agent the diffusion approach may be limited. they will be encapsulated with polymeric material.5nm .hyperthermia (US patent application 20050249817). A co-precipitation technique will be utilized in producing the particles . At x=0. Our research team will focus on producing magnetic nanoparticles that can be employed as imaging contrast agents and hyperthermia agents. The 10 . This bacteria will be administered intravenously to mouse bearing tumors assuming bacteria accumulate preferentially within the tumors (with tumor to normal ratios of 300-25. . For it. it can be conveniently eliminated from the body. The uniform heat generation around the particles needs their narrow size distribution. Gadolinium substituted Mn-Zn-ferrites ( Mn0.000:1) and persist in tumor tissue for more than 4 weeks . However. Previously the PI utilized ultrasonication technique to produce iron oxide particles with average diameter of 7nm and standard deviation of 2. The magnetic moment of these particles is proportional to the size of the particle with uniform image signal . The secondary research objectives are: 1. Uploading the Bacteria Strains with Nanoparticles: Salmonella strains YS1646 (synonymous to VNP20009) with a stable msbB deletion will be purchased from Vion Pharmaceuticals Inc.5Gd x Fe( 2 x )O4 ) is a suitable candidate that fits with the criteria. it is proposed to characterize the intake limits of the nanoparticles. polymeric or silica encapsulation during synthesis and acidic reduction. SEM and/or TEM microscopy will be conducted. If the particles are found toxic to the bacteria. Same approach will be utilized to synthesize the Gd-based nanoparticles. To perform insertion of the magnetic nanoparticles in the bacteria: There have been reports on attaching nanoparticles to microorganism. Other techniques to produce uniform particle distribution include centrifugation gradient. 21] and in silica in . optical microscopy. The approaches employed to attach nanoparticles to microorganism and impregnate the attenuated strains with nanoparticles are: a. . 17. To investigate the toxicity of the particles on the attenuated bacteria strains: The strains of bacteria will be incubated with magnetic nanoparticles according to the protocol reported by the PI in (US provisional patent application 60888343). In addition. The encapsulated particles can maintain the monodispersity utilizing techniques described elsewhere [23-27]. Encapsulation of nanoparticles with polymeric material will be done as described in [16. The material for producing the magnetic nanoparticles will be purchased from Sigma. The cells will be cultured according to the protocol by Clairmont et al.05 the compound reaches a Curie temperature (indicator of self-controlled heating under alternating magnetic field) of 325K. New Haven CT. Because of the VNP20009 sensitivity to a number of antibiotics..000 fold. 2. Gadolinium Diethylene-Triaminepentaccetic Acid (Gd-DTPA) is currently used as an imaging contrast agent.5 Zn0. rapid diagnosis of pathogen (US patent application 20060211061) and rapid diagnosis of AMI (US patent application 20060040412). it is environmentally friendly as it has difficulty to survive in free environment . the pathogenicity of this strain is less over 10.
The pellet will be sliced into thin sections using a micro-tome. In 1975. Interesting. 17. Blakemore discovered magnetotactic bacteria . which have an ability to synthesize fine (50–100 nm) intracellular membranebound particles consisting of magnetite or greigite (Fe3S4). murine tumors expressing L6 antigens were treated with L6 monoclonal antibodies conjugated to the microbial enzyme cytosine deaminase. The subsequent conversion of the prodrug 5-fluorocytosine to the cytotoxic agent 5-fluorouracil could then be followed with in vivo 19F spectroscopy . followed 1 day later by intratumoral injection with 5-fluorocytosine. This approach has been subsequently verified using colon cells stably transfected with yeast cytosine deaminase. and in colon tumor xenografts directly injected with attenuated Salmonella bacteria overexpressing cytosine deaminase. b. Label coated magnetic particles will be used for the protocol as reported elsewhere [4. In-take of plasmid coated particles. background 19F signals in cells and tissues are low. Fig. 21]. Because there are no naturally occurring fluorine-containing metabolites. 20. 1. The evaluation of the intake will be conducted using microscopy and fluorescent labeling of the coated particles.83 compared with the proton.  19 Magnetotactic bacteria Magnetotactic bacteria have got utility in the biotechnology field.attenuated bacteria will be grown in nanoparticles enhanced culture. 19 F is also a spin one-half nucleus with 100% natural abundance and a relatively high sensitivity of 0. Serial F MR spectroscopy spectra (13-minute time resolution) showing the conversion of 5-fluorocytosine to 5-fluorouracil in the presence of cytosine deaminase in a murine tumor. the magnetic 11 . Excellent reviews on the use of magnetotactic bacteria in this field have been recently reported . Following the induction phase a fixative will be applied to arrest the cells then collecting the pellet. The tumor was treated with an attenuated Salmonella typhimurium strain recombinant to provide cytosine deaminase. Investigation of the upper limit for the plasmid coated particles size will be conducted. In one example of the use of 19F MR to monitor enzyme activity associated with gene therapy. making this nucleus a potential candidate for the development of contrast agents for molecular imaging studies. Optical as well as electron microscopy will be conducted to evaluate the level of uploading of particles using direct diffusion. Magnetotactic bacteria form a heterogeneous group of Gram-negative prokaryotes with morphological and habitat diversity. The nanoparticles will be coated with plasmid and placed in the cell culture as the bacteria.
Following the successful completion of the project. antibodies and oligonucleotides . pursue commercialization prospects and seek external funding. their suspensions are very stable and the particles can be easily modified . candida and other bacteria. the PI is planning to file for patent application (provisional applications have been filed).nanocrystals are covered with an intracellular phospholipid membrane vacuole. Even the production of a protein-magnetite complex by genetically engineered magnetotactic bacteria Magnetospirillum sp. Due to the presence of the phospholipid layer the particles are biocompatible. Our goal is to capitalize the technology using salmonella. AMB-1 has been proposed recently . These magnetic nanoparticles have been used for the immobilization of a variety of enzymes. Bacterial magnetite nanoparticles obtained from magnetotactic bacteria after disruption of the cell wall and subsequent magnetic separation have been used for a variety of bioapplications. forming structures called magnetosomes. 12 . Future Work: This project constitutes the first proposal for the PI since joining the newly established center of research excellence in nanobioscience.
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Mater. R Kumar. synthesis and applications in glycoscience. 2005 .lucy.International Journal of Nanotechnology. 2005 .all 3 versions » PL Number. P Investigator.2006 . S Penadés . A Watterson.565. J Yoo. SJSSM Arrivo. R … . CP Leamon .all 2 versions » R Gref.… . G SusTech . S Hong.US Patent 5. 1996 . open .MD .2006 .Biochimica et Biophysica Acta (BBA)-General Subjects.all 2 versions » JA Reddy.all 6 versions » S Svenson. YP Number .peppas.Dendrimers in biomedical applications—reflections on the field star. VM Allagadda. 2006 . R Czerw. RS Langer .ingentaconnect.215.books. C Marquina.wiley. J … . D Serrate. 2005 Inderscience Amphiphilic polymers and methods of use thereof .doi.com Idea Development Award Proposal .freepatentsonline.net Superparamagnetic Colloids: Controlled Synthesis and Niche Applications* H Yang.com Web Search Targeting therapeutic and imaging agents to folate receptor positive tumors .Curr Pharm Biotechnol.Elsevier ONLINE PUBLICATIONS .Google Patents [BOOK] Nanotechnology in Cancer Therapy MM Amiji .org Glyconanoparticles: Types.google. G Lim.com 16 .mrs.freepatentsonline. R Fernandez. VS Parmar.all 3 versions » GSU Simon. biomedicine and material … M Jesús. S Soker.all 4 versions » JM De Teresa. Y Xia .all 2 versions » CK Colton. Y Minamitake.com From magnetoelectronic to biomedical applications based on the nanoscale properties of advanced … .Adv.com Cell scaffold matrices with incorporated therapeutic agents K Code. 2007 . DA Tomalia .Advanced Drug Delivery Reviews.Elsevier Biodegradable injectable particles for imaging . A Atala.
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