The motivation behind this project is to design or improve a cheaper gene gun that can help the world. The goal is to design a new low-cost gene delivery system that will allow Dr. Mahajan and University of Akron students conduct new research, with the aim of advancing society in many different fields. A few examples would be improving crops resistances to insects or harsh weather. This could happen by altering their genes to repulse insects or have stronger bases to have better resistance to the wind. In the medical world you could use gene therapy to help fight cancer or other diseases. The approach to this project is research, design and trial and error. The research will mostly be on how the gene delivery system drives DNA into cells using micro needles

Alolayan, Eid

Alyamani, Feras

Bader, Joseph

Nagelkirk, Connor

Wargo, Victoria

English

The University of Akron

The University of Akron IdeaExchange@UAkron Williams Honors College, Honors Research Projects The Dr. Gary B. and Pamela S. Williams Honors College Spring 2021 Gene Gun Research Project Victoria Wargo vrw15@uakron.edu Eid Alolayan ema62@uakron.edu Joseph Bader jwb89@uakron.edu Feras Alyamani fa48@uakron.edu Connor Nagelkirk cjn33@uakron.edu Follow this and additional works at: https://ideaexchange.uakron.edu/honors_research_projects  Part of the Mechanical Engineering Commons Please take a moment to share how this work helps you through this survey. Your feedback will be important as we plan further development of our repository. Recommended Citation Wargo, Victoria; Alolayan, Eid; Bader, Joseph; Alyamani, Feras; and Nagelkirk, Connor, "Gene Gun Research Project" (2021). Williams Honors College, Honors Research Projects. 1435. https://ideaexchange.uakron.edu/honors_research_projects/1435 This Dissertation/Thesis is brought to you for free and open access by The Dr. Gary B. and Pamela S. Williams Honors College at IdeaExchange@UAkron, the institutional repository of The University of Akron in Akron, Ohio, USA. It has been accepted for inclusion in Williams Honors College, Honors Research Projects by an authorized administrator of IdeaExchange@UAkron. For more information, please contact mjon@uakron.edu, uapress@uakron.edu.      SENIOR DESIGN PROJECT: GENE DELIVERY SYSTEM     By Connor Nagelkirk Eid Alolayan Feras Alyamani Joseph Bader Victoria Wargo   Final Report for 4600:471 Senior/Honor Design, Spring 2021 Faculty Advisor: Dr. Ajay Mahajan 3 May 2021     2  Introduction Abstract The motivation behind this project is to design or improve a cheaper gene gun that can help the world. The goal is to design a new low-cost gene delivery system that will allow Dr. Mahajan and University of Akron students conduct new research, with the aim of advancing society in many different fields. A few examples would be improving crops resistances to insects or harsh weather. This could happen by altering their genes to repulse insects or have stronger bases to have better resistance to the wind. In the medical world you could use gene therapy to help fight cancer or other diseases. The approach to this project is research, design and trial and error. The research will mostly be on how the gene delivery system drives DNA into cells using micro needles.             3  Tables of Contents Introduction                     2 - Abstract                              2 - Background                    4            Biology Aspect                    5 - Definition of Chloroplast                   5 - Location of Chloroplast                   5 Working Principles / Measurement                            7 - Working Principles of Syringe Gene Injector                7 - Measurements of a Lettuce Leaf                  8 Result                                        10 - Probability of Hitting the Chloroplast               10  Design                    11  - Simple Design Brief                 11 - Piezoelectric                  12 - 3D Printing                   16  Discussion/ Conclusion                 17  References                   18  Appendix 1                                     19 - Dimensional Drawings of Syringe Gene Injector              19  Appendix 2                                 21 - Figures of Syringe Gene Injector               21             4  Background The goals for the gene delivery system are to have a reliable and sturdy gene delivery that can go through many trials without damage or change in how the DNA is transferred. This is important so we can get accurate and repeatable results that will allow the users of the gene gun to study how the embedded DNA affects the host. The gene gun uses pressure from compressed C02 to fire heavy metal coated with DNA at a host’s cells. The metal punctures the cells, and the DNA is introduced into the cell as well. This allows the DNA to manipulate the cells. We have the opportunity to improve a design that is already built which allows us to focus on designing improvements or a better system for injection. Since we have a mostly complete gene gun, we are able to run longer trials on various design changes to collect more data before making decisions. The design goal is to have it replicate the results of the state of art gene gun as closely as possible. The current state of art gene gun is The Helios gene gun. The Helios gene gun is a convenient handheld device that uses an adjustable low-pressure helium pulse to sweep DNA, RNA, or biomaterial-coated gold microcarriers from the inner wall of a small plastic cartridge directly into target cells. The Helios cost approximately 30,000 dollars.          5  Biology Aspect Definition of Chloroplast Chloroplasts are the part of plant and algal cells that carry out photosynthesis, the process of converting light energy to energy stored in the form of sugar and other organic molecules that the plant or alga uses as food.  Figure 1, The shape of Chloroplast  Location of Chloroplast The chloroplast is a cell organelle that provides energy through photosynthesis and is only located in algal and plant cells. The middle and outer membranes of the chloroplast cell are the most advantageous locations to inject the genetic fluid through, where it is mostly enriched in chloroplasts.    6  Like mitochondria, chloroplasts are oval-shaped and have two membranes: an outer membrane that structures the chloroplast's exterior surface and an inner membrane that lies only underneath it. A thin intermembrane space of 10-20 nanometers exists between the outer and inner membranes. The stroma is the area inside the inner membrane. The inner membranes of chloroplasts are flat, while the inner membranes of mitochondria have several folds called cristae to absorb the surface region. Instead, the stroma of chloroplasts contains a number of small disc-shaped sacs known as thylakoids.  The thylakoids in vascular plants and green algae are arranged on top of one another, and a granum is a stack of thylakoids (plural: grana). Chlorophylls and carotenoids are pigments found in the thylakoids that capture light during photosynthesis. Photosystems are structures made up of light-absorbing pigments and other molecules such as proteins. The two types of photosystems           Figure (A)           Figure (B)                                  Figure (C) Chloroplast membrane protein densitometric tracings from the inner (A), middle (B), and outer (C) leaves of a lettuce plant.    7   Figure 2, A microscopic drawing the shows thousands of needles injected into a lettuce leaf to reach the chloroplast  Working Principles/ Measurements  Working Principles of Syringe Gene Injector  1. To start, align the far edge of the needles at the beginning of the 1x1 lettuce plant leaf - this will put the syringe injector in the start spot (zero displacements). 2. Apply an electric voltage to the piezoelectric to change the needles' orientation. As a result of the applied voltage, the needles would be displaced onto the lettuce leaf. 3.  fill the drug bottle's inlet with a liquidized drug.  4. Ascertain that the syringe plunger is fully extended. After that, manually push the syringe plunger to drive the drug into the lettuce leaf. 5. This will be repeated 10 times with 10 different distances between each repetition.      8  Measurements of Lettuce Leaf By measuring 10 different 1x1 inch lettuce pieces, using a Clapper device, the follow data was collected:   Table (1)   Unit  Micrometers Inch - Min  Inch - Max  Inch - Avg   Plant Cell Size  10 - 100 0.000393701 0.003937008 0.002165354 Cell Wall Size  0.1 - 10 3.93701E-06 0.000393701 0.000198819 Table (2) Note: Measurement of planes cell and the size of plane cell wall. Table No. 2        9  Unit  µm  Inch    Lettuce leaf size   654.05   0.02575   100%   654.05       0.026  Max  90%   588.65       0.023    80%   523.24       0.021    70%   457.84       0.018    60%   392.43       0.015    50%   327.03       0.013    40%   261.62       0.010    30%   196.22       0.008    20%   130.81       0.005    10%     65.41       0.003  Min  Table (3) Note: These data points were calculated by multiplying the average lettuce leaf thickness with a scale ranging from 10% – 100% for every 10%. Based on these calculations, we can select the needed amount of voltage to drive the piezoelectric actuator. Table No. 3  Unit  Inch Needle Length  0.03 Needle Inner Diameter  0.02 Needle Outer Diameter  0.03 Number of Needles in a row  31 Total Number of Needles 961 Table (4) Note: Measurement of Needle. Table No. 4  Material of Needles  Unit  Aluminum alloy  kg/m^3  Density  2712 Table (5)  Calculation  Unit No.961 - Needles Wight  0.000172 kg/m Needles Volume  0.000009 inch ^3 Table (6)    10  𝑊 = 𝜌 ∗ 𝜋(𝑑𝑜2 − 𝑑𝑖2)4 Equation (1) W = weight of empty pipe per unit length (kg/m, lb/in) 𝜌 = density of pipe material (kg/m3, lb/in3) do = outside diameter (m, in) di = inside diameter (m, in)  𝑉 =  𝜋 ∗ 𝑟2 ∗ 𝑙 Equation (2) V = Volume  𝑟 = Inner radius (Inch) 𝑙 = length (inch)   Results Probability of Hitting Chloroplast The probability of a single needle hitting the chloroplast of a plant cells is somewhere between 28% and 48%, depending on the species and size on the plant. This is due to the general size of the chloroplast ranging in about 5 to 7 micrometers long and can number from 1 to 100 in a single cell. The size of the cell also ranges dependent on the species. the calculation on these were done by averaging the size of the chloroplast multiplied to the average number of chloroplasts in a cell, then divided by the total size of the plant cell. Our high-performance    11  change was taken by having the larger chloroplast in the Higher count to the largest plant cell size.  Design  Figure 3, Syringe Gene Injector Simple Design Brief  The Syringe Gene Injector is a device that allows a large number of living cells to be injected with a genetic liquid drug material. Using 961, micro needles to drive the fluid flow into a lettuce plant leaf. This Mechanical approach will allow a large amount of genetic fluid to transfer into the plant cells while leaving the cells healthy enough to function normally. The    12  figure below will provide some information about Syringe Gene Injector parts and quantities of each part.  Figure 4, Parts of device & quantities  Piezoelectric   A major problem faced during this project was how to accurately position the microneedles inside the lettuce leaf. A high degree of precision would be needed, so we decided to use a piezoelectric actuator. The actuator will move the microneedles into the lettuce and then the liquid can be forced through the needles by hand. Piezoelectric actuators are capable of nanometer precision while also generating high amounts of force making them ideal for this design. Other benefits of using piezoelectric actuators include fast response times and a long cycle life. Operating a piezoelectric actuator requires both a signal generator and a voltage amplifier which will be discussed later.      13  Choosing the actuator was done using the data we gathered from measuring lettuce leaves, our data found the largest travel we would need is around 650 μm. To ensure our design could exceed this requirement an actuator with a travel range of 1000 μm would be chosen. The          P-602.8S0 Piezo Move High-Stiffness Linear Piezo Actuator fits this requirement and was chosen for our design. The actuator also can generate 100N of force which is more than enough to position the microneedle head. This actuator is a flexure-based actuator meaning, it is composed of several layer of piezo ceramic materials that bend which creates displacement vertical to the bend.   Choosing the amplifier and signal generator is dependent on several specifications of the chosen actuator. Several important factors were the voltage range and peak current requirement of the actuator. Fortunately, the manufacture of the actuator recommends several controllers that fit these requirements. The amplifier we chose is the E-610-S0 Piezo Amplifier / Servo Controller. This serves both the function of voltage amplifier and a servo controller eliminating the need for two separate pieces of equipment. The system will operate on a closed loop control system allowing for greater resolution of movement. A closed loop control system will take advantage of the built-in strain gauge sensors on the P-602.8S0.  Positioning the microneedle head using a piezoelectric actuator allows for different voltages to correlate between different positions of the microneedle head. The equation showing this relation is given below taken from the producer of the actuator’s website. The actuator we are using is an all-ceramic flexure actuator which is why this equation was chosen.     14    Several of the variables in this equation are not known to us, as they are inherent properties of the piezoceramic material, and the manufacturer does not list this information on their website. However, variables d, n, lf^ , hp^  are constant and will be the same at any voltage or displacement of the actuator. Next, we can set these variables all equal to a constant called x. This allows us to rewrite Equation (1) as Equation (2) shown below.   Lastly, we know the actuator will produce its largest displacement at the largest voltage within its operating range. From the data sheet of the actuator, we know max ∆L_bend=1000 μm and max V is 120 V. Putting these values into Equation (2) gives x=2.22e-5. This value can now be used in Equation (2) to calculate the voltage needed to reach a desired displacement. Results of using this formula from a range of 0 to 120 V are shown in Figure (1)     15    Figure (1) Displacement vs Voltage of Piezoelectric Actuator    Figure 5, The P-602.8S0 Piezoelectric Actuator      16   3D printing  3D printing or additive manufacturing is a process of making three dimensional solid objects from a digital file. We are using 3D printing to print our design for this project because it is cheap and with high quality by using Solid-Work. We are printing out the parts by using PLA (Polylactide) material. PLA material is highly recommended for 3D printer as it is easy to print with excellent surface finish, wide range of color options, high stiffness, biodegradable and environment friendly.   We are also going to use 3D printing to print the microneedles parts by using aluminum material because of the size of the needles are going to be super small. microneedles were observed to be shrunk and bent. So mechanically, microneedles should be strong. However, some of the polymers used to make microneedles don't have enough power to make arrays. Furthermore, for the needles as the famous facility has created a method for 3D printing high-strength aluminum. That’s why we are using aluminum for the needles. Furthermore, we are also printing the plunger and after we are going to print it, we will use a rubber material to go in the top of it. The benefit of the rubber Usually the needle pushes the rubber aside and it “self-heals.” The rubber is made from two types of materials: bromobutyl or chlorobutyl elastomer. Also, it is going to help us with the stability and will not be lose.       17  Discussion/ Conclusion Based on all research, we found that the best places to transform genes through lettuce are the outer & middle layers of lettuce cells. And that is because the outer and middle layers of cells contain the majority of Chloroplast (Chlorophyll) in lettuce. Our target and goal are to design a needle delivery system that transfers liquid drugs to lettuce leaf by injecting the drugs into the outer & middle layers of chloroplast in a lettuce cell. Our final model is the “Syringe Gene Injector” and allows a large number of living cells to be injected with a genetic liquid drug material. We will use 961 micro needles to inject the liquid. The needles are driven by a piezoelectric actuator for nanometer precision with high force. The probability of a single needle hitting the chloroplast of a plant cells is somewhere between 28% and 48%. In conclusion, the gene delivery system developed is an innovative design that still needs to be physically created and tested.            18  References L.P., P. (n.d.). P-602 PiezoMove HIGH-STIFFNESS Linear Piezo Actuator. Retrieved May 02, 2021, from https://www.pi-usa.us/en/products/piezo-actuators-stacks-benders-tubes/p-602-piezomove-high-stiffness-linear-piezo-actuator-202700/#specification Henriques, F., & Park, R. (1976, December). Development of the photosynthetic unit in lettuce. Retrieved May 02, 2021, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC431541/?page=2%E2%80%AF Editors, B. (2017, April 28). Chloroplast. Retrieved May 02, 2021, from https://biologydictionary.net/chloroplast/ Calculate weight of pipe. (n.d.). Retrieved May 03, 2021, from https://www.engineeringtoolbox.com/pipe-weight-calculation-d_745.html Derma, F. (2018, November 20). Pipe volume calculator. Retrieved May 03, 2021, from https://www.omnicalculator.com/construction/pipe-volume#:~:text=For%20a%20pipe%20use%20its,is%20fully%20filled%20with%20it). Metals and alloys - densities. (n.d.). Retrieved May 03, 2021, from https://www.engineeringtoolbox.com/metal-alloys-densities-d_50.html          19  Appendix 1   Syringe Gene Injector Frame    Piezoelectric Actuator    20   Syringe Plunger   Drug Inlet Bottle   Needles        21  Appendix 2    Isometric View of Syringe Gene Injector   Front View of Syringe Gene Injector    22   Side View of Syringe Gene Injector   Zoomed View of 961 Needles  

Gene Gun Research Project

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Gene Gun Research Project

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