Published on October 16, 2007
Artificial BloodThe blood of the future: Artificial Blood The blood of the future By Anjali Thakkar COSMOS 2007 Cluster 1 Mentor: Professor Paul Feldstein The History of Artificial Blood: The History of Artificial Blood Milk was one of the first substances used as a blood substitute in order to treat patients with Asiatic cholera. (Squires 1). After several patients died by receiving milk transfusions, other substances were discovered as potentials (Squires 1): Salt or saline solutions: used primarily as a plasma volume expander, rather than as artificial blood Hemoglobin isolated from red blood cells Animal plasma could be used as a substitute for human blood However, since many of the materials in animal plasma are toxic to humans, this posed a problem to using it as a substitute The problem of not having a workable substitute led to Ringer’s Solution… Ringer’s Solution: Ringer’s Solution Created by the physiologist Sydney Ringer in 1882 Ringer’s solution is a saline solution which is able to prolong the life of cells in the body Solution contains sodium chloride, potassium chloride, calcium chloride, and sodium bicarbonate: designed in the concentrations found in the human body to keep the heart pumping even outside of the body This solution resembles blood serum Often used to culture animal cells (Encyclopedia Britannica) Blood Grouping: Karl Landsteiner: Blood Grouping: Karl Landsteiner http://nobelprize.org/nobel_prizes/medicine/laureates/1930/landsteiner.jpg Karl Landsteiner was a world-renowned immunologist and pathologist in the 1900s. contributed to the world of medicine in the fields of anatomy, histology, and immunology 1909: Landsteiner classified human blood into four different groups: A, B, AB, and O Recognized the agglutinins in the blood This has become the basis for modern blood typing today received the Nobel Prize for Physiology or Medicine in 1930 (Nobel Foundation) Artificial Blood vs. Blood Substitutes: Artificial Blood vs. Blood Substitutes Ideal Characteristics of Artificial Blood: Ideal Characteristics of Artificial Blood Safe to use Compatible in the human body Able to transport and release oxygen where needed Storable and durable for longer time periods Is free of pathogens and toxins which would produce an immune system response in the human body (Squires 3). Problems Currently Associated with Artificial Blood and Blood Substitutes: Problems Currently Associated with Artificial Blood and Blood Substitutes Bodily immune systems may sometimes react negatively to the foreign blood that is inserted into the body (Goorha et al 46). Trauma/Shock Patients Since these patients are often frequent recipients of blood substitutes or plasma during surgery, it becomes challenging to understand which types of blood substitutes have affected which problem in the patient’s body (Winslow 2). Currently no real working source of artificial blood exists that can perform the multifarious tasks of real human blood cells (Goorha et al 46). Types of Blood SubstitutesPerfluorocarbons (PFC) emulsions: Types of Blood Substitutes Perfluorocarbons (PFC) emulsions Perfluorocarbons are derived from a group of hydrocarbons in which the hydrogen atoms are replaced by fluorine atoms. PFCs are chemically inert due to the strength of the carbon-fluorine bonds Used to create artificial blood during surgeries Process of production: Water, salts, and phospholipids surfactant are added and emulsified through high-pressure homogenization Purified through high temperatures of steam (Goorha et al 47-48). Common PFCs: Perfluorodecalin Perflubron Types of Blood SubstitutesPerfluorocarbons (PFC) emulsions: Types of Blood Substitutes Perfluorocarbons (PFC) emulsions Oxygenation: Saturation occurs with PFCs passively as oxygen molecules “dissolve into molecular cavities within droplets of the liquid” ("Types of Blood Substitutes”) Thus, the oxygenation of PFCs is directly related to the partial pressure of oxygen which is in contact with the PFC (See figure below) "Types of Blood Substitutes." eurobloodsubstitutes.com. 2007. Euro Blood Substitutes. 31 Jul 2007 <http://www.eurobloodsubstitutes.com/pfcInfo.htm>. Types of Blood SubstitutesPerfluorocarbons (PFC) emulsions: Types of Blood Substitutes Perfluorocarbons (PFC) emulsions Structure: Perfluorocarbon core Surrounded by a phospholipid surfactant (Werlin et al) A surfactant is “a substance that reduces the surface tension of the liquid in which it is dissolved” (Brown, useful definitions) http://biomed.brown.edu/Courses/BI108/BI108_2005_Groups/10/webpages/PFClink.htm Types of Blood SubstitutesPerfluorocarbons (PFC) emulsions: Types of Blood Substitutes Perfluorocarbons (PFC) emulsions Types of Blood SubstitutesHemoglobin-based Oxygen Carriers (HBOCs): Types of Blood Substitutes Hemoglobin-based Oxygen Carriers (HBOCs) Hemoglobin-based Oxygen Carriers were created as a mechanism to mimic the oxygen-carrying role of hemoglobin in the body, while still reducing the need for real human hemoglobin. Hemoglobin is a tetramer with two alpha and two beta polypeptide chains; each is bound to an iron heme group which successively binds to an oxygen molecule Hemoglobin’s heme bond allows it to have a higher affinity for oxygen, thus making it an excellent source of blood substitutes. (Goorha et al 46). Types of Blood SubstitutesHemoglobin-based Oxygen Carriers (HBOCs): Types of Blood Substitutes Hemoglobin-based Oxygen Carriers (HBOCs) Currently, experimentation is occurring to develop vesicles which carry the Hemoglobin before inserting it into the body as an HBOC There are several advantages to HBOC carrier vesicles Prevents the denaturation of the Hemoglobin The vesicles themselves are made of purified Hb and lipids, making them compatible with the human immune system Current Setbacks with Hb Vesicle Technology Materials such as nylon, gelatin, and gum arabic have been tried but the human immune system, specifically the reticuloendothelial system, removed the vesicle promptly as a natural bodily response. Later, the theory of phospholipid vessels was used to create a vesicle using phospholipids Researchers are trying to develop a way to create liposome sacks to carry the HBOCs (Goorha et al) Types of Blood Substitutes: Types of Blood Substitutes Goorha, Brig, and Maj Deb. "Artificial Blood." MJAFI 59(2003): 45-49. Types of Blood Substitutes: Types of Blood Substitutes Goorha, Brig, and Maj Deb. "Artificial Blood." MJAFI 59(2003): 45-49. This figure displays the different forms of blood substitutes that can be used to simulate the actions of a red blood cell Researchable Question: Researchable Question Can we make a blood substitute using Biotechnological techniques? In order to create a blood substitute that would effectively transport oxygen throughout the body, the hemoglobin gene would have to be inserted in a plasmid vector and multiplied in a specific medium. The hemoglobin gene would be inserted into the plasmid to carry the cloned gene into E. Coli cells This would allow for expression of large amounts of the hemoglobin protein Escherichia Coli P678-54: Escherichia Coli P678-54 The particular strain of the bacteria E. Coli P678-54 has the unique property of dividing abnormally – it forms a mother cell and a daughter cell (Marszalone et al 1219) The mother cell is similar to a regular cell in that it contains chromosomal DNA. However, it contains ALL of the cell’s DNA The daughter cell is achromosomal. It is smaller, and contains no chromosomal DNA. The daughter cell is often referred to as a “mini cell” Because E. Coli P678-54 has the property of dividing abnormally, it would be ideal if used as a Gene Delivery Vehicle, or GDV (Cohen et al 64). Since E. Coli P678-54 has a tendency to divide into mother cells as well as mini cells, these mini cells could be ideal for the creation of hemoglobin-based oxygen carriers (Giacalone et al 1). Method of HBOC production using E. Coli P678-54 Mini cells: Method of HBOC production using E. Coli P678-54 Mini cells In order to produce a hemoglobin-based oxygen carrier using E. Coli P678-54 mini cells, the hemoglobin gene must be inserted into the E. Coli bacterium. The general process to insert hemoglobin into E. Coli cells uses several techniques of biotechnology: PCR – to assemble the human hemoglobin gene Western Blotting – to ensure that the substance being produced really is the Hemoglobin protein Inoculation – to grow the E. Coli cells after transformation Electroporation – to open the E. Coli cell pores and allow the plasmid vectors containing the Hb gene to enter Blue/White Screening – to test whether or not the E. Coli cells have been successfully transformed with the Hb gene Gene Transformation – to incorporate the Hb gene into E. Coli DNA through plasmid vectors Enzyme digestion – to properly insert the Hb gene into the plasmid vector containing the lacZ indicator gene Experimental Design - Materials: Experimental Design - Materials Agar gel plate with Ampicillin antibiotic Plasmids with the lacZ indicator gene Micropipettes E. Coli P678-54 cells Human DNA Primers (to make complete PCR products of the human hemoglobin gene) Experimental Design – MethodsHemoglobin Gene Preparation: Experimental Design – Methods Hemoglobin Gene Preparation Preparation of Hemoglobin Obtain and purify hemoglobin DNA Use primers to cut hemoglobin DNA and remove introns Use modification methods to ensure that the cell free hemoglobin does not break down and lose its properties This newly assembled hemoglobin DNA can be used as a PCR product PCR Product: PCR buffer dNTP Hemoglobin primer mix Sterile water PCR (Polymerase Chain Reaction) Run the PCR product through a PCR machine Inserting hemoglobin into plasmid vectors Using a plasmid vector with the lacZ indicator gene, we can insert the hemoglobin DNA along with primers for both ends so that the hemoglobin can enter the plasmid This plasmid now contains the recombinant hemoglobin DNA Experimental Design – MethodsTransformation of E. Coli Cells: Experimental Design – Methods Transformation of E. Coli Cells The plasmid vectors are inserted into E. Coli P678-54 Insert the plasmid vectors into a solution containing E. Coli P678-54 Place the solution through “electroporation”: This allows the plasmid vectors to enter the E. Coli cells by opening up pores in the E. Coli Blue/White Screening Once the plasmid vectors have been inserted into the E. Coli cells, spread the transformed cells onto an agar plate with the Ampicillin antibiotic White colonies: represent colonies which have hemoglobin properly inserted into the plasmids the hemoglobin plasmid has successfully broken the lacZ gene region Blue colonies: represent colonies which haven’t been properly transformed: the lacZ gene displays the blue color; thus, colonies that show the blue color have the lacZ region still intact, meaning that the hemoglobin gene has not been incorporated into the region Experimental Design – MethodsMini Cell Separation: Experimental Design – Methods Mini Cell Separation Separation of Mini Cells from normal cells Suspend the solution by microcentrifuging it Mini cells will be suspended higher than the normal cells as they are less dense Pipette out the supernatant above the mini cells Pipette the mini cells without disturbing the normal E. Coli cells Experimental Design – MethodsTesting for Oxygen: Experimental Design – Methods Testing for Oxygen Testing transformed E. Coli minicells for oxygen content a simple way to test the transformed mini cells for oxygen binding to hemoglobin would be to check whether the cells are red or blue Oxygenated blood = red Deoxygenated blood = blue Check the Hemoglobin Saturation percentage (Wikipedia) This is a sigmoidal (non-linear) function which measures the partial pressure of oxygen In a healthy human being, 98.5% of the oxygen is bound to Hemoglobin, thus making it oxygenated 1.5% of the oxygen is bound to other liquids, making it inaccessible How to check Infrared absorption between oxygenated and deoxygenated blood varies highly, thus allowing hospitals and other clinics to differentiate between oxygenated blood and deoxygenated blood Experimental Design – MethodsHemoglobin Modification Methods: Experimental Design – Methods Hemoglobin Modification Methods Ensuring the stability of hemoglobin When hemoglobin is left outside a cell, it has the tendency to break into its individual parts, instead of remaining as an entire hemoglobin protein (“Types of Blood Substitutes”) Methods have been discovered to retain the stability of hemoglobin including: "Types of Blood Substitutes." eurobloodsubstitutes.com. 2007. Euro Blood Substitutes. 31 Jul 2007 Further Research: Further Research Since hemoglobin has been inserted into E. Coli cells, the body’s immune system will most likely develop antibodies to combat the foreign E. Coli cells that are inserted Option 1: the mini cells could be packaged in a liposome to trick the immune system (Li 3761). Option 2: we could find the gene in E. Coli that stimulates the production of antibodies and use gene silencing techniques to turn the gene off Works Cited: Works Cited Cohen, Amikam and W. D. Fisher. "DNA Isolated from Escherichia Coli Minicells Mated with F+ Cells." Proceedings of the National Academy of Sciences of the United States of America 61 (1968) 61-68. 17/07/2007. Giacalone , Matthew, and Gentile, Angela, and Lovitt, Brian, and Xu, Tong, and Sabbadini, Roger, and Surbur, Mark. "The Use of Bacterial Minicells to transfer Plasmid DNA to eukaryotic cells." Cellular Microbiology 8 (2006) 1624-1633. 16/07/2007 <www.blackwell-synergy.com/doi/pdf/10.1111/j.1462-5822.2006.00737.x>. Goorha, Brig, and Maj Deb. "Artificial Blood." MJAFI 59(2003): 45-49. Li, Shuliang. "Liposome-encapsulated actin–hemoglobin (LEAcHb) artificial blood substitutes." Biomaterials 26June 2005 3759-3769. <http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TWB-4DN13MC-1&_user=4421&_coverDate=06%2F01%2F2005&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000059598&_version=1&_urlVersion=0&_userid=4421&md5=795149aeb4f49af0ef50ad656ed4c02a>. Works Cited: Works Cited Marszalek, Piotr and Tian Tsong. "Cell Fission and Formation of Mini Cell Bodies by High Frequency Alternating Electric Field." Biophysical Journal 68 April 1995 1218-1221. 17/07/2007. Nobel Foundation, "Karl Landsteiner." Nobelprize.org. 2007. Nobel Foundation. 31 Jul 2007 <http://nobelprize.org/nobel_prizes/medicine/laureates/1930/landsteiner-bio.html>. “Ringer's solution." Encyclopedia Britannica. 2007. Encyclopedia Britannica Online. 23 July 2007 <http://www.britannica.com/eb/article-9063724>. Squires, Jerry E. "Artificial Blood." Science 295February 2002 1002-1005. 17/07/2007 <http://www.sciencemag.org/cgi/content/full/295/5557/1002>. "Types of Blood Substitutes." eurobloodsubstitutes.com. 2007. Euro Blood Substitutes. 31 Jul 2007 <http://www.eurobloodsubstitutes.com/pfcInfo.htm>. Werlin, Evan. "Current Synthetic Blood Products." biomed.brown.edu. April 2005. Brown University. 31 Jul 2007 http://biomed.brown.edu/Courses/BI108/BI108_2005_Groups/10/webpages/PFClink.htm Wikipedia, "Blood." Wikipedia.com. July 2007. Wikipedia. 31 Jul 2007 <http://en.wikipedia.org/wiki/Blood>.