UBSJ

University of Illinois

at Chicago

Bioengineering

Student Journal

Spring 2013

Vol. IV No. 1

CHIEF EDITOR

Cierra Hall

EDITORS

Benjamin Schwartz

Brynne Nicolsen

REVIEWERS

Helen Ashay

Jesse Gerringer

Forest Jacoby

Dan O’Neill

Sebastian Pernal

Hari Sreedar

Aishwarya Vaidyanathan

COVER ARTIST

Karan Kerwell

FACULTY ADVISOR

Professor Richard Magin

Contact:

bioejour@uic.edu

phone: (312) 996 – 2335 fax: (312) 996 - 5921

UIC Bioengineering Student Journal

Department of Bioengineering, University of Illinois at Chicago,

Science & Engineering Offices (SEO), Room 218 (M/C 063)

www.bioe.uic.edu

UBSJ IS A UNIVERSITY OF ILLINOIS AT CHICAGO BIOENGINEERING

STUDENT PUBLICATION

UIC Bioengineering Student Journal Spring 2013

Vol. IV No.1

Contents

Foreword

i

A TIMELINE OF BIOENGINEERING

Jesse Gerringer

1

ADVANCES IN DNA COMPUTING AND MOLECULAR PROGRAMMING

Mark Emmons

6

FULLERINES; POSSIBLE TO EVACUATE POST-ADMINISTRATION OR

ARE THEY JUST TOXIC?

Grant Hartung

9

REVIEW OF THE APPLICATIONS OF ULTRASONIC WAVES

Dan O’Neill

15

REVIEW OF METHODS OF DRUG DELIVERY TO THE POSTERIOR

SEGMENT OF THE EYE

Hari Sreedhar

20

EXTENDED MONITORING OF OSTEOGENIC DIFFERENTIATION

USING AN MRI-COMPATIBLE TISSUE CULTURE SYSTEM

Aishwarya Vaidyanathan

26

MEDICAL IMAGING IN FORENSIC SCIENCE

Marisa Doria

32

Call For Papers

38

Image Credits 39

i

Foreword

On behalf of the Bioengineering Department at UIC, I am happy to present this year’s volume of

the UIC Bioengineering Student Journal, a journal in which UIC Bioengineering students both

participate and manage themselves. UBSJ serves to unite students in the Department of

Bioengineering in their quests to develop not only skills related to writing and revising technical

written material, but also communication, cooperative, and organizational skills. Each article is

put through several rounds of peer review and finally an editorial board review. After final

approval, the article is accepted into the new volume. The stringency of this process helps to

ensure that students are able to graduate with the skills necessary to participate in the world of

journal publication and scientific article composition, in addition to their bioengineering skill

sets that they gain in their coursework.

I would like to thank Dr. Magin for allowing us this opportunity to improve ourselves, Luke for

assisting with website and printing concerns, the editorial board for their patient and meticulous

input, and the authors and reviewers for all their hard work.

This year UBSJ has begun issuing monthly reading recommendations in its Book of the Month

Club. Each month, one of the editors of the UIC Bioengineering Student Journal selects an

exciting book that relates to bioengineering, medicine and technology. Make sure to check the

UBSJ website each month for a new book to explore!

Cierra M. Hall

Editor-in-Chief

1

A TIMELINE OF BIOENGINEERING Jesse Gerringer jgerri2@uic.edu

Abstract This article presents a history of bioengineering. In order to analyze the history of bioengineering, it must be defined. Once defined, it is evident that bioengineering encompasses a wide range of sciences and engineering, thus making the history of bioengineering vast and complicated. Although bioengineering has its roots in ancient times, no significant advancements were made until the 14th and 16th centuries with the development of corrective lenses. Entering the 20th century, technological developments in bioengineering occurred decade after decade, especially in the field of genetics and cloning. As we progress through the 21st century, cellular and tissue engineering are becoming a major focus of bioengineering. By analyzing the history of bioengineering, it can be noted that humanity has progressed greatly and has potential for greater advancements. Keywords: bioengineering, history, prosthetics, spectacles, cells, PCR, cloning, tissue engineering

1. Introduction In order to analyze the development of bioengineering over time, it is essential to understand what bioengineering is. Unlike traditional engineering fields such as mechanical engineering, bioengineering applies the principles of engineering to biological systems [5, 16]. Integrating biology and engineering is difficult because biological interpretation is always changing. Therefore, the field of bioengineering is constantly adapting to fulfill the needs of society [5]. Bioengineering includes many topics such as genetic engineering, cellular and tissue engineering, biomedical engineering, neural engineering, and biotechnology. Engineering cellular life forms to carry out specific chemical or mechanical processes, improving drug delivery, creating prosthetics to help those who are disabled, and engineering organs in the lab for those in need of transplants are some of the many applications of bioengineering [5]. Figure 1. An example of one of the applications of bioengineering. Pictured is a microprocessor-controlled prosthetic leg [19].

With bioengineering defined, the development of the field over time can be analyzed. First, the pre-20th century advances in bioengineering will be examined, followed by specific eras of the 20th century. Finally, recent progress in bioengineering in the 21st century will be examined. 2. Pre-20th Century The time periods that are most notable for pre-20th century bioengineering advancements are ancient times and the 14th, 16th and 17th centuries. 2.1 Ancient Times While the technology of ancient times was primitive by today’s standards, recent studies have shown that there were some exceptions. One such exception is the prosthetic Greville Chester toe [6]. It was determined that the toe was fabricated around 600 BC, making it one of the earliest prosthetics discovered. The toe was found in Thebes and appears to have signs of wear from being attached to an Egyptian sandal. Even more remarkable than the Greville Chester toe is another prosthetic toe dating back to ancient Egyptian times. Estimated to have been used approximately 950-710 BC, this prosthetic toe was found not by itself, but instead attached to the owner’s foot [6]. The toe was composed of three parts: one part seemed to be made out of leather while the other two parts were made out of wood. Based on the structure of this prosthetic toe, the carver had a basic knowledge of anatomy. Even the

A Timeline of Bioengineering—J. Gerringer

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hinge seemed to resemble the flexion action of the metatarsophalangeal joint [6]. Figure 2. The prosthetic toe dating back to 950-710 BC [6]. 2.2 14th, 16th, and 17th Centuries As the human race progressed from ancient times, there was not many advancements in bioengineering until the 14th century. The most significant advancement related to bioengineering was the invention of eyeglasses. The first spectacles manufactured in the 14th century were basic in design and would be improved upon in the 16th century [4]. The spectacles produced in the 14th century featured mainly double convex lenses because hyperopia was the most common disorder. Hyperopia, also called farsightedness, is a condition in which the lens becomes unfocused, making near objects difficult to see [11]. Although the invention of eyeglasses improved the lives of many, the lenses were often imperfect due to errors in the manufacturing process. As the 16th century approached, the popularity of spectacles increased. The manufacturing process had improved, and concave lenses, to aid those with myopia, had also emerged. Myopia, also called nearsightedness, is a condition in which far objects are difficult to see. By applying the concepts of physics and engineering techniques, scientists were able to create a product to address the shortcomings of the biological function of sight [4]. Figure 3. The pictures above show how the lenses found in spectacles correct vision. The eye on the left represents a person with hyperopia. The light is focused behind the eye, making close objects difficult to see. The double convex lens properly focuses light to the retina. To the right is an image of a myopic eye. The light is focused in front of the retina, so objects far away are difficult to see. The concave lens focuses light to the retina [2].

Thanks to the development of spectacles, scientists could now experiment with lenses. This led to studies involving magnification. By 1590, the first microscope was invented by Dutch lens maker Hans Janssen [3]. While Janssen’s microscope was simply two lenses placed in a tube, his invention has led to light microscopes that can magnify almost 1000 times today. Following the invention of the microscope, Robert Hooke made the famous discovery of cells. In the 1660s, Hooke used microscopes to analyze over 60 specimens, such as sponges, wood, and seaweed [8]. While analyzing cork under the microscope, he described different compartments, or “cells.” Bioengineering, or cellular engineering specifically, is based on living cells. Without the discovery of cells, neither bioengineering – nor biology itself – would have progressed to the extent which it has. 3. The 20th Century In regards to the advancement of bioengineering, the 20th century can be divided into three parts: the first half of the 20th century, the 1980s, and the 1990s. 3.1 The First Half of the 20th Century At the turn of the 20th century, scientists knew that living beings were composed of cells. With the aid of recent studies in genetics, scientists were beginning to unravel the mystery of life and were developing new techniques that would become crucial to bioengineering. In 1907, Ross Harrison was able to perform a successful in vivo cell culture of frog nerve cells at Johns Hopkins University [1]. Cell culturing involves isolating cells from specific tissues and growing them in the lab. By successfully performing a cell culture, Ross Harrison enabled scientists to perform experiments on specific cells in an organism. Another significant advancement in bioengineering took place in Manchester, England in 1914. By the end of the 19th century, scientists in cities like Manchester were trying to solve the problem of waste, specifically sewage. It was well known that sewage carried disease and many scientists in the world were working towards a cleaner environment in cities. In Manchester, bacteria were used in the sewage treatment process by allowing the bacteria to decompose the contents of the sewage [15]. For the first time, people were using microorganisms to carry out chemical processes for the benefit of the human race. This form of sewage treatment, known as

A Timeline of Bioengineering—J. Gerringer

3

“activated sludge,” is still used today and is vital towards sanitation in cities around the world [15]. The 1940s was a decade full of discovery in both biology and bioengineering. During this time, DNA was determined to be the genetic material within all cells. In 1943, Oswald Avery conducted exp eriments based on studies performed in the 1920s by Frederick Griffith [21]. Griffith’s experiment involved injecting mice with Type III virulent Pneumococcus. This type was found to be lethal. Another strain of Pneumococcus was injected, Type II non-virulent, and the mice survived. After proving these two cases, Griffith killed the Type III Pneumococcus with heat so when injected, the bacteria would not be lethal to the mice. However, when the Type II bacteria were injected along with dead Type III bacteria, the mice would die. When Avery conducted the same experiments in 1943, he was able to deduce the cause of this – DNA tests for Type III turned up positive [21]. This proved that genetic material could be passed from one strain to another. Through further experimentation it was determined that DNA was being transferred, thus proving DNA was the source of the genetic code [21]. With this knowledge, scientists could now perform genetic experiments pertaining to inherited traits, opening up many applications. Like the first half of the 20th century, the 1980s saw significant advances in the field of genetics. 3.2 The 1980s In 1980, the Supreme Court ruled that engineered life forms could be patented [10]. This court case arose due to increased manipulations of DNA. One of the most important inventions of the 1980s was that of the Polymerase Chain Reaction (PCR) process. This process was invented by Dr. Kary Mullis in 1983; however, it would not come to full fruition until the early 1990s. The goal was to make multiple copies of the same strand of DNA. To do this, the DNA strands would be heated in order to split them in two. Then the primer needed for the target sequence would be added along with the enzyme DNA polymerase [7]. This would allow for the synthesizing of the target sequence on a massive scale. PCR technology would become crucial in both the biotechnology and genetic fields. 3.3 The 1990s In 1990, the first federally approved gene therapy treatment was performed. Gene therapy is the transfer of genes into a patient in order to treat a

genetic disease [20]. While the concept sounds simple, in reality it is quite difficult. A notable example is suicide genes in gene therapy. For the treatment of tumors, for example, this type of therapy would include transferring the Tumor Necrosis Factor alpha gene (TNF-α) [20]. This gene causes tumor cells to undergo a premature death. The concept was that tumor-infiltrating immune cells would enter the tumor cells with TNF-α and transfer the gene to the tumor cell causing the premature death [20]. Figure 4. This is a diagram describing the PCR temperature cycle. The temperature is raised to melt the DNA strands and then lowered to let primers anneal. Finally, the temperature is set to 72°C to allow the polymerase to extend the primers [12]. At the end of the 1990s another application of bioengineering was discovered: cloning. In 1997, Dolly the sheep was the first animal cloned from adult cells [14]. Using cells from a frozen tissue sample from a six year old sheep, many attempts were made to transfer the nucleus to an egg cell in order to initiate development. Out of 277 transfers, Dolly was the only successful sheep to come to term [13]. Because of Dolly, scientists around the world began racing to clone animals, and in the past decade have done so at higher success rates [18]. So far, the nuclear transfer process is the only process used in cloning adult animals [18]. On a basic level, the somatic cell nuclear transfer (SCNT) beings with selecting a cell from a part of the body. In the case of Dolly, mammary gland cells were used. The nucleus of an egg cell from another organism is replaced with that of the somatic cell. Stimulation, typically via electric shock, initiates cell development. After the embryo develops to a specific point, it is then placed in a third organism where the embryo can develop to term. However, of all the animals that have been cloned, this method is only successful up to 20% of the time [18]. Into the 21st century, cloning

A Timeline of Bioengineering—J. Gerringer

4

experiments are still being performed. The goal is to increase efficiency by improving the SCNT method. One theorized method is using another round of cell transfer. This time, pluripotent stem cells (cells with the ability to develop into many differentiated cells) would be added in order to promote development [18].

Figure 5. Outline of three different methods of nuclear transfer cloning [18]. 4. The 21st Century A key advancement in bioengineering during this time period has been in cellular and tissue engineering. Skin replacements have become a reality and technologies for repairing other tissues are already well underway [9]. Tissue engineering helps bypass many difficulties encountered in therapies and organ transplants. Tissue engineering involves using tissue extracted from the patient and seeding it in the lab using scaffolds. It may hold the promise of “replacing living tissue with living tissue designed and fabricated to meet the individual defects” [17]. A main reason why tissue replacement using these methods is highly effective is that the body does not reject the cells, as they were originally obtained from that individual [17]. In order to successfully engineer tissues in the laboratory, it is crucial to have efficient bio-reactors –systems or devices that can support an active

biological environment – and cell-friendly scaffolds [17]. A difficulty in culturing somatic cells is that some proliferate slowly. To get around this, totipotent stem cells – cells with the ability to develop into all living cells for a specific individual – are needed. Similar to cloning, therapeutic cloning is a more promising application of tissue engineering. Like cloning, an egg cell’s nucleus would be replaced with the nucleus of a somatic cell from the host. Once this embryo develops, the stem cells are removed and can be developed into specific cells for use in the individual [17]. Figure 6. The basic outline of therapeutic cloning [17]. These totipotent stem cells have the potential to develop into nearly any type of cell in the body. The cells can be cultured and chemically signaled to develop into the specific cell needed and injected into the host. While this aspect of tissue engineering is simple, the aspect of growing organs in the lab is much more difficult. This is mainly due to the fact that organs require much more complicated scaffolds than that of simple tissue. Research in this area has gone to the extent of applying inkjet printers to growing organs. They have been able to accomplish this by using a cell suspension as the “ink” of the printer and then printing the cells onto disks in the formation that is needed [14]. 5. Conclusion Bioengineering is a field that brings the principles of engineering and biological systems together on the same interface [5]. Over the course of human history, advancements in bioengineering have led to the improvement of human health and the understanding of genetics. Ancient Egyptians were working on prosthetics centuries before any other civilization [6]. Spectacles in the 14th and 15th centuries helped correct the vision of near and far-sighted population,

A Timeline of Bioengineering—J. Gerringer

5

and even today, many people depend on eyeglasses in order for their eyesight to function properly [4, 11]. During the 20th century, scientists began to understand the composition of living beings and realized the potential of using microorganisms to their benefit [15]. Towards the end of the 20th century, the understanding of DNA led to advancements in replicated specific sequences, as seen with PCR [8]. Also, cloning an adult animal became a reality [13]. As we progress into the 21st century, cellular and tissue engineering are proving to have numerous applications in improving the health of the general public. Patients will no longer run the risk of rejection of tissues and organs because they are derived from their own cells [17]. The future of bioengineering is very promising and has the potential to fix many problems related to human health. 6. References 1. Abercrombie, M.; Ross Granville Harrison;

Biographical Memoirs of Fellows of the Royal Society; 7, 110-126, 1961

2. “Astigmatism, Hyperopia & Myopia.”

Children’s Hospital Boston, Web 1 November 2012 <http://www.childrenshospital.org/az/Site1517/mainpageS1517P0.html>.

3. Ball, C.S.; The early history of the compound

microscope; Bios, 37, 51-60, 1966 4. Bardell, D; Eyeglasses and the discovery of the

microscope; The American Biology Teacher, 43, 157-159, 1981

5. Endy, D.; Foundations for engineering biology;

Nature, 438, 449-453, 2005 6. Finch, J; The art of medicine: the art of ancient

prosthetic medicine; The Lancet, 377, 548-549, 2011

7. Flannery, M.C.; Biotechnology and

bioengineering; The American Biology Teacher, 60, 464-467, 1998

8. Gest, H.; Homage to Robert Hooke; Perspectives

in Biology and Medicine; 52, 392-399, 2009

9. Griffith, L.C., A.J. Grodzinsky; Advances in biomedical engineering; JAMA; 285, 556-561, 2001

10. Hughes, S.S.; Making dollars out of dna; Isis, 92, 541-575, 2001

11. Ilardi, V; Eyeglasses and concave lenses in

fifteenth-century Florence and Milan: new documents; Renaissance Quarterly, 29, 341-360, 1976

12. Kubista, M., J.M. Andrade, M. Bengtsson, A.

Forootan, J. Jonák, K. Lind, R. Sindelka, R. Sjöback, B. Sjögreen, L. Strömbom, A. Stahlberg, N. Zoric; The real-time polymerase chain reaction; Molecular Aspects of Medicine, 27, 95-125, 2006

13. McLaren A.; Cloning: pathways to a pluripotent

future; Science; 288, 1775-1780, 2000 14. Nakamura, M., A. Kobayashi, F. Takagi, A.

Watanabe, Y. Hiruma, K. Ohuchi, Y. Iwasaki, M. Horie, I. Morita, S. Takatani; Biocompatible inkjet printing technique for designed seeding of individual living cells; Tissue Engineering; 11, 1658-1666, 2005

15. Pickstone, J.; Manchester’s history and

Manchester’s medicine; British Medical Journal; 295, 1604-1608, 1987

16. Riley, M.R.; Introducing journal of biological

engineering; Journal of Biological Engineering, 1, 1-3, 2007

17. Shieh, S., J.P. Vacanti; State-of-the-art tissue

engineering: from tissue engineering to organ building; Surgery; 137, 1-7, 2005

18. Thuan, N.V., S. Kishigami, T. Wakayama; How

to improve the success rate of mouse cloning technology; Journal of Reproduction and Development; 56, 20-30, 2010

19. Thurston, A.J.; Paré and prosthetics: the early

history of artificial limbs; ANZ Journal of Surgery, 77, 1114-1119, 2007

20. Tiberghien, P.; Use of suicide genes in gene

therapy; Journal of Leukocyte Biology, 56, 203-209, 1994

21. Vigue, C.L.; Oswald Avery and dna; The

American Biology Teacher; 46, 207-211, 1984

6

ADVANCES IN DNA COMPUTING AND MOLECULAR PROGRAMMING

Mark Emmons memmon3@uic.edu

Abstract

The creation of nanoscale computers, assembled piece-by-piece from strands of deoxyribonucleic acid, is a growing field within nanotechonology. A variety of different methods have been implemented that allow for the use of DNA to solve computational problems. Using DNA is advantageous in that it allows for the implementation of parallel computing, or computing of several operations at one time, via many separate strands of programmable DNA as well as a significant increase in bit density. These computational strands have been successfully used to create many simple programs, such as solving simple mathematical equations, deciphering and encrypting images, and more advanced logic computations. Recently scientists have demonstrated that DNA can be used to store binary information and be re-sequenced with 100% accuracy. Programmable DNA is an exciting, new possibility in the field of illness diagnosis and treatment. Properly-programmed DNA could be potentially used not only to identify afflictions, but also to release drugs at illness sites. The idea of programmable “smart-drugs” created from DNA has led to research of in vivo methods of imaging, diagnosis, and treatment.

Keywords: DNA computing, nanotechnology, molecular programming, DNAzyme, smart-drugs, molecular automaton, DNA storage

1. Introduction

The possible applications of Deoxyribonucleic Acid (DNA) are infinite. The basic units of DNA are the four base pairs – adenine, cytosine, guanine, and thymine (referred to as A, C, G, and T, respectively). These four base pairs are arranged into sequences which code for the organism’s phenotypes [1]. In recent years, experiments with DNA have expanded to solving computational problems. Professor Leonard M. Adleman was the first person to establish the possibility of DNA computing in 1994 with a proof-of-concept experiment in which he solved the NP-complete (nondeterministic polynomial time) Hamiltonian Path problem with a simple biological experiment [1]. Lipton expanded upon this work by proving that DNA could solve any NP-complete problem, more efficiently than electrical computers due to its ability to run in parallel [2]. One year later, Dan Boneh et. al. discussed the different possible methods of using DNA to solve computational problems, including formula comparisons, DNA circuits, and nanoscale Turing Machines [3].

2. Methodology

Boneh et. al compiled a list of numerous laboratory operations to show how computations could be

carried out upon DNA [3]. These operations included extract, length, pour, heat, cool, amplify, and cut. The authors proposed that by carrying out a combination of the operations mentioned, DNA could be used to solve any satisfiability problem. “Pouring” refers to combining the contents of two test tubes. “Heating and cooling” refer to the separation and rebuilding of double strands by changing the temperature of the system in which the DNA resides. “Cutting” is achieved with restriction enzymes to allow for excision of DNA segments at specific points [3].

To extract a specific segment of DNA, a complementary single strand is created and bound to a magnetic bead. The DNA of interest is melted and spread over a bead matrix. Strands containing the segment will bind, while the others will be washed away. The amplification of DNA is achieved by simple polymerase chain reaction (PCR) where the DNA is melted, combined with a primer, and then duplicated using the enzyme DNA polymerase until a satisfactory amount of the segment is achieved. Separation of the strands by length is done by gel electrophoresis. The molecules are placed in a well of agarose gel and given an electrical charge. Larger segments of DNA will move more slowly through the gel, allowing segments of specific lengths to be isolated from the mix for further analysis and computation [4].

Advances in DNA Computing and Molecular Programming—M. Emmons

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2.1 DNAzymes

J. Elbaz et. al. from The Institute of Chemistry at the Hebrew University of Jerusalem created a library of molecular logic gates for DNA computation [5]. The logic gates were constructed out of catalytic nucleotides known as “DNAzymes” to create a unified basis for completing computations from DNA. DNAzymes were initially synthesized by Ronald R Breaker and Gerald F Joyce in an attempt to prove that, despite never occurring in nature, catalytic DNA strands that function in the same manner as ribozymes could be artificially manufactured in a lab environment to perform similar reactions to their RNA counterparts [6]. The functional site of the DNAzyme synthesized by Breaker and Joyce featured a segment, rA·, of RNA that was necessary to form a more favorable binding site for the Pb2+ the cleaving reaction was dependent on. Joyce would later develop an all-DNA enzyme that could cleave almost any RNA strand in the presence of Mg2+ [7]. This enzyme contains a functional site 15 units long in the middle and is flanked on each side by recognition arms, the segments that attach to the RNA, that can be changed to allow for attachment to different strands of RNA.

The logic gates developed at The University of Jerusalem work by combining DNA enzymes and their substrates into one “computational unit” when the correct priming sequence is recognized by the enzyme [5]. The computational unit is composed of two subunits, the “input module” and “processing module”. The input module is represented by the recognition arms of the enzyme and the input strands from the DNA. The processing module is represented by the active sites of the enzyme which operate on the DNA. The substrate, if recognized by the processing arms, is cleaved and the enzyme remains unchanged. A substrate that is not recognized by the enzyme “logic gate” will remain the same.

Using the DNAzyme library proposed by Elbaz et. al. is advantageous over the more simplistic methods developed by their forerunners because enzymes are unchanged after a reaction, allowing for continued use in future circuits [1, 2, 3, 5]. Based on this, a simple XOR gate was made using a system of DNAzyme subunits, a fluorescent substrate, and two input gates labeled I1 and I2. The inputs and substrate were read by the DNAzymes and if I1 or I2 were present, an active DNAzyme is formed and the substrate fluoresces, signaling “true” or “1”. If neither inputs are read, then nothing will happen and the substrate will not fluoresce. Likewise, if both inputs are read, then a non-fluorescent complex is

formed, triggering a “false” or “0”. Using similar methods, the authors were able to create other common logical expressions such as AND and OR gates.

3. Applications

Adleman concluded that it could be possible, in the future, for scientists to synthesize their own custom-made enzymes [1]. After doing so, Santoro and Joyce used their DNA enzyme in a test against HIV-1 at five different loci. Each one was cleaved as expected, but at varying efficiencies depending on the binding sites used [7]. Ezziane discussed the possibility of using DNA “smart chips” in attempts to diagnose illness based on the recognition of gene expression [8]. The effectiveness of these smart chips, however, is questionable as they are prone to noise from external factors. Ezziane asserts that there is an array of possibilities for practical applications of DNA computers that future experiments will provide.

3.1 Smart Drugs

Benenson et. al describe a device that operates in vitro as a diagnostic and in some cases treatment molecule for levels of mRNA expression to aid in the treatment of cancer [9]. The molecule operates on the scale of almost a trillion computers per microliter and works under the principles as Elbaz et. al’s logical XOR gate with an additional output module accompanying the input and computational modules [5, 9]. The computational module is a molecular automaton similar to a Turing Machine that exists in two states, either “yes” or “no”. As it reads a string of mRNA from left to right, it increases or decreases the probability that the indicator for transformation is present. As the molecule reads the strand it can transform from the “yes” state (its starting position) to “no,” or stay the same. If the indicator probability becomes large enough, then the molecule changes to the “no” position. Once in this position, the molecule cannot transition back to the “yes” state, and a positive diagnosis is said to have been made. Once a positive diagnosis is made, one of two automatons releases a single strand of DNA that works as a drug to either suppress the expression of the mRNA molecules, or encourage further expression.

3.2 DNA Storage

Recently, researchers Church, Gao, and Kosuri managed to store 700 terabytes of information on to a single gram of DNA [10]. This method was made possible by the increased ease with which DNA can be synthesized and sequenced using current

Advances in DNA Computing and Molecular Programming—M. Emmons

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technologies. Now, by representing each nucleic acid as a bit of information (A and C correlate to “0” and T and G correlate to “1”), information can be stored in units of DNA at one bit per cubic nanometer rather than the current resolution of one bit per 1012 cubic nanometers [1]. Further demonstrating the effectiveness of DNA for information storage, researchers at the European Bioinformatics Institute managed to store a variety of different files, including PDF, MP3, ASCII text, and more onto DNA using a base-3 method of encoding [11]. Each byte of information was transcribed to a five or six digit number in base-3 (0, 1, and 2), then assigned an appropriate base pair. The DNA was shipped from the US to Germany and properly decoded with 100% accuracy.

4. Conclusion

The idea of a non-electrical computer has been nearly forgotten in a time when almost any piece of information can be accessed at whim. But the principle which makes modern computers possible is being applied on a molecular level to create new solutions for computational problems, diagnosing and treating illnesses, and storing information. Since Adleman first solved the Hamiltonian Path Problem the methods for using DNA as a nanoscopic computing device have advanced first to the “discovery” of DNA enzymes that could be designer-made to cleave virtually any RNA molecule, and then to molecular automatons that could diagnose and treat diseases [3, 5, 9]. It is clear that DNA will play a significant role in future bioengineering and nanotechnology developments.

5. References

1. L. M. Adleman, “Molecular Computation of Solutions to Combinatorial Problems,” National Science Foundation, 1994

2. Lipton et. al “Using DNA to Solve NP-Complete Problems,” Science, 1995

3. D. Boneh, C. Dunworth, R. J. Lipton, J. Sgall, “On the Computational Power of DNA,” National Science Foundation, Princeton University, 1995

4. E. M. Southern “Detection of Specific Sequences Among DNA Fragments Seperated by Gel Electrophoresis,” Journal of Molecular Biology, vol. 98, pp. 503-508, 1975

5. J. Elbaz et. al. “DNA Computing Circuits Using Libraries of DNAzyme Subunits,” Nature Nanotechnology, vol. 5, pp. 417-422, 2010

6. R. R. Breaker, G. F. Joyce, “A DNA Enzyme that Cleaves RNA,” Chemistry & Biology, vol. 1, no. 4, pp. 223-229, 1994

7. S. W. Santoro, G. F. Joyce, “A General Purpose RNA-Cleaving DNA Enzyme,” The National Academy of Sciences of the USA, vol. 94, pp. 4262-4266, 1997

8. Z. Ezziane, “DNA Computing: Applications and Challenges,” Nanotechnology, vol. 17, no. 2, 2006

9. Y. Benenson et. al. “An Autonomous Molecular Computer for Logical Control of Gene Expression,” Nature 429, pp. 423-429, 2004

10. G. M. Church, Y. Gao, S. Kosuri, “Next-Generation Digital Information Storage in DNA,” Science, doi: 10.1126, pp.1-2, 2012

11. N. Goldman et. al. “Towards Practical, High-Capacity, Low-Maintenance Information Storage in Synthesized DNA,” Nature 494, pp. 77-80,

 

9

FULLERENES; POSSIBLE TO EVACUATE POST-ADMINISTRATION OR ARE THEY JUST TOXIC?

Grant Hartung grantrem@yahoo.com

Abstract The medical field is quickly expanding and with the discovery of nanoparticles (quantum dots, carbon nanotubes, fullerenes, gold and silver nanoparticles, etc.) there lies a vast field of unsolved medical problems. Fullerenes have capabilities to be free radical scavengers at a more potent rate than vitamin E, for example, by up to 4 fold. Fullerenes are also able to jump the blood-brain barrier, scavenge free radicals to stabilize them, return to the normal blood stream, and are excreted via the excretory system. Fullerenes have also been proven to be better or worse in their scavenging capabilities based two main things; the distance between the fullerene core from the functional polar heads (closer leading to more aggressive scavenging) and the polarity of the entire molecule. The focus of this review, though, is not nanoparticles, but rather to assess the biological reaction to the particles post-medication.

Keywords: Fullerene, Buckminsterfullerene, Bucky Ball, Nanoparticle, Biofiltration

1. Introduction

In the field of bioengineering, there exists a series of subcategories. Of these subcategories there exists the category of nano-biotechnology. Nano-biotechnology takes into account all kinds of fields including but not limited to molecular biology, biochemistry, quantum chemistry, quantum mechanics and even macro-biology. Together these fields produce many different types of products that are valuable and unique which may hold the answers to many of the most challenging biological problems of today's world.

Fullerenes are a type of carbon-based molecule. This is important to notice because all current carbon-based nanoparticles (except diamondoids) have a core that is chemically comprised the same as graphite. Graphene and fullerenes (including C60 and carbon nanotubes both single and multi-walled) are both comprised of a ring of carbon atoms bonded with alternating single-double bonds. These bonds form hexagonal carbon formations and pentagonal (5-sided) carbon formations to create a ball-shaped configuration in fullerenes.

Fullerenes have very special characteristics due to the fact that they are a hollow sphere. This allows them to encapsulate other molecules in order to transport them around the body. This makes them a good candidate for drug administration. It is also possible to break the double bonds in order to attach other molecules to the fullerenes. They are potentially very specializable, including making them polar (or water-

soluble) and making them tissue-specific by size and chemistry.

Another important characteristic of the C60 fullerene is that its diameter is 0.71 nm from nucleus-to-nucleus (entire molecule diameter, not distance between adjacent atoms). Having such a small diameter allows it to be highly mobile in the body and be able to pass through many different membranes (including the blood-brain barrier) in order to accomplish its function. Given that the average red blood cell is about 10 µm, this allows the fullerenes to be able to even jump the membrane into individual cells - something that can be extremely useful.

One of the main focuses of this paper is on the reaction of the body to "invaders" which can be anything the body isn't used to having inside of it which it then attempts to encapsulate and break down. This is a part of the immune system's response to things such as dirt particles, virus-infected cells, bacteria in the blood stream, or even C60 fullerenes if improperly prepared. In some cases, even raw metal shavings having gotten into the body for some reason and been attacked by the immune system. The first reaction of the immune system is the same no matter what type or nature the invader is. This is a response called the "innate" immune system response. This response is designed to kill all invading particles and if the invading particle is a mutated cell (changed in any way that is observable by the body), to kill that cell also. The innate immune response to infection or invasion is to rally neutrophils. Neutrophils are not

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the only part, but they are the main contributor to factors such as reactive oxygen species proliferation and cellular membrane degradation which leads to cell death and have been noticed frequently in nanoparticle research in-vivo (this is to say it was tested inside a living animal as opposed to in-vitro which is in a lab only).

The second type of immune response is called "adaptive" immune response. This is the response of such items as T and B cells which are designed to kill foreign viruses outside of cells and kill infected and mutated cells in a more specialized and unique way for each invader. Another type of adaptive immuno response, of importance in this paper, is that of macrophages. Macrophages are cells that encapsulate foreign bodies and attempt to degrade them over time through such processes as peroxidation, which will be described later on.

2. Advances in Nanotechnology

It has proven difficult to test both the in-vivo toxicity of nanoparticles such as fullerenes, quantum dots and carbon nanotubes due to the agglomeration of the nanoparticles into micrometer-sized clumps. This proves problematic as many in vitro studies maintain a salt-free environment whereas in vivo there are numerous natural salts present and capable of facilitating such agglomeration. It has been shown that the effects of these particles is very different between their nano-scale building blocks and their micrometer sized compliments [1, 2, 3, 6, 8], thus a study that tests the ability to keep the nanofibers in their respective nano-scale form such that they may accomplish their tasks would be useful.

An optimum method for deaggregation of microparticles into their nanoparticle composites was investigated in a study by Seong C. Kim et. al. [5]. A phosphate-buffered saline (PBS) solution, human serum albumin (HSA), and Tween 80 were all tested as pre and post-sonication mediums with sonication being the main deaggregation method. Sonication is the transfer of sound energy (through sonic pulses) into the material in order to deaggregate nanoparticles. The final result rendered an optimum dislocation patterning when PBS was mixed with HSA or Tween 80 in water and added to the particles post-sonication. The study was focused on TiO2 nanoparticles due to their elevated levels of aversion to dislocation methods but multiple nanoparticles were tested under the same conditions (both single-walled and multi-walled nanotubes, diesel nanoparticles, and even ZnO).

The zeta potential is the surface potential of any given particle in a given medium. Since the zeta potential holds the same sign convention (negative or positive) on any given particle, all particles have the same polarity and any 2 adjacent particles will repulse. So having a zeta potential closer to 0 will allow for easier agglomeration because the particles will not repulse each other but rather will easily clump together. It was noted that the particles had a significant zeta potential (>1 or <-1 mV) when dispersed in distilled water. It was also noticed that the zeta potential became less negative when prepared with the HSA, PBS or Tween 80 serums (which is favorable for dislocation). The experiment rendered two ranges of zeta potentials that would result in a stable dispersion (above 30 mV and below -30 mV per particle) when the pH and the concentration of electrolytes in the system are confined.

3. Advances in Fullerenes:

A study by Boushehri et al [4] explores the use of a C60 fullerene derivative as targeted drug delivery. This was a treatment of injection of the particles into the blood stream in a rat subjected to hypoxia-induced tissue damage (a heart attack during which affected tissues increase in acidity levels). They utilized an ionized isotope of magnesium which could be delivered to doxorubicin-induced damaged heart muscle. This type of damaged heart muscle simulates hypoxia-induced tissue damage. The particular fullerene chosen is the iron containing porphyrin monoadduct of the classical C60 molecule known as Porphyllerene-MC16 (PMC16). It was noted that the fullerene acted as a “smart particle” and released the Mg2+ in response to increasing acidity of its medium, a phenomenon experienced by hypoxia-related tissue damage [14]. When the PMC16 molecule reaches level of increased acidity it releases the Mg2+ molecule which creates an overproduction of ATP and reverses the affects of the hypoxia (lack of oxygen in the tissue) and rejuvenates the tissue back to proper health. There has been a direct link in this study between the PMC16-treated samples and a reduced hypoxia-induced detriment. An important note is that the nanoparticles only last in the heart for a few days and are filtered out of the blood stream in a matter of days as well, as proven by previous research [3, 4, 7, 9, 10]. This allows for fewer complications involved with post-treatment. During this experiment there was reported a “total lack” of absorbance of the particles into such “trapping” tissues as kidneys, liver, lungs, and

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skeletal muscles. It is also noteworthy that through mediating the dose to manipulate the engineered tissue-specific particles, there is no need to engineer an application that is target specific.

The overproduction of ATP through the Mg2+ – dependent metabolic process was, in fact, ignited in a tissue-specific manner. This provides significant evidence that particular derivatives (PMC16, C3, D3 etc) of the C60 molecule can be designed by engineers. It also means C60 particles can entrap molecules to be released under such conditions that we know to be biologically realistic. It is due to this that the derivatives of C60 can potentially be used as a medical treatment process. This is helped by the note that the particles were able to be excreted from the body in a normal biological filtration cycle as with food (only a few day process).

It is known that the double bonds between carbon atoms on fullerene cages (of which there are 30 for C60 fullerenes, 27 for C3 and D3 derivatives) can be broken and the energy absorbed by lipid radicals to stabilize them. The innate problem with natural C60 (a non-derivative C60 particle) is that the molecules agglomerate and block blood vessels. After they agglomerate, the particles get attacked by the body's immune system [15-18]. Polar C60 molecules (such as C3 and D3) are prevailing as they do possess strong antioxidant effects yet do not agglomerate in such a manner as natural fullerenes (due to their hydrophilic nature).

A study by Wang et al [10] takes a closer look into a different use of fullerenes; radical scavenging abilities and why different derivatives of C60 fullerenes are able to differ in their scavenging efficacies. This study introduced superoxide and hydroxyl radicals (naturally generated via xanthine-zanthine oxidase and Fenton reactions [19-21]) to the cell cultures to simulate in vivo lipid peroxidation and measured the control of the peroxidation when fullerene derivatives were added. The amount of lipid peroxidation was measured through membrane leakage of staining dies that were administered into the cells prior to the experiment.

This study by Wang attempts prove a link between the distance between the polar end and the reactive protons of the C3 and D3 fullerene derivatives and the resulting difference in radical scavenging efficacies. The results had shown that C3 derivative had a 5-fold increase in interaction strength between the molecule and the lipid bilayer when compared to D3. Further assessment indicated that the ability for C3 to penetrate into the lipid bilayer membrane was 14+/-4

Angstroms. Such a small distance allows ROS scavenging inside cells.

In a study by Dugan et. al. [9], Hydroxyl radicals were shown to be entirely eliminated by both C3 and D3 isomers with fullerene concentrations as low as 4 µM. That is almost two orders of magnitude lower than the scavenging capabilities of other free radical scavengers (such as vitamin E) [9]. With an increase of one order of magnitude in fullerene concentration, superoxide anions were entirely scavenged as well. After being exposed to N-Methyl-D-aspartic acid (NMDA, an amino acid derivative which mimics a neurotransmitter glutamate) or α-amino-3-hydroxy-5-methyl-4-isoxanozolepropionic acid (AMPA, a type of neurotransmitter glutamate), neuronal death occurs. In this experiment, a significant dose-dependent decrease in cortical neuron death was observed when being treated with fullerenes [10]. It was also noted that C3 was significantly more successful (more potent and more effective) than the D3 isomer, consistent with the previously mentioned study by Wang et al. The concentration and dose required to achieve full protection against free radical injury to cells varied depending on the nature and severity of the ROS proliferation. It was also noticed that the C3 isomer (D3 was not tested) was able to reduce neuronal death after deprivation of oxygen and glucose for 45-60 minutes which reflects an injury through NMDA receptor initiated cell damage and death. The results of this study conclusively that the C3 isomer of C60 is the more capable free radical scavenging [9,10].

A study by Quick et al. [7] measured reactions to C3 C60 and compared different mice over different age lengths, body weights, tested memory, and learning capabilities between young untreated mice, older treated mice, and older untreated mice. The important note about this study is that the C60 molecules were distributed to the subjects by dissolving the molecule into the everyday drinking water and despite their ability to jump the blood-brain barrier, were able to be passed from the body within a couple of days as routine ingestion of the body.

It was first tested whether or not the lifespan increased between the C60 induced mice and the control mice of both genders. There was no considerable difference between lifespan between genders, however there was an 11% increase in the mean longevity for the group treated with C60 as opposed to the control group. This was attributed to the reduction in oxidative stress based on previous research linking oxidative stress to natural aging processes [11]. Also based on previous research was

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the conclusion that ample oxidative stress was induced by the proliferation of ROS in the brain, which could be countered by the ROS scavenging affects of the C60 derivative used in this study [10].

The effects of spatial learning and memory were also tested through a series of previously described maze tests [12]. The tests in the set were between the young mice and the old mice from both treated and control groups. The first test was done on dry land while the second was done in water. It was noticed that the trial time for the initial battery was significantly shorter in the younger and the aged C60 treated mice than the aged control group of mice. It was also noted that the second battery of tests had a large decrease in early time trials only in the young mice. The tests were performed until the mice all had similar trial times and had shown that it took considerably more trails for the non-treated aged mice than any of the other sample sets to achieve the goal time in the routines. These results signify an increase in spatial learning and memory functions which are attributed, again, to the ROS scavenging aspects of the C60 particles.

This study is novel in that, aside from calorie restriction processes [13], it is the first successful approach to lengthening the lifespan of mammals. Previous research in the former category (calorie restriction methods) have shown an increase in lifespan in rats very similar to ours, giving rise to the notion that lifespan of humans can be well reflected by experiments performed on rats. There is also speculation as per the correlation between these two aging-related phenomenon. Previous research on calorie restriction returned that the younger the subject is when the trials begin, the greater the effect of the increased lifespan: something that might be able to be applied to the results of this study. In order to provide evidence that the longevity of life is related to oxidative stress, dihydroethidium oxidation was measured through confocal microscopy immediately after death post-injection [7]. The results had revealed that there were much higher levels of superoxide formation in the older mice when compared to the younger mice, proving that oxidative stress is relative to age in a variety of tissues, including the brain.

In order to assess the most effective tissue specificity, a study by Baker et al. [2] assesses 3 sets of rats (control, nano fullerenes, and micro fullerenes) which were injected with natural C60 fullerenes and then, after each of the 3 standard time intervals passed, one mouse from each group was taken, and analyzed via individual organs. When the fullerenes were first

injected, and before the rats were sacrificed, the only marked observed difference in the rats were nasal and retinal discharge which cleared up the next day. Histopathologic examination of over a dozen organs did not reveal any exposure-related microscopic lesions [2]. The main statistically significant changes that were observed were a decrease, although minimal, in the red blood cell count, monocyte count, eosinophil count, platelet count, albumin concentration, an increase in bile acids, and creatine kinase (CK) for the microparticle group. In nanoparticle, the blood glucose was greatly increased. There was no statistically significant change in cytology analysis or in the fluid cytokines of the bronchoalveolar lavage (BAL) yet the protein concentrations significantly increased in the nanoparticle group. It was noted however, that some of the BAL fluid macrophages collected from both nano and microparticle samples had shown traces of globular, brown intracytoplasmic pigment within them which are likely an artifact (an incongruity left by experimental imperfections) left by encapsulated fullerene molecules by white blood cells (intra histiocytic) [2]. In order to accurately create airborne fullerenes that could be inhaled in a measurable fashion, a process was done to procure an aerosol version of the fullerenes [22] and upon x-ray spectroscopy and HPLC, it was determined that the aerosol version of the fullerenes matched the solid state in every way. It was proven in this study that the exposure over 10 days to aerosol fullerenes did not lead to extensive toxicity in the rats[2]. The lung assessments revealed that the particles at different times in the lung decreased as a function of time (in terms of micrograms of fullerenes per lung sample). It was also reported that for the nanoparticle group, the adsorption onto the lung tissue was 47% higher than that of the microparticle group [2]. The deposition fraction, percent of total sample that was fixated onto the lung tissue alone, was 14.1% for nanoparticle and 9.3% for microparticle tissues (taken at 10 days) [2]. It was also reported that none of the particles in any group of rats showed traces of fullerenes in the red blood cell samples. The weights of the organs were effectively unchanged from the beginning to the end of the experiment in all groups and none of the tissues had shown any lesions or other signs of physical abuse. The differences experienced in the red blood cell and platelet count between the nanoparticle and microparticle tissues when compared to the control was very slight (3% or less [2]) and due to the short length of the experiment, long term in vivo toxicology cannot be completely assessed from this experiment alone. The changes in white blood cells,

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monocytes, and eosinophils were reduced in both fullerene groups by significant amounts as well when compared to the control. 4. Discussion

Lipid peroxidation is one of the most hazardous phenomena to happen to a cell. Oxidative stress, created naturally by ROS through neutrophils as part of the immune system, can create lipid peroxidation and can be generated from in-vivo implementation of any particle if incorrectly prepared.

Fullerenes are powerful tools and if they are prepared in their water-soluble form, they are not attacked by neutrophils even with a small diameter of less than 1 nm. They also have capabilities of becoming target-specific. Without being attacked by the immune system, they are also able to be passed through the body without detrimental effect (through the kidneys and liquid waste excretion). It has also been proven that the specificity of fullerenes can be tailored such that in extreme cases of medication delivery they are able to be initiated via environmental factors specific to the tissue of interest. This makes them otherwise non-toxic to the body in tissues not being medicated.

Because water soluble fullerene derivatives (C3 being the best described in this report) can be excellent radical scavengers throughout the body and even across the blood-brain barrier, fullerenes have proven themselves to be the most comprehensive and capable particle that can increase reasoning functions over time and lengthen lifespan in mammals. With the ease of administration and inability to elicit side-effects, this particle has extremely valuable possibilities.

The filtration from the body of any fullerene is best described by the surface chemistry and hydrodynamic diameter. The preferred size range is actually inside of 4nm and 200 nm to be passed without much trouble but at larger radii the particles begin to have the same problems as that of the micron sized particles (attack from the neutrophils eliciting ROS followed by full encapsulation by the macrophages).

The second important attribute to assess is the size of the material being questioned. If the material happens to be a quantum dot, made up of a core and an exterior, the entire diameter must be smaller than 500 nm and larger than 4 nm in order to be passed through the body via normal functions. If the polar end is too far from the core, the fullerene core cannot perform any functions. On the same note, if the polar

end (a required part of a successful nanoparticle) is in charge of accomplishing the medication parameters, it can be too long itself and change the shape of the particle. In this situation, the particle will no longer be a sphere and the problems associated with carbon nanotubes (becoming lodged in the lipid bilayer of cells or being agglomerated within the cytoplasm of cells) will begin to take place.

5. References 1. S. Fiorito, A. Serafino, F. Andreola, P. Bernier,

“Effects of fullerenes and single-walled carbon nanotubes on murine and human macrophages” Carbon 44 (2006), 1100-1105

2. Gregory L. Baker, Amit Gupta, Mark L. Clark, Blandina R. Valenzuela, Lauren M. Staska, Sam J. Harbo, Judy T. Pierce, and Jeffery A. Dill “Inhalation Toxicity and Lung Toxicokinetics of C60 Fullerene Nanoparticles and Microparticles” Toxicological Sciences 101(1) (2008), 122-131

3. Gunnar Damgard Nielsen, Martin Roursgaard, Keld Alstrup Jensen, Steen Seier Poulsen and Soren Thor Larsen, “In Vivo Biology and Toxicology of Fullerenes and Their Derivatives” Basic & Clinical Pharmacology & Toxicology 103 (2008), 197-208

4. Seyed Vahid Shetab Boushehri, Seyed Nasser Ostad, Saeed Sarkar, Dmitry A. Kuznetsov, Anatoly L. Buchachenko, Marina A. Orlova, Bagher Minaii, Abbas Kebriaeezadeh, Seyed Mahdi Rezayat, “The C60-Fullerene Porphyrin Adducts for Prevention of the Doxorubicin-Induced Acute Cardiotoxicity in Rat Myocardial Cells” Acta Medica Iranica 48(5) (2010).

5. Seong C. Kim, Da-Ren Chen, Chaolong Qi, Robert M. Gelein, Jacob N. Finkelstein, Alison Elder, Karen Bentley, Gunter Oberdorster, David Y H Pui, “A nanoparticle dispersion method for in vitro and in vivo nanotoxicity study” Nanotoxicology 4(1) (2010), 42-51.

6. Yuanyuan Su, Fei Peng, Ziyun Jiang, Yiling Zhong, Yimei Lu, Xiangxu Jiang, Qing Huang, Chunhai Fan, Shuit-Tong Lee, Yao He, “In vivo distribution, pharmacokinetics, and toxicity of aqueous synthesized cadmium-containing quantum dots” Biomaterials 32 (2011), 5855-5862.

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7. Kevin L. Quick et al. "A carboxyfullerene SOD mimetic improves cognition and extends the lifespan of mice" Neurobiology of aging 29 (2008), 117-128

8. Hak Soo Choi, Binil Itty Ipe, Preeti Misra, Jeong Heon Lee, Moungi G. Bawendi, and John V. Frangioni "Tissue- and Organ-Selective Biodistribution of NIR Fluorescent Quantum Dots" Nano Lett. 9(6) 2009

9. Laura L. Dugan, Dorothy M. turetsky, Cheng Du, Doug Lobner, Mark Wheeler, C. Robert Almli, Clifton K.-F. Shen, Tien-Yau Luh, Dennis W. Choi, and Tien-Sung Lin "Carboxyfullerenes as neuroprotective agents" Neurobiology 94 (1997), 9434-9439

10. I Chen Wang, Lin Ai Tai, Don Dar Lee, P. P. Kanakamma, Clifton K.-F. Shen, Tien-Yau Luh, Chien Hong Cheng, Kuo Chu Hwang “C60 and Water-Soluble Fullerene Derivatives as Antioxidants Against Radical-Initiated Lipid Peroxidation” Journal of Medicinal Chemistry 42 (22) (1999), 4614-4620

11. Schriner SE, Linford NJ, Martin GM, Treuting P, Ogburn CE, Emond M, Pinar CE, Warren L, Wolf N, Remmen HV, Wallace DC, Rabinovitch PS, "Extension of murine life span by overexpression of catalase targeted to mitochondria". Science 308(5730) 2005,1909–11

12. Hartman RE, Izumi Y, Bales KR, Paul SM, Wozniak DF, Holtzman DM. "Treatment with an amyloid-beta antibody ameliorates plaque load, learning deficits, and hippocampal long-term potentiation in a mouse model of Alzheimer’s disease". J Neuroscience 25(26) (2005), 6213–20

13. Bellush LL, Wright AM, Walker JP, Kopchick J, Colvin RA." Caloric restriction and spatial learning in old mice". Physiological Behavior 60(2) (1996); 541–7

14. Wallace KB, Starkov AA. “Mitochondrial targets of drug toxicity.” Annu Rev Pharmacol Toxicol 40 (2000), 353-88

15. Ruoff RS, Tse DS, Malhotra R, et al. "Solubility of C60 in a variety of solvents." J Phys Chem, 97 (1993),3379-83

16. Oberdӧster E, "Manufactured nanomaterials (Fullerenes, C60) induce oxidative stress in the brain of juvenile largemouth bass."

Environmental Health Perspect, 112 (2004),1059-62

17. Sayes CM, Gobin AM, Ausman KD, et al. "Nano-C60 cytotoxicity is due to lipid peroxidation." Biomaterials 26 (2005),7587-95

18. Isakovic A, Markovic Z, Nikolic N, et al. "Inactivation of nanocrystalline C60 cytotoxicity by γ-irradiation." Biomaterials 27 (2006), 5049-5058

19. Tien, M.; Svingen, B. A.; Aust, S. D. "An Investigation into the Role of Hydroxyl Radical in Xanthine Oxidase-Dependent Lipid Peroxidation." Arch. Biochem. Biophys. 216 (1982), 142-51

20. Fukuzawa, K.; Tadakero, T.; Kishikawa, K.; Mukai, K.; Gebicki, J. M. "Site Specific Induction of Lipid Peroxidation by Iron in Charged Micelles." Arch. Biochem. Biophys. 260 (1988), 146-52

21. Gutteridge, J. M. C. "The Role of Superoxide and Hydroxyl Radicals in Phospholipid Peroxidation Catalyzed by Iron Salts." FEBS Lett. 150 (1982), 454-58

22. Gupta A, Forsythe WC, Clark ML, Dill JA, Baker GL. "Generation of C60 nanoparticle aerosols in high mass concentrations." Journal Aerosol Sci. 38 (2007), 582-603

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REVIEW OF THE APPLICATIONS OF ULTRASONIC WAVES Dan O’Neill

doneil5@uic.edu

Abstract Defects inside the body are a growing issue in the medical world that continues to ravage and potentially kill patients every year. There is a shortage of effective treatments to remove tumors, kidney stones, or cancerous tissue from within the body. Almost all effective methods of tumor or cancer removal require invasive surgery or chemotherapy, which leaves the body scarred or damaged. These treatments also can be ineffective, causing the patient to die from the tumor. Surgery can also be a high risk to the patient for tumor, kidney stone, and cancer tissue removal. To address this issue, researchers in California are using ultrasonic waves focused at one point to destroy defects within the body, without any scarring to the body. This new method, often referred to as the “sound bullet,” enables doctors to easily rid a patient of any tumors, kidney stones, or cancerous tissue without major surgery, effectively reducing recovery time and leaving the body unharmed. Keywords: Sound Bullet, Chemotherapy, Malignant, Benign, Nonlinear Acoustic Lens, Ultrasonic Imaging

1. Introduction

There are many classifications of tumor cells. Two of the most common classifications are malignant and benign tumors. Benign tumors are not cancerous and do not metastasize [6]; however, they can still be dangerous. Metastasis is the ability of tumor cells to migrate inside the body or infect other areas of the body [11]. Benign tumors can grow inside the body, pressing on vital organs and disturbing natural bodily functions. Malignant tumors are tumors composed of cancerous cells that can migrate inside the body, infecting other tissue [6,11]. These tumors are removed either with chemotherapy or invasive surgery. Cancerous tissues typically follow the same guidelines as tumors. Cancerous tissue can migrate inside the body through metastasis [11]. There can be many risks to invasive surgery such as infections or common diseases. Radiation can be used in chemotherapy, where a patient is exposed to radiation to destroy tumor or cancer cells. The radiation passes through the body, destroying the tumors, but also destroys healthy cells. These treatments have no guarantee of success because tumors can reappear and cancer can still survive treatments and migrate inside the body [6]. Kidney stones are calcium deposits in which there is no main procedure for removal. Similar to tumors, they can also reappear after they have been removed [4]. This means multiple treatments can be involved on identical kidney stones. The most common way to remove a kidney stone is to break up the stone and expel it naturally. There

are other less common ways to remove a kidney stone but, for cost reasons and risk of infection, they are used less frequently. When the kidney stone is expelled naturally the body pushes it out naturally over time, causing extreme discomfort to the patient [4]. The discomfort caused by a kidney stone can last for two weeks and discourage an individual from urination because of the pain. There are few processes that ease the removal process and the pain involved is almost unavoidable if not treated with pain killers.

Overall, these processes can be expensive, time-consuming, and uncomfortable for the patient. The sound bullet is being developed to utilize ultrasonic waves as an alternative cost-effective treatment of tumors. The sound bullet can destroy tumors deep inside the body without any radiation exposure or invasive surgery. Cancer can be located and destroyed in multiple parts of the body with surgical precision without any actual surgery. With the development of this new procedure, recovery time is minimized, infection or radiation poisoning is avoided, and cost is reduced significantly because the need for nurses, doctors, and surgical personnel is reduced.

This article will reveal the potential impact that the sound bullet may have on the medical field. The sound bullet will revolutionize removal surgery and its procedures, by creating a more cost effective, safe, and quicker procedure.

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2. Traditional Removal Methods

2.1 Invasive Surgery Invasive surgery is used for many reasons including muscle repair, tissue removal, and bone replacement or repairs. Invasive surgery is a last resort option because it leaves the patient open for hours at a time, leaving the body subjected to physical scarring and the risk of infection. Infections can happen in the wound itself or other areas of the body such as the lungs or bladder [3].

Modern technology has reduced the physical impact of invasive surgery on the body. More advanced instruments have allowed for smaller incisions into the body, leaving smaller or unnoticeable scarring. However, this is not always the case for tumor removal. Sometimes a significant cut must be made into the body for retrieval of a tumor. Areas in the body that require invasive surgery could lead to problems such as blood loss, blood clots, and breathing issues during surgery [3].

However, invasive surgery cannot be used to remove all tumors. Because a tumor can grow anywhere inside the human body, the tumor can grow inside the brain or lungs where traditional invasive surgery cannot be performed without serious risk to the patient. Invasive surgery can be a life-changing event for tumor patients, but it can also leave a deformed scar and keep the patient in the hospital for weeks at a time.

2.2 Radiation Treatment

Radiation treatment is another alternative for tumor removal. Radioactive electromagnetic energy is controlled by an electromagnetic energy field that passes photons through the body. These photons are transmitted at the speed of light. This gives radiation its ability to burrow deep inside the body through the spaces in the aligned molecules, enabling access tumor or cancerous cells inside the body where invasive surgery would be too much of a risk to the patient’s health [8]. However, radiation treatment is typically avoided when possible, due to its unwanted side effects. During the process of treatment, the patient becomes very ill and weak and starts to lose hair. This is because the radiation not only kills the tumor or cancer cells inside the body, it also kills the healthy cells used for daily bodily functions. The overall experience of the patient is painful. Radiation that is applied to the prostate or the brain can cause disabilities. Brain damage and rectal complications are highly likely as well. The treatment can leave the patient paralyzed in some extreme cases [8].

Radiation treatment is a highly effective option for tumor removal, but can leave the patient in pain and with possible permanent damage to their body. 2.3 Kidney Stone Removal

Kidney stones are calcium deposits in the kidney. Kidney stones can be formed from a lack of urine, citrate, magnesium, and zinc in the bladder and an increase in calcium, uric acid, and phosphate [4]. With the most common form of treatment, kidney stone removal is almost all-natural and very uncomfortable. If the stone is too massive for the urinary tract, the patient may be given a solution to help break down the stone to a lesser size to be passed with more comfort [4]. However, the pain, in most cases, is unavoidable for the patient. The patient is encouraged to drink gallons of water so that they may simply urinate until the stone is passed through their urinary tract. The passing of the stone through the urinary tract can cause harmful side effects such as exhausted pain, blood, urinary tract infection, or renal insufficiency, also known as kidney failure [4]. Among these side effects, the kidney stone can be too massive to pass through the urinary tract of after being treated with the solvent. In this case, invasive surgery is the only other way to remove the stone safely. Kidney stones can occur in anyone, regardless of sex or age. About 13% of men and 7% of women will suffer from kidney stones with a 50% chance of a reoccurring stone [4]. Thus it is likely that the patient will experience discomfort or pain again and can severely damage the quality of life of the patient.

3. Theory

3.1 Ultrasonic Waves

Ultrasonic waves are sound vibrations that pass through liquids, gases, and solids at a frequency that is above the threshold of human hearing. These sound waves have frequencies ranging from 20 kHz up to a 1MHz [3]. Animals have been using ultrasonic waves to navigate since their creation. Bats use high-pitched sound waves that produce vibrations in the air, a technique called echolocation. Fish also use ultrasonic waves to create vibrations in water called sonar. Sonar vibrates water molecules, sending out a wave that bounces off objects. Both bats and fish utilize these waves as they pass through less dense media such as water or air to recognize more dense objects such as, e.g. cave walls or even other animals. After the waves hit a denser object, the wave then returns and is interpreted by the animal into an image.

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Ultrasonic waves can be used to also pass through solid media. The stiffer is the medium through which the wave propagates, the less the wave is attenuated. At the boundary between two media of different stiffness, some of the ultrasonic energy is transmitted through while some is reflected back to the wave source. This form of ultrasonic waves is used in ultrasound transducers, the most well-known medical device that utilizes ultrasonic waves. Among other things, ultrasonic waves penetrate the stomach of a pregnant woman to create cross sectional views of a fetus. A diagram of waves is displayed in Figure 1.

Figure 1. The electromagnetic spectrum of inferred waves, ultraviolent waves, radioactive waves, etc. [1].

The waves pass through the less dense medium of the stomach and bounce off the more dense medium of the fetus inside pregnant women. The waves are then interpreted into images, allowing mothers to see their children before they are born.

3.3 The Sound Bullet

Researchers at the California Institute of Technology are doing significant work in the field of biomedical engineering developing the sound bullet. The sound bullet was inspired through Newton’s cradle, a popular desk prop displayed in Figure 2. It was discovered that the steel spheres have the ability to create high frequency waves that can be used to pass through solid objects. The sound bullet features 21 aligned parallel spheres in 21 rows and columns. The spheres are impacted at one end as the wave transfers through the steel spheres [9]. The waves generated by the strike travel down the line of steel spheres and into the nonlinear acoustic lens, generating a single nonlinear acoustic wave. The wave is nonlinear because it leaves the sphere in all directions opposite the strike. Controlling each strike allows the waves to have high amplitude that gives the wave its destructive force.

Figure 2. Newton’s Cradle, desk prop [2].

Figure 3 conceptually explains the production of the ultrasonic wave and how it is focused. The conceptual diagram demonstrates where the focused waves can be consolidated to produce heat [5]. The speed of the waves is controlled by the repetition of strikes at the far end of the machine as the waves are produced at the close end. The wave passes through each ball in the row and then through a nonlinear acoustic lens which applies the wave at a focal point. Although the steel balls create a powerful nonlinear acoustic wave, the wave is unusable unless focused by the nonlinear acoustic lens as shown in Figure3.

 

Figure 3. A conceptual drawing of the sound bullet shows the waves as they enter and leave at the focus point. The white represents the harmless waves, while the gold represents the area in which the waves can be utilized to create heat [9].

The nonlinear acoustic lens can be compared to a magnifying glass that can take light rays and focus them on one point. The focused light rays degrade the substance from the heat that is created, sometimes resulting in the burning of the substance. The sound bullet is similar to that idea because it uses an acoustic lens to focus single high-powered waves to destroy the substance. With repetition, the waves can create heat at the focal point without heating other areas [9]. The ultrasonic waves can be used destroy

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other objects that either lie inside the object or just beyond it [9].

4. Applications of Ultrasonic Waves as an Alternative Treatment

4.1 Ultrasonic Imaging

Traditional methods of visualizing the inside of the body include using either magnetic resonance imaging (MRI) or X rays. Both an MRI and X rays pass radiation through the body and then onto detectors. MRI is the improved method of radiation X rays, utilizing less radiation so is non-ionizing [8]. An MRI uses magnetic energy and radio waves to create images of the interior of the body [7]. Non-ionizing radiation is radiation that does not create enough charge to do harm to surrounding tissue [8]. MRI applies magnetic fields through the human body to obtain cross-sectional views of the body’s interior. The benefit of MRI is that it can penetrate inside the body more easily. MRI is currently the leading method used to view the interior of the body with the cross sections images used for medical diagnoses.

Ultrasonic imaging is already used to view the interior of the body. Modern ultrasounds use piezoelectric crystals that, when charged, create high-frequency waves of around 10 MHz. The ultrasonic waves created from the sound bullet form a single wave with large amplitude that is pulsed similarly to that of an ultrasound [10]. The frequency is controlled based on how many times the row is struck, as opposed to the traditional charged particle wave used in other methods, such as the MRI. This allows the wave to be precisely controlled. This imaging modality can, then, exceed the imaging abilities of a traditional ultrasound or an MRI, and with the same non-ionizing high powered waves as an MRI.

4.2 Tumor Removal

The sound bullet can provide a safe and more accurate alternative to invasive surgery or radiation treatment for removing tumors. The sound bullet utilizes ultrasonic waves to reach areas where the traditional methods could not reach without harming the patient as shown in Figure 4. Because the body is never cut open, there is no risk of infection, morbidity, or mortality commonly associated with invasive surgery. The physical damage and negative side effects inflicted through radiation treatment can also be avoided. Ultrasonic waves can be passed through the body instead of charged particles, destroying only the targeted tissue and leaving the

surrounding tissue unharmed. The potential surgical accuracy used by the sound bullet can act as an alternative to subjecting the body to harmful radiation.

 

Figure 4. A diagram of the sound bullet being utilized to target a defect inside a human head. The spheres represent the machine while the red inside the brain represents the heat accumulating on the affected area by the ultrasonic waves. The yellow areas in the head are waves passing safely through the brain [5]

The average recovery time for a patient in invasive surgery is 1 to 4 weeks [3]. The average radiation treatment is about 6 to 8 weeks, varying depending on the severity of the tumor [8]. They both require a significant amount of time, which is costly to the patient. The sound bullet is a potential solution to this problem. The ultrasonic waves can be applied to find and destroy tumors in one session, compared to previous tumor removal methods where tumor recognition and removal are two separate and independent processes. Unlike radiation treatment, the ultrasonic waves can pass through the body without harm to the surrounding tissue, allowing for tumor removal to only take one session, thus saving the patient time and money.

4.3 Kidney Stone Removal

The same ultrasonic waves that can be used to pass through the body to destroy tumors can be applied to kidney stones. Ultrasonic waves will be passed harmlessly into the kidney, to be focused on the calcium deposits in a method similar to that of an ultrasound. If the calcium deposit is broken into smaller pieces, it may be able to pass harmlessly out of the body. Because of the potential abilities of this procedure, the risks of urinary tract infection and the risk of infection can be avoided. With the calcium deposit destroyed inside the body, a tremendous amount of pain can also be avoided because the

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calcium deposit will pass through the urinary tract as dust.

4.4 Cancer

Because cancer can metastasize inside the body, like malignant tumors, it is not easily removed [11]. Cancer is typically diagnosed and destroyed as two separate procedures similar to tumors and can sometimes take multiple sessions to find to be destroyed. If radiation treatment is used, it often takes months for the cancer to be destroyed and in some cases it never is. The sound bullet can be applied as a potential solution. Because the sound bullet can utilize ultrasonic waves to create cross sectional views of the interior of the body and destroy defects, cancer can be destroyed in one treatment. This means that even if the cancer metastasizes, the sound bullet can potentially locate cancer multiple times and thus destroy each lesion in one session. Because radiation treatment is avoided, the recovery time is nearly nonexistent and the procedure time is minimized.

5. Conclusion

The sound bullet has many applications through the already vast functions of ultrasonic waves. They not only help provide solutions to modern medical procedure problems, but also provide a solution on an individual patient level. Ultrasonic waves, focused through a nonlinear lens, are given a new role in aiding the medical field in treating patients. Whether the sound bullet is being applied to a tumor or kidney stone, the patient’s overall experience can potentially be improved. Cancer patients can rid themselves of a cancerous lesion without exposing their bodies to the effects of radiation treatment. With the patient’s experience potentially improved, an alternatively economical solution can be applied to modern costly and risky procedures. The sound bullet provides fewer doctor visits, fewer treatments, no risk of infections, reduced pain, and shorter recovery time. Since patient time is reduced, hospitals can become more efficient, saving them time and money. With the potential vast applications of the ultrasonic waves, the sound bullet can be used to revolutionize the health care industry for both patient and hospital.

6. References:

1. Krakowski, A.C., L.A., Kplan, Auerbach:

Wilderness Medicine; Elsevier; 2011, 294-312 pp.

2. Clay Dillow. Sound bullets Inspired By Newton’s Cradle Could Sink Subs, Pummel Underground Bunkers, Destroy Tumors. Popsci. http://www.popsci.com/science/article/2010-04/sound-bullets-could-sink-subs-pummel-underground-bunkers-destroy-tumors

3. Lee, D.H., S.Y. Kim, S.Y. Nam, S.H. Choi, J.W. Choi, J.L. Roh; Risk factors of surgical site infection in patients undergoing major ongological surgery for head and neck cancer; Oral Oncology, Vol. 47, 528-531 pp., 2011

4. Schade, G.R., G. Faerber; Primary Care in Office Practice; Elsevier; Vol. 37, 2010

5. Niederer, P.F., Basic Engineering for Medics and Biologists; Amsterdam; IOS Press BV; 2010, 320 pp.

6. Abeloff, M.D., J.O. Armitage, J.E. Niederhuber, M.B. Kastan, W.G. McKenna; Clinical Oncology; 2013, 233-239, 471-488 pp.

7. Rajiniti Prasad, R., N. Verma, , A. Sirvastava, B. K. Das, O. P. Mishra; Magnetic resonance imaging, risk factors and co-morbidities in children with cerebral palsy; Journal of Neurology; 2011, 471-478

8. Abeloff, M.D., K.A. Vallis, J.E. W.G. McKenna; Clinical Oncology; 2008, 471-488 pp.

9. Spadoni, A., C. Daraio; Generation and control of sound bullets with a nonlinear acoustic lens; Procedures of the National Academy of Science; 2010. 1-5 pp.

10. Jiang, X., K. Snook, T. Walker, A. Portune, R, Haber, X. Geng, J. Welter, W.S. Hackenberger; Single Crystal Piezoelectric Composite Transducer for Ultrsound NDE Applications; SPIE; 2008, 1-9 pp.

11. Geiger, T.R., D.S. Peeper; Metastasis Mechanisms; Elsevier; 2009, 294-303 pp.

 

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REVIEW OF METHODS OF DRUG DELIVERY TO THE POSTERIOR SEGMENT OF THE EYE

Hari Sreedhar hsreedhar7@gmail.com

Abstract Drug delivery to the posterior segment of the eye is important for the treatment of many degenerative ocular diseases. However, conventional methods of drug delivery, such as intravitreal injection, periocular injection, and systemic administration, are hampered by their low efficiency in targeting the back of the eye, their invasiveness, the adverse side effects accompanying them, and/or the discomfort they cause to the patient. Hollow and solid microneedles offer a new option in this field, and promise to provide a safe and effective alternative for drug delivery to the posterior segment of the eye, especially through the suprachoroidal route. Another attractive option is the use of trans-scleral iontophoresis, a non-invasive process in which an electric current is used to force ionized drugs across the sclera and into the eye. Keywords: Microneedle, Posterior Segment of Eye, Intravitreal Injection, Periocular Injection, Suprachoroidal space, Iontophoresis, Trans-scleral Iontophoresis

1. Introduction Many debilitating diseases are associated with the posterior segment of the eye. Conditions such as age-related macular degeneration, for example, frequently lead to visual impairment [2]. Even as new drugs are formulated for treatment of these illnesses, a continuing problem remains in the issue of drug delivery to the posterior segment of the eye. In order for treatment to work as effectively as possible, the drug must be delivered as safely and as efficiently as possible to the back of the eye. A major obstacle in drug administration to the posterior segment of the eye is that many methods of ocular drug delivery are far less effective at delivering drugs to the posterior segment, as compared to the anterior segment [2]. The effectiveness of conventional methods of drug delivery to the back of the eye is limited by such factors as inefficiency of drug delivery, invasiveness, adverse side effects, and patient discomfort [2]. The two newer approaches investigated here offer non-invasive, effective methods of targeted drug delivery to the posterior segment of the eye: microneedles and iontophoresis. Microneedles, which are needles of micrometer sizes, were originally used to deliver drugs to the skin [5]. However, they are now being explored as a safe, efficient way to administer drugs to the posterior segment of the eye. Iontophoresis is a method of moving drugs that are ions through a membrane using an electric current [2]. This method is non-invasive, and also offers an effective method of drug delivery to the posterior segment of the eye.

2. Conventional Methods of Drug Delivery to the Posterior Segment of the Eye Drug delivery to the posterior segment of the eye is inherently more challenging than drug delivery to the anterior segment. Many options for ocular drug delivery have limited effectiveness at targeting the back of the eye, or carry a risk for unwanted complications or discomfort. 2.1 Topical Administration Topical administration of a drug using eye drops is generally effective for administering drugs to the anterior segment of the eye. This process is minimally invasive; patients can conduct the procedure on their own. However, the distance that the drug must diffuse across the vitreous humor in order to reach the back of the eye is large. Therefore, this method is not suited to drug delivery to the posterior segment of the eye because only a very low concentration of the dose reaches the back of the eye [2]. 2.2 Systemic Administration Systemic administration of a drug to the ocular tissues involves getting the drug into the bloodstream via injection or oral administration [2]. In this way, it can dissolve from the systemic circulation into the eye, including the tissues of the posterior segment

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[2]. This method of drug administration is especially well suited to the choroid (the vascular layer of the eye, between the sclera and retina) because of the large number of blood vessels in these tissues [2]. A disadvantage of this method lies in the fact that it carries the drug not only to the tissues of interest but also to other tissues all throughout the body. Since only a small amount of blood flows to the posterior segment of the eye, drug delivery via systemic administration requires high drug concentrations in the blood. This increases the risk of adverse side effects. [2] 2.3 Intravitreal Injection The posterior segment of the eye can be more specifically targeted by directly placing a drug into the vitreous humor (the transparent gel filling the center of the eye) and letting it dissolve toward the retina and other posterior sections of the eye [7]. The drug is injected directly into the eye with a needle (See Figure 1), where is can dissolve through the vitreous humor [2, 7].

Figure 1. Illustrates the intravitreal injection of a drug into the eye. The needle is actually used to penetrate into the vitreous humor, so the released substance comes closer to the back of the eye. The drug can then diffuse through the vitreous humor and reach tissues of the posterior segment of the eye. [6]  The problem with this direct approach is that it is highly invasive, and can have complications. Intravitreal injections can result in drug delivery to undesired tissues, and the procedure carries a risk of endopthalmitis (inflammation of interior ocular tissues) [7]. 2.4 Implants Another option for targeted drug delivery to the back of the eye is through the use of an extended-release

implant placed in the vitreous humor [8]. The released drug can then diffuse to tissues of the posterior segment of the eye. After treatment, biodegradable implants do not need to be removed from the eye [2]. While this approach allows for drug delivery over an extended period of time, putting it into the eye requires invasive surgery with risks of complication [7]. In addition, there may be toxic reactions to implants that are kept in the eye for long periods of time [1]. 2.5 Periocular Injection  Another approach to targeting the posterior segment of the eye is through periocular injections, which involve depositing the drug just at the outside surface of the eye [7]. This method is less invasive and carries less risk of complication than intravitreal injection [2].

Figure 2. Illustrates a sub-Tenon’s injection, one kind of periocular injection. As shown, the drug is not delivered directly into the vitreous humor, but deposited near the eye’s outside surface, from where is can diffuse into the eye. [4] However, the less invasive nature of this method comes along with a reduced effectiveness. Some amount of the drug is lost because of movement during the injection, and the targeting effectiveness with this procedure is limited [4, 7]. 3. Microneedles An attractive alternative to the above mentioned methods is the use of microneedles, which are micrometer-sized needles, employed either alone or arranged in arrays [5]. Made of materials such as silicon, glass, and stainless steel, these needles typically have lengths around 500 µm - 750 µm in order to pierce through the sclera, whose mean thickness is 600 µm [1, 5, 8].

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Figure 3. Brightfield microscopy of a stainless steel microneedle shown next to a penny for comparison [5]. 

 

There are two types of microneedles that can be used to deliver a dose of a drug: solid (coated with the drug) or hollow. 3.1 Coated Microneedles Solid microneedles or arrays of solid microneedles can be used to deliver drugs to the eye. These are first coated with a drug, and then used to penetrate the sclera. The drug can then quickly dissolve off the microneedle(s) and into the eye [1, 5].

Figure 3. Microneedles coated with (A) sodium fluorescein, (B) fluorescein-labeled bovine albumin, and (C)

fluorescein-labeled plasmid DNA. Also shown is (D) a 50-microneedle array coated with sodium fluorescein [5].

3.2 Hollow Microneedles Hollow microneedles are another option for ocular drug delivery [7, 8]. They have been used before in other settings, such as for the administration of insulin and the influenza vaccine to humans [7]. Factors such as needle length and applied pressure affect the efficiency of ocular drug delivery with hollow microneedles [7].

Figure 4. A hollow microneedle, far smaller than any conventional hypodermic needles. Scale bar is 5 mm [8].

3.3 Drug Delivery Routes to the Posterior Segment of the Eye 3.3.1 Suprachoroidal Route for Drug Delivery The suprachoroidal space is a potential space, or a space which exists only when fluid builds up or is injected. It exists between the sclera and the choroid, going all the way around the eye [7]. This space has recently been investigated as a route for the delivery of drugs to the back of the eye. Drugs injected into the suprachoroidal space near the corneal limbus (at the border of the cornea and sclera) can flow circumferentially around the eye through this space [7].

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Figure 5. Brightfield microscopy of frozen eye cross- sections. In (a), the suprachoroidal space is merely a potential space between the sclera and the choroid, as there is nothing in it. In (b), the space is filled with pink sulforhodamide B that has been injected into it. Scale bar is 500 µm [7].

Experimentation with a hollow microneedle in rabbit eyes and human cadaveric eyes have demonstrated that this method can be used to efficiently target the posterior segment of the eye without adverse side effects [7,8]. Substances injected into the suprachoroidal space have a concentration at least 10 times higher in the back of the eye than in the front, as determined with the injection of fluorescently tagged compounds and an ocular fluorophotometer [8]. 3.3.2 Intravitreal Route with Microneedles Microneedles can also be used to deliver drugs by letting them dissolve through the vitreous humor to the posterior segment of the eye. However, they should be of a length adequate to penetrate the sclera, whose thickness in humans ranges from 400 µm – 900 µm, depending on position [1].

Figure 6. Drug Delivery Route to the Posterior Segment of the Eye [1]. Injection near the equator of the eye is ideal because the sclera is thinnest near this point, and it has been demonstrated that scleral thickness is inversely proportional to permeability [1]. A microneedle length of over 400 µm is necessary to penetrate both the bulbar conjunctiva (which lines the eye over the eye over the sclera) and the sclera at least near the equator of the eye [1]. Having penetrated the bulbar conjunctiva and the sclera, the microneedle could deposit drug in the anterior uvea (the uvea is the middle tissue coat that surrounds the eye), from which the dose could dissolve through the vitreous humor to the back of the eye [1].

4. Iontophoresis Another minimally invasive approach that promises to be effective in treating diseases of the posterior segment of the eye is iontophoresis. Iontophoresis is a process that drives drugs across a membrane using a low electric current [2]. The membrane of interest in this case is the sclera. Since it is more permeable to drugs of higher molecular weight than the cornea, and has a greater surface area, the sclera is a more appropriate route for the purposes of drug delivery to the posterior segment of the eye [1, 2]. 4.1 Device Construction The basic components of an iontophoretic device are a DC power source, an electrode attached to a component in contact with the eye, and an electrode connected to ground [3]. There are two major classes of devices for ocular iontophoresis, distinguished by the means in which contact is made with the eye. Some devices have an eye cup placed into contact with the eye that carries the drug in solution in a small reservoir (See Figure 7). The electrode with the same charge as the drug is inserted into the reservoir so as to be immersed in the solution. [3]

Figure 7. Iontophoretic device with an eye cup. The eye cup has a reservoir in which the drug is contained. An electrode is immersed in the reservoir. [3]

The second type of Iontophoretic device does not use an eye cup to contact the eye. The electrode runs instead to a hydrogel matrix. This gel is saturated with drug solution, and used as the probe to touch the eye [3].

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Figure 8. Device for ocular iontophoresis with a drug-saturated hydrogel matrix [3]. 4.2 Transport Mechanism Understanding iontophoresis begins with the fundamental fact that ions of the same charge repel each other [2]. An electrode of the same charge as the drug, in the part of the device put near the eye, is used to push the drug through using electrorepulsion [3]. However, even neutral substances have improved delivery across a membrane with the help of an electric current [3]. In addition, the application of an electric current also increases tissue permeability [3]. 4.3 Effectiveness in Targeting the Posterior Segment of the Eye Trans-corneal and corneo-scleral iontophoresis (the names refer to the membranes through which the drug is forced) are not very effective at delivering drugs to the back of the eye, because of difficulties in forcing drugs through the three layers of corneal tissue: the epithelium, stroma, and endothelium [1, 2]. The alternative, trans-scleral iontophoresis is different in that it involves placing the iontophoretic device over the sclera and not over the cornea. This route is capable of delivering drugs to the retina through the choroid [3]. 5. Advantages of Microneedle and Iontophoretic Approaches Microneedles and iontophoresis show promise as agents for drug delivery to the posterior segment of the eye. They also hold several advantages over conventional methods of drug delivery to the back of the eye in terms of their minimal invasiveness, simplicity, and comfort for the patient.

Figure 9. Drug delivery to the posterior segment of the eye via trans-scleral iontophoresis, which is able to force more drugs through to the retina by working on the sclera as opposed to the cornea [3]. 5.1 Minimal Invasiveness and Simplicity of Use Microneedle-based drug delivery is significantly less invasive than methods such as direct intravitreal injection and ocular implants. This conveys a number of benefits to those who require drug administration to the back of the eye. First, the relative simplicity of a microneedle injection, which needs only penetrate the sclera, makes it easier to perform than other methods such as intravitreal injection [7]. This means that microneedle treatments can be administered by less-skilled caregivers, which promises to reduce the amount of time patients must wait or the expense they must go through in order to receive routine drug administration for diseases of the back of the eye. Second, reducing the need for highly trained and experienced healthcare practitioners to administer drugs will make treatments for posterior segment diseases more accessible in countries with fewer resources. Even if trained physicians are not readily available in an area, other caregivers can perform the microneedle injections with basic training. Ocular iontophoresis also carries some of the same benefits. Like microneedle delivery, iontophoresis is minimally invasive, not requiring any injections or local anesthetic. This process is also easy to use, as the applicator needs only to be placed on the eye [2]. In both cases, if periodic visits to the doctor can replace the need for a timed-release implant and the associated surgery, treatment of posterior-segment diseases can become more affordable.

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5.2 Safety The small size of microneedles and the fact that they need only to pierce the sclera should minimize any tissue damage as well as any complications resulting from ocular injection [5]. When compared to systemic administration, microneedle-based approaches also reduce the risk of adverse side effects [5]. Similarly, iontophoretic approaches have only occasional mild side effects [2]. Iontophoresis does not carry the risk of complications involved with intravitreal injections and implants. 5.3 Comfort While the level of patient comfort may not directly influence the effectiveness of a drug delivery, it is still an important issue to consider. Even though procedures such as intravitreal injections can be conducted safely and sterilely, the prospect of any large needle entering the eye automatically evokes fear and anxiety in patients. Microneedles must still penetrate part of the eye, but their small size limits the pain they cause [5]. In addition, a tiny microneedle, from a patient’s point of view, should be less frightening than a regular-sized needle. Iontophoresis has the advantage of not requiring any kind of injections into the eye at all. This should make the method more acceptable to patients [2]. The idea of placing the applicator on the eye may even be compared to the use of a contact lens, which is generally seen as non-threatening, and thus avoid unsettling patients. 6. Conclusion Effective drug delivery to the posterior segment of the eye is critical for the treatment of many debilitating diseases, but many conventional methods of ocular drug delivery are inefficient at reaching the back of the eye (eye drops, periocular injections). Others are accompanied by adverse side effects (systemic administration), or carry risks because of being very invasive (intravitreal injection, slow-release implants). Microneedles offer a safer and more effective way to deliver drugs to the back of the eye, especially when delivered through the suprachoroidal route. Whether coated or hollow, they carry less risk of complication and are capable of efficient local delivery of drugs to the posterior segment of the eye with minimal complications. Trans-scleral iontophoresis is also an excellent option, because of its good ability to deliver drugs to

the posterior segment. With no injection and minimal invasiveness, this process is also easy to use and acceptable for patients. Both of these techniques promise methods of posterior segment drug administration that will be more comfortable and more accessible to patients with debilitating ocular disease all around the world. 7. References 1. Abser, M. N. Solid Silicon Microneedles for

Safe and Effective Drug Delivery to Human Eye. Journal of Electrical Engineering (the Institute of Electrical Engineers, Bangladesh) 33(2): 28-34, 2009

2. del Amo, E. M. and A. Urtti. Current and Future

Opthalmic Drug Delivery Systems: a Shift to the Posterior Segment. Drug Discovery Today 13: 135-143, 2008

3. Eljarrat-Binstock, E. and A. J. Domb.

Iontophoresis: A Non-Invasive Ocular Drug Delivery. J. Control. Release 110: 479-489, 2006

4. Fanelli, J. L. The Use of Injections in Primary

Eye Care. Review of Optometry Online 2002 5. Jiang, J., H. S. Gill, D. Ghate, B. E. McCarey, S.

R. Patel, H. F. Edelhauser, and M. R. Prausnitz. Coated Microneedles for Drug Delivery to the Eye. Investigative Ophthalmology and Visual Science 48(9): 4038-4043, 2007

6. Meyer, C. H., A. Fung, S. Saxena, and F. G.

Holtz. Steps for a Safe Intravitreal Injection Technique: A Look at How European and American Approaches Compare. Retinal Physician 2009

7. Patel, S. R., A. S. P. Lin, H. F. Edelhauser, and

M. R. Prausnitz. Suprachoroidal Drug Delivery to the Back of the Eye Using Hollow Microneedles. Pharmaceutical Research 28(1): 166-176, 2011

8. Patel, S. R., D. E. Berezovsky, B. E. McCarey,

V. Zarnitsyn, H. F. Edelhauser and M. R. Prausnitz. Targeted Administration into the Suprachoroidal Space Using a Microneedle for Drug Delivery to the Posterior Segment of the Eye. Investigative Ophthalmology and Visual Science 53(8): 4433-4441, 2012

 

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EXTENDED MONITORING OF OSTEOGENIC DIFFERENTIATION USING AN MRI-COMPATIBLE TISSUE CULTURE SYSTEM

Aishwarya Vaidyanathan avaidy7@uic.edu

Abstract The extracellular matrix (ECM) is highly dynamic. Understanding its biochemical and mechanical properties is necessary for successful tissue regeneration. Magnetic Resonance Imaging (MRI) has been used as a tool to investigate the magnetic relaxation response of water in the ECM by noninvasively measuring the changes in spin-lattice (T1) and spin-spin (T2) relaxation times with sub-millimeter resolution, thereby overcoming the shortcomings of optical microscopy, micro CT etc. The long imaging period involved in analyzing the tissue engineered scaffolds containing cells renders the tissue "dead" within the magnet. To maintain cell viability for an extended period of time, an MRI-compatible culture system was designed for continuous serial monitoring of the osteogenic differentiation in tissue-engineered constructs. We hypothesized that this culture system would permit cell viability when placed within the magnet without compromising on the behavior of the cells or the integrity of the MRI scanner. The MRI-compatible tissue culture system was found to be successful in maintaining the morphology and viability of the cells within constructs, but the behavior of the constructs cultured in the MRI-compatible housing was significantly different from that of the constructs cultured in conventional tissue culture system. Keywords: MRI-compatible culture system, osteogenic differentiation, tissue-engineered constructs

1. Introduction Tissue engineering requires development of functional biomaterials. In the past, materials that remained inert and provided mechanical strength after implantation were considered biocompatible. But as the demands of the medical devices and tissue substitutes changed, so did the definition of biocompatibility. Currently, a biocompatible material is viewed as a material that can interact with the surrounding environment to direct and control tissue response in a desirable way [8]. In order to achieve this control and direction over tissue response in vitro, scientists have been studying cell behavior, biomaterials and the microenvironment of the cells in vivo so that they can mimic the cell microenvironment and try to emulate it within the engineered constructs [7]. A main player in directing cell behavior is the cell microenvironment, consisting of cells, the signals emanating from them, and the extra cellular matrix (ECM). ECM is mainly comprised of modeling and remodeling proteins such as collagen, laminin and proteoglycans containing growth factor binding sites that play a significant role in cell migration, proliferation and differentiation. Major changes occurring during the tissue engineering process are dictated by the ECM. Hence, monitoring the ECM

provides valuable information for successful tissue regeneration [7].

Magnetic Resonance Imaging (MRI) has been used o gain insight into the way water in ECM goes to a lower energy state (relaxation) after being excited by RF pulses by measuring the changes in spin-lattice (T1) and spin-spin (T2) relaxation times [10]. The entire process requires that the sample be kept in the presence of a strong magnetic field. Based on the nature of the magnetic dipole moments and spins within the sample, a net magnetization vector is present which can be excited by Radio Frequency (RF) pulses. Relaxation is the process by which nuclear spins within the sample that are excited by an RF pulse return back to their ground energy state. This process consists of two independent events: longitudinal relaxation and transverse relaxation.

A number of studies have been done that prove the ability of MRI to detect changes in tissue-engineered constructs over time and to correlate MRI parameters with the composition of the ECM [9]. As cell-biomaterial interaction progresses within the tissue-engineered constructs, changes in the ECM composition, tissue hydration, or water/lipid ratios occur, which in turn are reflected in MRI parameters or as contrast in weighted images [9]. As MRI is a non invasive technique, it

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is possible to make several measurements repeatedly on the same sample.

The long imaging period involved in analyzing the tissue-engineered constructs is detrimental to the cells within them. The cells are removed from the optimum conditions that are maintained in the incubator and placed within the bore of a magnet. This largely affects the viability of the cells within the constructs. Hence the prospect of using the MRI-analyzed constructs as an implant within an animal, or the possibility of further characterization of the constructs is lost. To maintain cell viability for an extended period of time, an MRI-compatible culture system was designed for continuous monitoring of tissue-engineered constructs. We hypothesize that this culture system will permit cell viability when placed within the magnet without compromising the behavior of the cells or the integrity of the MRI scanner. This altered system would enable the MRI-analyzed constructs to be characterized further.

2. Materials and Methods 2.1 Hydrogel Preparation A 1:1 collagen-chitosan copolymer was used to prepare three-dimensional constructs that were seeded with Human Marrow Stromal Cells (HMSCs) to enable differentiation into osteoblasts following protocol published by Ravindran et al [7]. Briefly, collagen Type I and chitosan solutions in acetic acid were used along with 5% (w/v) NaHCO3 and Hank’s Balanced Salt Solution (HBSS) to maintain osmotic pressure within the scaffolds. HMSCs were seeded at the concentration of 2 x 106

cells/mL. A predetermined volume of 1 M Sodium Hydroxide solution (determined by titration) was added to the monomer mixture to raise the pH and initiate polymerization. This monomer mixture was incubated for 45-60 min in an incubator at 37 0C and 5% CO2. The polymerized constructs were maintained in four different conditions for 28 days: a) in conventional tissue culture dishes with growth media, b) in our designed MRI-compatible culture system with growth media, c) in conventional tissue culture dishes with osteogenic differentiation media and d) in our new MRI-compatible culture system with osteogenic differentiation media. The osteogenic differentiation media in group c) and group d) was formulated by the addition of � glycerophosphate, dexamethasone and ascorbic acid to the growth media.

2.2 MRI-compatible culture system

The new culture system consisted of a magnetic susceptibility-matched Ultem plug platform (Doty Scientific, SC, USA) placed within a 10 mm MRI tube on which three constructs were placed (Figure 1). The constructs were maintained in this modified MRI-compatible culture system and monitored using 11.7 T Bruker microimager bi-weekly for 28 days. HEPES buffer (2.4 g/L) in the nutrition media was used to maintain the pH of the culture independent of CO2 atmosphere and a variable temperature control unit installed in the probe of the MRI scanner, and maintained the samples being analyzed at a constant temperature of 370C.

Figure 1: MRI-compatible culture system with an inset representative MSME image of the tissue-engineered constructs.

2.3 Magnetic Resonance Imaging

The 11.7 T Bruker microimager uses a 56 mm vertical bore magnet (Oxford instruments, UK) and a Bruker DRX-500 MHz Avance Spectrometer (Bruker Instruments) controlled by a Silicon Graphics SG12 and a Bruker imaging software Paravision 4.0. The system consists of a Bruker triple axis gradient system with a maximum magnetic field gradient of 200 G/cm and micro 5 imaging probes. All experiments were performed using commercial Bruker 10 mm RF saddle coils in an imaging probe equipped with a variable temperature control unit in which the culture system was maintained at a constant 370C throughout the imaging experiments. Experiments were performed in the presence of standard 10 mM Copper sulfate solution, following tuning and matching. Standard MSME pulse sequence was used to measure T2 relaxation time of the scaffolds, with 32 linearly-spaced echoes, TE 5 ms, TR 5000 ms, measured on axial slices of thickness 1mm on a FOV 1cm x 1cm, with frequency and phase encoding matrix size of 128x128, amounting to a spatial resolution of 78 �m x 78 �m [4]. In MSME, multiple echoes are

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generated by repeated application of combination of slice-selective RF pulses. The time constant with which the signal obtained from each echo decays is T2 or transverse relaxation. T2 relaxation occurs when spins lose energy by interacting with other spins and reach ground state. Standard RAREVTR pulse sequence was used to measure T1 relaxation time of the scaffolds. T1 relaxation occurs when spins transfer excess energy to the surrounding lattice and reach ground state. RAREVTR was performed with 8 variable repetition times ranging from 104.6 ms to 15000 ms and with TE 11.45 ms. Other scan parameters remained the same as MSME sequence parameters. Raw MRI data were processed using custom-written MATLAB code to calculate T2 and T1. Three constructs cultured in the conventional tissue culture dishes were removed every week and were analyzed using MRI. Serial measurements were performed on the same constructs cultured in the MRI-compatible system twice every week.

2.4 Microscopy

A fluorescence-based live-dead assay was performed after 28 days to compare the cell survival in constructs maintained in the new culture system model to the cell survival in constructs maintained in conventional tissue culture dishes. Cell morphology and orientation within the three-dimensional environment were determined by using a fluorescent analog of Phalloidin which binds to actin filaments within cells and imaged using Zeiss LSM 510 and 710 confocal microscopes. Z stack of the constructs was performed in which a set of images of planes at varying depths from the surface of the sample is obtained and was reconstructed using Zeiss imaging software. Von Kossa staining for mineral deposits and Safranin O staining for proteoglycan deposits were performed on cryosections from constructs fed with osteogenic media cultured in both conventional and MRI-compatible culture system.

3. Results

3.1 Assessment of cell viability

After four weeks of culturing the collagen-chitosan constructs in the respective culture groups, fluorescence based live-dead assay was performed to reveal dead cell population in the constructs. Figures 3a and 3b show representative images from live-dead assay of constructs cultured in the conventional system and the MRI-compatible

system. Cell counter macro in ImageJ software was used to quantify the red dead cells [6]. A comparison chart of red dead cells in the conventional and MRI-compatible housing is shown in Figure 2. The Y axis represents the number of dead cells present in 320x430 �m2 which was the FOV during the light microscopy imaging process.

Confocal microscopy to visualize the morphology of cells within the constructs was performed. Representative images were depth-coded using Zeiss software (Figures 3c and 3d). It is evident that the morphology of cells in both conventional and MRI-compatible system was identical, where the cells retained branched and extended nature.

Figure 2: Comparison of number of dead cells, as counted from live-dead assay images using ImageJ.

3.2 Assessment of cell behavior by studying mineralization

Figures 3e and 3f show representative images of Von Kossa stained cryosections made from constructs fed with osteogenic media cultured in the conventional system and the MRI-compatible culture system. The black phosphate deposits occurring as a result of mineralization appeared dense and concentrated in the constructs from the conventional culture system, while the deposits appeared scattered and diffuse in constructs from the MRI-compatible system. Safranin O staining was performed to reveal proteoglycan deposits as a result of osteogenesis in constructs cultured in the conventional and MRI-compatible housing for four weeks. The representative images are shown in Figures 3g and 3h. The proteoglycan deposits are much higher in the constructs cultured in

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conventional culture system than in the constructs cultured in MRI-compatible culture system.

3.3 MRI assessment of constructs

The T2 trends of constructs fed with normal and osteogenic media cultured in the conventional culture system are shown in Figure 4a. The constructs exhibited a decreasing trend over a period of four weeks. The T2 trends of the constructs maintained in normal and osteogenic media, cultured in the MRI-compatible housing are shown in Figure 4b. Contrasting the decreasing trend exhibited by constructs cultured in conventional system, no specific trend was observed in these constructs. It is notable that the T2 of constructs maintained in osteogenic media is lower than the T2 of constructs maintained in normal growth media at all time points in both systems.

Figure 3: Panels a, c, e, g represent results of constructs cultured in conventional culture system. Panels b, d, f, h represent results of constructs cultured in MRI-compatible culture system

4. Discussion

We have developed a simple but elegant way to maintain cell viability within constructs while performing MRI analyses by satisfying three basic requirements for cell viability: temperature regulation, pH regulation and nutrition supply. The present study was performed to assess the suitability of the MRI-compatible housing to culture tissue engineered constructs. All experiments were performed with the aim of drawing a comparison between the results shown by constructs grown in conventional tissue culture system and the constructs grown in the MRI-compatible housing.

Figure 4: T2 trends of constructs cultured in conventional system (a) and MRI-compatible culture system (b)

The results from live-dead assay and actin-stained confocal microscopy images reveal that the MRI-compatible housing has been successful in maintaining the viability and morphology of the cells within the constructs. But the cell behavior of constructs cultured in the MRI-compatible housing was not similar to that of constructs cultured in conventional culture system. The extent of ECM remodeling as exemplified by proteoglycan

a)

b)

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remodeling and mineralization was far less pronounced in the MRI-compatible culture system than in the conventional system, pointing to low osteogeneesis in the MRI-compatible system. In the conventional system, an increase in macromolecular deposition and mineralization is the reason behind the observed decrease in T2. When there is an increased deposition of macromolecules like proteins and proteoglycans, a hydration layer is formed along the surface of the macromolecules, constricting the free movement of protons, causing a reduction in T2 [5]. The absence of any trend in T2 in constructs cultured in the MRI-compatible housing is attributed to inadequate ECM deposition accompanied by scattered and diffuse mineralization.

Three phases constitute the process of osteogenic differentiation. The mesenchymal cells proliferate by forming and reforming strong focal adhesions and begin to differentiate to osteoblasts. Next, they secrete and accumulate ECM proteins and express Alkaline Phosphatase (ALP). As a result of ECM deposition and ALP expression, complex mineralization occurs. Matrix mineralization is influenced by expression of specific ECM proteins (BMP-7, DMP-1 etc.) which initiate complex mineralization. For mineralization to occur, the cells must have secreted and expressed adequate ECM proteins in their maturation phase [3].

It becomes clear that the above events took place in the conventional culture, leading to a decreasing trend in T2, but did not take place in the MRI-compatible housing and hence no decrease in T2 was observed over four weeks. This can be explained by the constant perturbations in the orientation of the constructs cultured in the MRI-compatible culture system. The orientation of the cytoskeleton inside the cell can dictate the orientation of the ECM produced [1]. In the conventional culture, the constructs are not disturbed by any external motion; hence the cells remain in a particular orientation, enabling them to synthesize ECM properly and promote subsequent mineralization. In the case of the MRI-compatible culture system, the constructs rest on the Ultem plug placed inside the MRI tube. The constructs are constantly subjected to motion and disturbances in an attempt to remove bubbles or during transport to the MRI scanner. This affects the direction and orientation of ECM synthesis, consequently affecting the dense mineralization.

5. Conclusions

MRI has once again proven to be useful in monitoring the progression of osteogenic differentiation in collagen-chitosan based tissue-engineered constructs. The collagen-chitosan based tissue-engineered constructs used in this study have been shown to support osteogenic differentiation and subsequent mineralization, which can be detected by MRI as a shortening of T2 over four weeks.

The developed MRI-compatible tissue culture system has been successful in maintaining the cell viability and morphology but has not been successful in supporting osteogenic differentiation. This is believed to be due to the absence of control over the orientation of the constructs in the MRI-compatible housing, hence perturbing ECM deposition and mineralization.

We have identified orientation as an important parameter influencing the cell behavior in the developed MRI-compatible housing. A possible development would be to design the MRI-compatible housing with grooves or cassettes to hold the tissue-engineered constructs in place, thereby enabling the cells within to synthesize ECM along their cytoskeletal direction and pave the way for subsequent mineralization.

6. References

1. Albert B, Bray D, Lewis J, Raff M, Roberts K, and Watson J, Molecular Biology Of The Cell, 3rd Edition, Garland, New York; 1994.

2. Hennig J, Nauerth A and Friedburg H; Rare Imaging: A Fast Imaging Method For Clinical MR; Magnetic Resonance in Medicine; 3(6): 823-33; 1986.

3. Lian JB and Stein GS; Development of the osteoblast phenotype: molecular mechanisms mediating osteoblast growth and differentiation; The Iowa Orthopaedic Journal; 15:118-40; 1995

4. Pai A, Li X, And Majumdar S; A Comparative Study At 3T Of Sequence Dependence Of T2 Quantification In The Knee; Magnetic Resonance Imaging; 26(9), 1215-20; 2008

5. Peptan IA, Hong L, Xu H and Magin RL; MR Assessment Of Osteogenic Differentiation In Tissue-Engineered Constructs; Tissue Engineering; 12(4):843-51; 2006

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6. Rasband WS; ImageJ; National Institutes Of Health, Bethesda, MD, http:rsb.info.nih.gov=ij=, 1997–2007, 2007

7. Ravindran S, Song Y and George A; Development Of Three-Dimensional Biomimetic Scaffold To Study Epithelial–Mesenchymal Interactions; Tissue Engineering: Part A;16(1): 327-42; 2010

8. Ricci JL and Terracio L; Where Is Dentistry In

Regenerative Medicine?; International Dental Journal 61 (Suppl.1): 2–10; 2011

9. Xu H, Othman SF, Magin RL; Monitoring Tissue Engineering Using Magnetic Resonance Imaging; Journal Of Bioscience And Bioengineering; 106(6), 515–27; 2008

10. Xu H, Othman SF, Hong L, Peptan IA, Magin RL; Magnetic Resonance Microscopy For Monitoring Osteogenesis In Tissue-Engineered Construct In Vitro; Physics In Medicine And Biology; 51(3): 719–732; 2006

 

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MEDICAL IMAGING IN FORENSIC SCIENCE Marisa Doria

mdoria2@uic.edu Abstract The discovery of X-radiation by Wilhelm Rontgen in 1895 was the start of the medical imaging field that is known today. Medical imaging techniques are not only used for medical diagnostics, but also in forensics to help solve criminal investigations. This article is an overview of the common medical imaging techniques used in forensics, court cases in which medical imaging techniques were used, and relevant research that has been conducted. There are numerous methods of medical imaging; x-rays and computed tomography (CT) scans are the two most common techniques. In many cases, the most critical use of medical imaging is identifying the victim and the cause of death. Keywords: medical imaging, forensics, medical diagnostics, x-rays, computed tomography, court cases

1. Introduction Forensic imaging has been a critical source in many high-profile criminal and civil cases. The word “forensic” comes from the Latin “forensis” meaning "of or before the forum" [13]. Forensic imaging techniques can often help prove someone’s innocence or guilt. Forensics is a multidisciplinary field; the branch that deals with legal issues is known as forensic medicine or legal medicine. This field is most commonly associated with cases involving robbery, kidnapping, rape, assault, or murder [5]. It is often important to know the age, sex, ethnicity, and stature of the suspect in order to come to an understanding of the crime that was committed and to capture the culprit behind the crime [6]. Prosecutors often rely on medical imaging to close cases and sway the jury in their favor. The use of medical imaging techniques in forensics has brought great attention to the public, and researchers to continue to use these technologies in their investigations. Medical imaging can be useful to show victim’s internal injuries. Using a variety of techniques, a forensic scientist can visualize everything from broken bones to organ damage. In some cases, the face of a deceased victim has been reconstructed by using skeletal features. By reconstructing the victim’s face, scientists have been able to reveal the victim’s identity [4]. Medical imaging has advanced from basic 2-D imaging to more advanced 3-D imaging, allowing medical examiners to obtain more accurate information of the injuries or cause of death [7]. Imaging is now commonly used in legal cases around the world and has been popularized by many television shows. Bones were once commonly

studied, but this was an extensive and time-consuming process. The removal of all tissue to study the bone could also cause the destruction of other vital evidence, and in some cultures the removal of all tissue is prohibited. Investigators needed a non-invasive procedure to gather physical evidence [1]. Although medical imaging was not initially accepted in many courtrooms because of its lack of precedence, it is now a reliable source that most judges and courts accept [5]. 2. Review of Medical Imaging Methods There are many different medical imaging techniques that exist today, and some are extensions or further developments of previous methods. The techniques most commonly used in forensics are x-rays, computed tomography (CT) scans, and magnetic resonance imaging (MRI). 2.1 X-rays The German physicist Wilhelm Rontgen discovered X-rays in 1895. Rontgen was experimenting with cathode radiation, and discovered the x-ray by performing many different experiments with “invisible light” that created shadows of images on film. The first known x-ray is that of Röntgen’s wife’s right hand with a ring on it, as shown in Figure 1 [14].

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Figure 1. First x-ray performed by Röntgen on his wife’s hand. The x-ray made the bones visible [14]. The different shades of black, white, and grey shown on the x-rays films correspond to the attenuation, or the weakening, of the x-ray signal by different materials in the body. Bone typically appears brightest; this is due to its increased density attenuating the signal more than other tissues. Most soft tissues in the body have an attenuation coefficient similar to each other, which makes it hard to differentiate between them on x-ray films. Therefore, x-rays are typically used to see the bones and fractures that may have occurred in the body as shown in figure 2 [6]. X-rays are an effective method to reveal blunt trauma to a victim. Blunt trauma can cause cracks or fractures in the bones.

Figure 2. A picture of how a broken bone may be visualized on an x-ray film [10]. The main disadvantage of x-rays is the inability to obtain the exact location of an ossified object or fracture in the body. X-rays only create “shadows” of what is imaging, so x-rays are unable to show the depth of the object or injury [6]. X-rays are also used for dental purposes. In many cases, unknown victims can be identified through dental x-rays and records such as the x-rays in figure 4 [5]. This technique of identifying the victim

through dental records is used when the victim cannot be identified due to external damage.

Figure 3. Dental x-rays of a victim [10]. 2.2 Computed Tomography Computed tomography (CT) scans, a method of producing a three-dimensional image from x-rays, were developed by Dr. Alan Cormack and British engineer Sir Godfrey Hounsfield from existing x-ray technology [2]. CT scaners are widely used today, but because of their high expense and high radiation dose, they are mostly used to further investigate abnormalities seen in x-rays [10]. A CT scan is essentially a cross-sectional image of the body [6]. A CT scanner is shown in figure 4.

Figure 4. A typical CT scanner used in most medical facilities. CT scans of the head can show internal bleeding and skull fractures as shown in figure 5. Blood is denser compared to soft tissue, making it easily identifiable in CT scans. The lungs can also be scanned; in a healthy lung, the lung tissue appears dark, but not as dark as the air within the lung [6].

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Figure 5. A CT scan of a normal brain. The figure on the left shows the distinctions of different parts/areas of the head when a CT scan is performed [9]. 2.3 Magnetic Resonance Imagining The most recently developed medical imaging technique is magnetic resonance imaging (MRI) shown in figure 6. Paul Lauterbur captured the first MRI images in 1973 [6]. The MRI is most commonly used to visualize soft tissues within the body. Unlike CT scanners that use x-rays to form an image, an MRI scanner uses magnetic fields and radio waves to create the images of the soft tissue [11].

Figure 6. A typical MRI scanner used in medical facilities [11]. MRIs show maps of the spatial distribution of different types of tissue. An MRI scanner is composed of strong magnets; therefore, patients with metallic form implants are advised to not have one performed due to the probability damage to the implants. In MRIs, air and bone do not give a strong signal, so these areas appear black in scans as shown in figure 7. It is also imperative that patients do not move during the scans for a better resolution. MRIs can show tumors in the brain, torn ligaments in joints, lesions in livers, cartilage, and more. One of the main

disadvantages of MRI scanners is their high expense and long time to conduct the scan [6].

Figure 7. MRI scan of the human head. The soft tissue is seen in much greater detail [12]. 3. Uses in Forensics Forensic imaging has become a vital part of investigations of a wide variety of crimes. Although medical imaging in the late 1800s caused controversy, Judge LeFerve ruled that x-ray imaging, which was modern science at the time, could be submitted as evidence [5]. 3.1 Court Cases Lawyers began using x-rays as forensic evidence in the courtroom shortly after their discovery by Röntgen. Once the other medical imaging techniques were developed, they also started to be used in courtrooms [5]. 3.1.1 Holder vs. Cunning In the case of Holder vs. Cunning, Holder was accused of attempted murder by shooting Cunning in the leg in 1895. The bullet could not be found upon physical examination, and the wound healed. Cunning continued to have complications where he was shot, so he submitted himself to have an x-ray performed. The x-rays revealed the bullet located between the fibula and tibia. With this evidence, Holder was convicted and sent to prison [5].

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3.1.2 President Theodore Roosevelt President Roosevelt was shot in the chest while running for his third term. President Roosevelt’s coat, suit, shirt, and manuscript of his speech helped reduce the impact of the bullet. An x-ray showed that the President had been shot in the right side of the chest, and the bullet was embedded in his rib. The bullet was left in the president because of the fear of further complications. 3.1.3 Motor Vehicle Accidents Motor vehicle accidents are a common occurrence, and external signs are often not indicative of internal damage. CT scans like the ones in figure 8 are done to obtain a better understanding of internal damage. Clavicles are often broken due to seatbelts during motor vehicle accidents.. This usually occurs due to high impact collisions. When a fractured clavicle is suspected, a CT scan of the thorax is performed like in figure 9 [7].

Figure 8. CT slices of the injuries of a motor vehicle victim. (a) The location of the broken bone. b) A side view of the broken bone. (c) The area of the patient that was scanned. (d) The hematoma on the outside of the victim [7].

Figure 9. This CT scan shows the fractured clavicle of a victim of a motor vehicle accident.

3.2. Research Medical imaging research has been conducted since the technology was first developed. This research is performed to further understand how medical imaging can be used in forensics. Medical imaging methods are investigated to validate their functionality and reliability. 3.2.1 Superimposing Skull on Image In 2001, Matsui et al introduced a new method to determine the facial features of a deceased victim based on skull structure [4]. Pictures of the victim, taken both before and after death and from multiple angles, were loaded into a database. Major anatomical points were used to computationally match the skull with the ante-mortem image an example is shown in figure 10.

a) b) Figure 10. (a) The digitized skull is superimposed over the ante-mortem image, then (b) the major anatomical points are shown. Matsui et al showed a few inconsistencies, but with further investigation of DNA analysis, a match was made to identify the victim [4]. 3.2.2 Cause of Death Although post-mortem CT scans are often conducted during investigations, there are different causes of death that can and cannot be diagnosed using CT scans. Kasahara et al conducted a study in which they tried to determine exactly which causes of death can and cannot be diagnosed with CT. The group analyzed 439 cases in which autopsies were already conducted to investigate in their study the results are shown in table 1. If the cause of death could not be determined by the autopsy, the case was automatically taken out of the study. In some cases, forensic imaging is suggestive of cause of death, but needs to be interpreted by the medical examiner to come to conclusion of the cause of death [3]. Table 1. Results once the CT was conducted

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Kasahara et al showed that hemorrhage was easily diagnosed using CT scans, while cardiac ruptures typically require interpretation by an expert because the ruptures are not evident in CT scans. Traumatic deaths are easily diagnosed due to the fractures of bones and lesions. Drowning and asphyxiation are considered suggestive because signs are not always visible. Cases of burning, hypothermia, and drug overdose are not typically analyzed through CT scans; their diagnoses are usually obtained using other medical methods. 4. Other Uses In addition to determining the cause of death, identifying the victim is one of the main objectives of criminal investigations. Investigators often begin with attempting to determine the victim’s gender in order to narrow down the identification process. In most cases, it is easier to differentiate between male and female skeletons after puberty. Differentiating between male and female pre-pubescent victims is possible, but it is more cumbersome and has to be combined with other techniques such as DNA analysis. The male skeleton is typically heavier and bigger than that of a female. Characteristics of the bones, such as dimensions of the femur and femoral head, can also help differentiate between male and female victims [5]. These dimensions can be obtained through medical imaging techniques such as x-rays or CT scans. In bullet wounds, the exact location of the bullet can be pinpointed in the body. In many cases, the forensic specialist can infer the angle at which the bullet entered the body and the angle the body was positioned at the time of impact [5]. Medical imaging is not only used in obtaining images of the body but also images of documents. In the past, parchment was used for legal documents. By taking an x-ray of the paper, thinner areas would indicate that the paper had been tampered with. The Food and Drug Administration uses x-rays to visualize the adulteration of food. CT scans have also been used to see the contents of mummies such as age, sex, hidden valuables, and injuries [5]. Additionally, many people come in contact with medical imaging while traveling. In some airports in the United States, x-rays have been implemented to

detect if travelers are carrying illegal objects that were not sensed by the metal detectors. Medical imaging has had a significant impact on the forensics of criminal investigations. MRI is beginning to be used more frequently in forensic applications, but it is still expensive to perform [5]. This is likely the reason that MRI scans are only gradually being accepted within the forensic field. CT scans are more expensive than x-rays but less costly than MRIs. The elevated cost of CT scans is compensated by the more accurate portrayal of the inside of the human body. However, there is a limit to the application of these techniques in forensics. Currently, a medical examiner is required to analyze the data, conclude the cause of death, and determine if it was accidental or intentional. Using medical imaging techniques, an examiner can visualize broken bones or internal bleeding, and can often determine the shape of the weapon that was used. Despite the numerous advances in medical imaging technology, a specialist is still required to analyze the data and extrapolate the necessary information. Most forensic specialists do not rely on only one method to conclude the victim’s cause of death. In addition to methods described previously, many examiners also use toxicology reports and DNA analysis [5]. 5. Future Medical imaging will continue to change over time with new discoveries in technology. Forensics examiners could use machines that are more transportable, so that bodies could be imaged without moving them. This could lend a more accurate understanding of the crime scene, and bodies could always be reimaged should more precise information be required. There is always more medical imaging that can be used in forensics. Once CT scans and MRIs become more accessible and affordable, there will be in increase in their use in the field of forensics. Technology has rapidly advanced in the last few decades, and only time will tell what techniques will be developed in the future.

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6. References 1. Brough, A., G. Rutty, S. Black, and B. Morga.

Post-mortem computed tomography and 3D imaging: anthropological applications for juvenile remains. Forensic Sci Med Pathol. 8: 270-279, 2012.

2. Davis, L. CT Scan. eMedicine Health. Web. 2012. http://www.emedicinehealth.com/ct_scan/article_em.htm.

3. Kasahara, S., Y. Makino, M. Hayakawa, Y.

Yajima, H. Ito, and H. Iwase. Diagnosable and non-diagnosable causes of death by postmortem computed tomography: A review of 339 forensic cases. Legal Medicine. 14: 239-245, 2012.

4. Matsui, K. Digital Imaging in Forensic Medicine. Digital Color Imaging in Biomedicine. 73-76. 2001.

5. Romans, L. Forensic Radiology. University of Michigan Hospitals. Web. 2012. http://www.cewebsource.com/coursePDFs/forensicRadiology.pdf.

6. Smith, N.B. and A. Webb. Introduction to Medical Imaging Physics, Engineering, and Clinical Applications. Cambridge: Cambridge University Press, 2011.

7. Urschler, M., A. Bornik, E. Scheurer, K. Yen, H. Bischod, and D. Schmalsteig. Forensic Case Analysis: From 3D Imaging to interactive Visualization. IEEE: Computer Graphics and Applications: 79-87.

8. Weaver, J. Protein Jab Mends Broken Bones. Nature. Web. 2010. http://www.nature.com/news/2010/100428/full/news.2010.209.html

9. Case 1 – Normal CT Scan. Crash. Web.

http://www.crash.lshtm.ac.uk/ctscanlarge.htm. 10. Dental X-rays. Smile By Design. Web. 2011.

http://smilebydesignny.com/cleanings-prevention/dental-x-rays.

11. How Have the Advances in Medical Imaging Affected Health Care?. MRI. Web. 2012. http://ts-1.eee.hku.hk/ccst9015sp12/medical-imaging/medical-imaging-technology/mri-scan/.

12. Sagittal MRI Scan. FMRIB Center. University of

Oxford. Web. 2012. http://www.fmrib.ox.ac.uk/education/fmri/images/sagittal_scan.jpg/view.

13. Shorter Oxford English Dictionary (6th ed.), Oxford University Press, 2007.

14. The Discovery of X-rays. NDT Resource Center. Web. 2012. http://www.ndt-ed.org/EducationResources/HighSchool/Radiography/discoveryxrays.htm.

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CALL FOR ARTICLES - Spring 2013

Mission The mission of the journal is to develop the art of scientific writing among students.

Students may submit articles that discuss original research or review research published

elsewhere. This allows students to hone their writing skills without being limited by a

lack of a data to present. The journal also provides students with an opportunity to be

involved as editors and reviewers. This gives students an overall appreciation of the

processes involved in disseminating scientific findings. The journal finally serves to

expose the reader to current trends in the bioengineering spectrum.

Scope Completed research projects are not necessary for publication. Articles are intended to

document research accomplishments to date. It is expected that many of the articles that

appear in the journal will later be expanded to full-length studies and published

elsewhere. Publication in the UBSJ will not preclude later publication of the results in a

more complete presentation. Submissions can range from original research articles and

technical reviews to book reviews relevant to bioengineering. Letters to the editor are

also welcome.

Submission Process & Guidelines

We have setup a Bioengineering Student Journal Blackboard site to streamline

submissions and make the process as transparent as possible.

1. Single author.

2. The student must be a current UIC Bioengineering undergraduate/graduate student.

3. Only review and research articles may be submitted.

4. Appropriate credit must be cited and permission must be given when applicable.

5. Papers are typically around 4-6 pages long and must be formatted according the

template available on Blackboard.

6. The criteria of acceptance shall be based on volume of papers received, relevance,

subject mastery, organization, appropriate documentation etc.

7. Please refrain from submitting any material that might involve copyright restrictions.

This journal is for learning purposes only. Papers already submitted elsewhere, in-press,

or already published may not be resubmitted here.

8. All submissions will be peer reviewed and the author will be informed in advance if

their article is selected for publication.

9. Submissions are to be made as both .doc and .pdf files. The files shall be named in the

following format:

AuthorName_AuthorLastName_AbbreviatedArticleTitle_VersionMonthDate

If you have any questions please contact:

bioejour@uic.edu

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FRONT

“Stained Rat Cortical Neurons and Glia.” Image courtesy of EnCor Biotechnology Inc. 2013.

< http://www.encorbio.com/Album/Chickenabpics.htm>