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he

Pre-Summer Preparation work

• Was introduced to the Bowman-Birk Inhibitor(BBI) by Dr. Aaron Goodarzi, who is a radiooncology researcher at the Charbonneau Cancer Institute at the University of Calgary

• BBI: a protease inhibitor derived from soybeans, which was found to have radioprotective properties by a lab in Germany headed by Dr. Klaus Dittman (Dittmann K, Loffler H, Bamberg M, Rodemann HP. Bowman-Birk proteinase inhibitor (BBI) modulates radiosensitivity and radiation-induced differentiation of human fibroblasts in culture. Radiother. Oncol. 1995;34:137–143.)

• Decided on doing the BBI as our radioprotective peptide of choice; found the sequence of the truncated or modified version of the BBI which has been shown to retain its functions as a radioprotector (Dittmann KH, Gueven N, Mayer C, Rodemann HP. Characterization of the amino acids essential for the photo- and radioprotective effects of a Bowman-Birk protease inhibitor-derived nonapeptide. Protein Engineering. 2001;14:157–160.)

• The whole BBI protein (70 a.a.) can cause problems in blood clotting; 9 a.a. Sequence avoids that issue, because the section of the peptide that acts as a protease inhibitor is cut off

• Previous literature has also suggested a potential molecular pathway by which BBI works. (Gueven N, Dittmann K, Mayer C, Rodemann HP. Bowman-Birk protease inhibitor reduces the radiation-induced activation of the EGF receptor and induces tyrosine phosphatase activity. Intl. J. Radiat. Biol. 1998;73:157–162.) BBI mostly works in Non-Homologous End Joining pathways, which is expected as that is the most prevalent mode of DNA double stranded repair in cells
• Looked into other potential peptides that we can use in case the BBI does not work out: KTI, Glutathione, antioxidants

• KTI: another radioprotective peptide that has been found to be effective in protecting against certain types of radiation that the BBI does not (Van den Hout, R.; Pouw, M.; Gruppen, H. Inactivation kinetics study of the Kunitz soybean trypsin inhibitor and the Bowman−Birk inhibitor. J. Agric. Food Chem. 1998, 46 (1), 281−285.)
• Glutathione: a reactive oxygen species “sink” that quenches reactive oxygen species by binding to its lone electron. Mostly works near the mitochondria of the (Chatterjee A. Reduced Glutathione: A Radioprotector or a Modulator of DNA-Repair Activity? Nutrients. 2013;5(2):525-542. doi:10.3390/nu5020525.)
• Antioxidants: works by a variety of pathways (Weisse J. F. and Landauer M. R., Radioprotection by Antioxidants. Annals of the New York Academy of Sciences. 2000; 899: 44–60. doi:10.1111/j.1749-6632.2000.tb06175.x)

• Also looked at current other methods of radioprotection (doubles as market research for the application of our device) (Kamran, M. Z., Ranjan, A., Kaur, N., Sur, S. and Tandon, V. (2016), Radioprotective Agents: Strategies and Translational Advances. Med. Res. Rev., 36: 461–493. doi:10.1002/med.21386)

• Currently some radioprotectors have been proposed for cancer radiotherapy (ex. synthetic thiol-containing compounts, amifostine) but uptake by the public and medical community has not been great as side-effects were undesirable and drug benefits were not well defined
• NASA Biocapsule - we did not know about this the time when we came up with our project, but after some searching around we stumbled apon this on the internet. Comparable technology to our device, unsure about the progress/research that has been done on this as NASA is very secretive (http://sservi.nasa.gov/articles/nasa-breakthrough-could-save-millions-lives/; http://www.medgadget.com/2012/02/nasa-biocapsule-implant-diagnoses-and-treats-diseases-without-human-intervention.html )

• Were unsure about the specific potential application of our peptide: it was a debate between cancer radiation prevention (as radiotherapy is known to cause secondary cancers, and certain existing literature stated that BBI has radioprotective effects only on p53+ cells, which about 60% of all cancers are p53-) (Dittmann KH, Gueven N, Mayer C, Ohneseit P, Zell R, Begg AC, Rodemann HP. The presence of wild-type TP53 is necessary for the radioprotective effect of the Bowman-Birk proteinase inhibitor in normal fibroblasts. Radiation Research. 1998;150:648–655.), or space application

• We realize these applications would call for very different designs for our device as well as different methods of testing, but those differences exist mostly in the more refined details of our project, which would not affect our preliminary work
• Decided that we needed a deadline by which to decide the application of our project: End of June (tentative)

• Planned out some experiments we wanted to do to test viability of the various peptides: Clonogenic survival assay with both cancer cells as well as primary cells

• We had a few variations on the clonogenic survival: thought about doing high throughput assays with 96 well plates, dual plating with cancer and primary cells to see interactions, comparison of survivability of cancers vs. primary cells with and without BBI treatment

• Expected results: BBI is expected to protect our primary cells against radiation while not protecting our cancer cells as much as the primary cells. Meaning that the surviving fractions of our cell lines treated with BBI will be higher than that of cell lines not treated with BBI, and the surviving fractions of primary cell lines treated with BBI will be higher than that of cancer cell lines treated with BBI

May 3, 2016

Dr. Goodarzi Interview

• We met with Dr. Goodarzi to talk about potential assays and our prospects for the summer
• Dr. Goodarzi confirmed the viability of our clonogenic survival assay in testing the effectiveness of BBI and mBBI, but he also suggested a few more assays we can use, namingly the H2AX assay (Mariotti LG, Pirovano G, Savage KI, Ghita M, Ottolenghi A, Prise KM, et al. (2013) Use of the γ-H2AX Assay to Investigate DNA Repair Dynamics Following Multiple Radiation Exposures. PLoS ONE 8(11): e79541. doi:10.1371/journal.pone.0079541) and the Flow cytometry assay

• H2AX: Can use fluorescent tags to tag H2AX foci (histone proteins that localize around double stranded breaks) to detect the number of double stranded breaks post irradiation with or without BBI.  Using fluorescent microscopy to view double stranded breaks post irradiation, we can blind count the number of detected foci at various timepoints within 24 hours of irradiation to see if the cells treated with BBI are able to repair their double stranded breaks (marked by the decrease of fluorescent foci) at a faster rate comared to cells that are not

• Dr. Goodarzi had also suggested that we use non-cancer cell lines as well as cancerous cell lines to test the difference in radiation protection in cancer cells versus non-cancerous cell lines
• Listed a number of cell lines that will be able to provided for us, including a variety of cancerous cell lines as well as normal cell lines

May 4, 2016

Laboratory introduction and techniques week

• Made and Autoclaved agar (1L)

• 1% tryptone (10g)
• 0.5% Yeast extract (5g)
• 1% NaCl (10g)
• 1.5% Agarose (15g)

• Poured plates of LB agar with 3 different types of antibiotics:

• Amp (stock 100 mg/mL; final 100 ug/mL)
• Kan (stock 50 mg/mL; final 50 ug/mL)
• Chlor (stock 50 mg/mL; final 30 ug/mL)

May 5, 2016

Laboratory introduction and techniques week

• Results: Cultured competent E.Coli as well as Chlor-resistant E.Coli on each type of Agar plate(s)

• Growth of a lawn of red-coloured bacteria (Chlor-resistant culture on Chlor plate & Chlor-resistant culture on Amp plate)
• Growth of a lawn of white/yellow- coloured bacteria (competent E.Coli culture on Amp plate) --> contamination (?)
• No growth observed on other plates

• Results: Liquid Culture of normal as well as Chlor-resistant E.Coli (no antibiotic added to liquid culture)

• Growth of white/yellow bacterial cultures on the bottom of all tubes

• Made 50mM CaCl2 (5mL of 1M CaCl2, 95mL ddH2O) & 50mM CaCl2 with 15% Glycerol (5mL 1M CaCl2, 15mL Glycerol, 80mL ddH2O) for making competent E.Coli --> placed in 4°C fridge

• Inoculated 2 tubes of 5 mL LB with old (#10) competent E. coli cells
• Inoculated 2 tubes of 5 mL LB with new (C3037) competent E. coli cells (Used steril 5 mL LB as negative control) --> incubated at 37°C O/N, shaking (200 rpm)

May 6, 2016

Laboratory introduction and techniques week

• The overnight cultures of competent E. Coli (#10 and C3037) were subcultured in 49ml of LB. There were four subcultures total: 2xC3037 and 2x#10. Cultures were incubated at 37.5°C and shaken at 200rpm for 4 hours. The cultures were grown to an optical density of 0.647 (within the range of 0.4-0.6)

• Chemical comptenece

• Cultures were spun down for 7 minutes at 3000rpm and 4°C
• 12.5ml of cold 50mM CaCl2 was added and the pellet resuspended
• Cells were then incubated on ice for 10 minutes
• Cells were spun down again at 1500rpm for 2 minutes at 4°C
• Cells were resuspended in 2ml of 50mM CaCl2 with 15% glycerol
• Transfer to microcentrifuge tube in 100μl aliquots
• Storage at -80°C

• The rest was left up to chassis team to document and discuss results

May 9, 2016: EXPERIMENTAL GOALS FOR THE SUMMER - DISCUSSION

BBI Efficiency

• test in human cell lines
• requires positive/negative control

Cytotoxic treatment

• induce DNA damage from something other than radiation/determination of mechanism
• H2AX assay

Circuit Function

• testing construct components
• promoters and RBS
• test with reporter genes
• BBI expression
• use mass spec/MALDI to measure peptide amount
• use rtQ-PCR as a backup

Excretion (overlap with Chassis)

• similar assay to BBI excretion

May 10, 2016

• Researched the demand for a radioprotective strategy in space exploration, found that NASA and the CSA are trying to further research efforts in this area, making it an important issue to address (http://www.space.com/21353-space-radiation-mars-mission-threat.html)
• Similar products are currently under development by NASA called the biocapsule (http://www.americaspace.com/?p=13700)

• The developments of the biocapsule technology is unknown in the public, so we do not know how similar the biocapsule is from our proposed project

May 11, 2016

Peptide Sequences

• Compiled the mBBI amino acid sequences found in literature searches, and also put in some of our own additions to prime the peptide for secretion and delivery, as well as to accommodate our own assays
• Reverse translated them into genetic sequences for E.Coli as well as B. Subtilis

Common Identifier	FASTA	E. Coli	B. Subtilis	D. Radiodurans
(S) 9mer	CALSYPAQC	TGC-GCG-CTG-AGC-TAT-CCG-GCG-CAG-TGC	TGC-GCA-CTG-TCA-TAT-CCG-GCA-CAA-TGC	UGC-GCC-CUG-AGC-UAC-CCC-GCC-CAG-UGC
(V) 9mer	CALVYPAQC	TGC-GCG-CTG-GTG-TAT-CCG-GCG-CAG-TGC	TGC-GCA-CTG-GTT-TAT-CCG-GCA-CAA-TGC	UGC-GCC-CUG-GUG-UAC-CCC-GCC-CAG-UGC
(S) 7mer	ALSYPAQ	GCG-CTG-AGC-TAT-CCG-GCG-CAG-TGC	TGC-GCA-CTG-TCA-TAT-CCG-GCA-CAA-TGC	GCC-CUG-AGC-UAC-CCC-GCC-CAG
BBI protein	SACKSCICALSYPAQCFCVDIT	UCC-GCG-UGC-AAA-UCC-UGC-AUU-UGC-GCG-CUG-UCC-UAU-CCG-GCG-CAG-UGC-UUU-UGC-GUG-GAU	TCA-GCA-TGC-AAA-TCA-TGC-ATT-TGC-GCA-CTG-TCA-TAT-CCG-GCA-CAA-TGC-TTT-TGC-GTT-GAT-ATT-ACA
Peptide #1 BBI	KSCICALSYPAQCF	AAA-AGC-TGC-ATT-TGC-GCG-CTG-AGC-TAT-CCG-GCG-CAG-TGC-TTT	AAA-TCA-TGC-ATT-TGC-GCA-CTG-TCA-TAT-CCG-GCA-CAA-TGC-TTT	AAG-AGC-UGC-AUC-UGC-GCC-CUG-AGC-UAC-CCC-GCC-CAG-UGC-UUC
Peptide #2 - NLS	GPKKKRKVKSCICALSYPAQCF	GGC-CCG-AAA-AAA-AAA-CGC-AAA-GTG-AAA-AGC-TGC-ATT-TGC-GCG-CTG-AGC-TAT-CCG-GCG-CAG-TGC-TTT	GGC-CCG-AAA-AAA-AAA-AGA-AAA-GTT-AAA-TCA-TGC-ATT-TGC-GCA-CTG-TCA-TAT-CCG-GCA-CAA-TGC-TTT	GGC-CCC-AAG-AAG-AAG-CGC-AAG-GUG-AAG-AGC-UGC-AUC-UGC-GCC-CUG-AGC-UAC-CCC-GCC-CAG-UGC-UUC
Peptide #3 BBI HA	KSCICALSYPAQCFYPYDVPDYA	AAA-AGC-TGC-ATT-TGC-GCG-CTG-AGC-TAT-CCG-GCG-CAG-TGC-TTT-TAT-CCG-TAT-GAT-GTG-CCG-GAT-TAT-GCG	AAA-TCA-TGC-ATT-TGC-GCA-CTG-TCA-TAT-CCG-GCA-CAA-TGC-TTT-TAT-CCG-TAT-GAT-GTT-CCG-GAT-TAT-GCA	AAG-AGC-UGC-AUC-UGC-GCC-CUG-AGC-UAC-CCC-GCC-CAG-UGC-UUC-UAC-CCC-UAC-GAC-GUG-CCC-GAC-UAC-GCC
Peptide #4 BBI NLS-HA	GPKKKRKVKSCICALSYPAQCFYPYDVPDYA	GGC-CCG-AAA-AAA-AAA-CGC-AAA-GTG-AAA-AGC-TGC-ATT-TGC-GCG-CTG-AGC-TAT-CCG-GCG-CAG-TGC-TTT-TAT-CCG-TAT-GAT-GTG-CCG-GAT-TAT-GCG	GGC-CCG-AAA-AAA-AAA-AGA-AAA-GTT-AAA-TCA-TGC-ATT-TGC-GCA-CTG-TCA-TAT-CCG-GCA-CAA-TGC-TTT-TAT-CCG-TAT-GAT-GTT-CCG-GAT-TAT-GCA	GGC-CCC-AAG-AAG-AAG-CGC-AAG-GUG-AAG-AGC-UGC-AUC-UGC-GCC-CUG-AGC-UAC-CCC-GCC-CAG-UGC-UUC-UAC-CCC-UAC-GAC-GUG-CCC-GAC-UAC-GCC

May 12, 2016

• Asked for peptide quotes from a multitude of peptide synthesis companies to find the cheapest and best synthesis deal

May 14-15, 2016: Lethbridge Workshop

Imagine, Design, Create – Cesar Rodriguez (cesar,.rodriguez@med.fsu.edu)

• imagine: Write it down!

• Cell based therapeutics is the third pillar of medical therapeutics (small molecule → proteins/antibiotics → cells)
• If[molecule]>x THEM produce therapy (molecule→senser→procduct→activator→product)
• Sense environment; produce effect (ex. Smell bread→salivate)

• Design: design specification→simulation modification

• Apple omnigrapho
• Microsoft visio
• Cell modeller
• Jupyter (anaconda package)
• Neuvidiaflex

• Create: Make your prototype

• Gen9 (DNA synthesis company)
• Twist (DNA synthesis)
• Cloudlab (do your experiments in a remote lab)

Policy & Practices in DIY Bio and iGEM – David Lloyd

• Core of HP: Society←→ Lab

• Social sciences based questions
• Follows a methodology
• Demonstrates real world application
• Meaningful impact on your labwork
• Discover things you didn’t know before

• Outreach  P&P (outreach is put out; P&P is bring in)
• Read the judge’s notebook on website COMMUNICATE! TARGET AUDIENCE!

• Make it obvious how you fulfill the criteria
• Make sure you differentiate P&P vs. outreach

• Be careful of survey design!

• If you are doing a survey, make sure you make it statistically relevant
• Good example: Gender Study (Paris Bettencourt 2013)

Mathematical Modelling for SynBio – Brian Ingals

• Physical vs. conceptual
• Mathematical modesl can be mechanistic (description) or predictive (make inferences/extrapolation)
• Mass action kinetics

• Chemical rxn: X→P
• Rate constant: k=[P]/[X]
• Etc.

• Programs that can be used

• COPASI
• MATLAB
• XPPAIIT
• Mathematica

• Separation of time scales; phase plane analysis

• Processes slow → treated as frozen in time
• Processes fast → treated as occurring instantaneously

Wiki & Visual Design in iGEM – Patrick Wu

• Design vs. Art (not the same thing)

• Design: communicate the same thing to everyone (USABILITY is key!)
• Art: Can have different interpretations to different people

• Content, accessibility, visual hierarchy, grid layout to organize info
• Interpret data for your audience! Figure captions and descriptions
• Summary page for judges is good for check-boxing

May 16, 2016

• Decided on BioBasic as our peptide synthesis company and ordered all of the peptides
• Ordered full length BBI as well as KTI (as a backup peptide) from Sigma Aldrich
• Wait for ~3 weeks for peptides to come!

June 6, 2016

• Dr. Goodarzi had suggested the idea of reducing our peptide; in the original structure of BBI, our truncated section contains 2 cysteines (which means the possibility of cysteine bridges) (Voss, R.-H., Ermler, U., Essen, L.-O., Wenzl, G., Kim, Y.-M. and Flecker, P. (1996), Crystal Structure of the Bifunctional Soybean Bowman-Birk Inhibitor at 0.28-nm Resolution. European Journal of Biochemistry, 242: 122–131.doi:10.1111/j.1432-1033.1996.0122r.x)
• Literature supports that reduction of our peptide would not affect its radioprotective effects (Gueven N, Dittmann K, Mayer C, Rodemann HP. The radioprotective potential of the Bowman-Birk protease inhibitor is independent of its secondary structure. Cancer Letters. 1998;125:77–82.)
• We chose to use DTT as our reducing agent from literature search

June 10, 2016

Clonogenics 0.1 – cell splitting protocol

• take HTC116 cell line with >50% confluency
• wash cell line twice with 1mL of DMSO
• add 1mL 37C trypsin and incubate for 4 minutes
• add 4mL of media (McCoy's with 10%FBS and 0.5%pen-strep) to each of 4 new plates
• add 3mL media to plate from step 3 to quench trypsin, pipette up and down to separate the cells (ideally we have no clumps in the cells but have them individually suspended in solution
• add 1mL of the cells from step 5 to each plate done in step 4, making sure to seed the cells all over the plate
• incubate at 37C with 5% CO2 to use for next time (over the weekend)

 

June 13, 2016

Clonogenic 0.1 – solvation of peptide

• dissolved our heptamer peptide (ALSYPAQ) in McCoy's Media, our peptide was not visible to the naked eye after adding McCoy's Media, so it's soluble(?). We then did a serial dilution with the peptide solution to make varying concentrations

concentration	volume of peptide solution	volume of media	total volume
1200μM	dry peptide	10mL	10mL
150μM	1mL from 1200μM	7mL	8mL
100μM	4mL from 150μM	2mL	6mL
50μM	2mL from 100μM	2mL	4mL

We got scolded...

** Remember for next time/actual experiment: only 2-10μL of drug/peptide treatment (negligable amounts) should be added to cell lines to do experiment instead of incorporating the peptide into our media

 

Clonogenic 0.1 – Cell counting and seeding protocol

• a ~50% confluent cell line of HCT116 cells
• removed media, washed twice with 25°C DPBS
• add 1mL of 37°C trypsin, incubated for 6min in 37C, 5% CO2 incubator
• quench effects of trypsin by adding 3mL media (McCoy's Media with 10% FBS and Pen-Strep) pipetted up and down to suspend cells
• put 4mL of solution containing cells into autoclaved 15mL falcon tube and centrifuged at 2500rpm for 5min at 25C
• removed all media, leaving only pellet of cell
• resuspended cells with 1mL media (removed 50μL to look at under the microscope to make sure clumping of cells is minimal)
• pipette 10μL of cell mixture and mixed with 10μL of typan blue; pipetted onto cell counting slide (10μL in each of well A and B)
• put the slide in cell counter:

• Well A: live cells 9.15x105 cells/mL (69% live cells)
• Well B: live cells 7.04x105 cells/mL (81% live cells)

 

Clonogenic 0.1

• The discrepancy between the two wells is quite big, meaning that we did not mix the cell mixture well before counting. Ideally we would want to work with cell cultures that are 85-100% live cells, so we should be careful of cell death (leaving trypsin on for less time, more cogniscent of bubbles etc) in coming experiments.
• But for the sake of practice we decided to seed the cells from the cell counting experiment (average of well A&B: 809500 cells/mL) by plating 6 plates of each of the following cell counts:

cell count/mL	cell solution volume	media volume	total volume
2000	37μL from 809500cell/mL solution	15mL	~15mL
1000	6mL from 2000cell/mL solution	6mL	12mL
500	4mL from 1000cell/mL solution	4mL	8mL

• 1mL of each solution was added to 4mL media on the plates. (6 plates for each cell count)
• the plates were incubated at 37C and 5% CO2 (will check up on them Friday) OUR CANCER BABIES!!!

 

 June 14, 2016

Clonogenic 1.0

• BBI Treatment of HCT116 Cells - 7mer (ALSYPAQ)

• Time of Application: 10:55 AM
• Time of Irradiation: 4:10PM

**Plates were treated with BBI by Sid** (see put peptide in solution entry from Monday 6/13)

Plate #	Cell Density	BBI Concentration	IR Dosage
1-6	500 cells	0	0 Gy
1-3	1000 cells	10 microMolar	5 Gy
4-6	1000 Cells	30 microMolar	5 Gy
1-6	2000 cells	0	5 Gy

 

June 15, 2016

Clonogenic 0.1

• Made 500mL fix solution (3% acetic acid, 8% methanol, 89% water)
• stain solution (0.2% crystal violet, 10% formalin in PBS)

• could not find formalin to make stain solution, we will get some from Nick Jette, but in the meantime we ordered some

 

June 16, 2016

Clonogenic 0.1

• Some of our stock cells look very overgrown, so we split some of the less confluent ones, and we are going to throw away the ones that are more overgrown

• Looked at the cells we decided to irradiate and treat. The 1000 plates (treated with BBI and irradiation) looked very strange under the microscope as it had little specks that were not our cells. We suspect it is because our peptides were not soluble in McCoys media and clumped up in solution. So we will try to dissolve the other peptides in DMSO first before applying to our cell cultures. In the meantime, we are trying to salvage our heptamer by centriguging to see if the peptides will form a pellet so that we can test a few more things on it before we toss it out.

June 17, 2016

Clonogenic 0.1 – Fixing and Staining protocol

• Our staining solution (06/15 entry) was not up to par to the standards as it did not look dark enough. The suspicion is that our crystal violet was not a the right concentration to begin with (as our crystal violet came in solution form instead of powder form), so we will go back and search up the initial concentration of our crystal violet. In the mean time, this practice will be done with crystal violet provided to us by the Susan-Lees Millers lab.
• Media was removed from plates
• 1mL fix solution was added (see entry on June 15); let sit for 2 min, then removed
• 1mL stain solution was added (see entry on June 15); let sit for 5 min, then removed
• deionized water was added to each plate (covers surface of the plate) to rinse the cells, removed

Clonogenic 0.1

• looked at stained colonies

• the 500 plates had more colonies than the other 2 types of plates
• 1000 plates had very little to no colonies
• 2000 plates had about the same amount of colonies than 500 plates, but the colony sizes are much smaller

 

 June 20, 2016

Clonogenic 0.2

• before we start to do our official trials, we needed to do another practice to familiarize ourselves with techniques

• Learned that the P1000 pipettes were very effective in breaking up cells

• counted and seeded 48 plates with varying cell counts (x6 plates for each manipulation)

Drug (DMSO) Treatment Volume	0Gy Radiation	5Gy Radiation
0μL	sd. 500	sd. 2000
3μL	sd. 500	sd. 1000
6μL	sd. 500	sd. 1000
9μL	sd. 500	sd. 1000

 

June 21, 2016

Clonogenic 0.2

• Treated cells with varying volumes (Table15) of filter-sterilized DMSO as a carrier control as well as irradiated all our plates.
• Now to just wait for our babies to grow in the incubator!

 

Clonogenic 0.2 - For Thursday (10AM)

• More cell count and seeding practice
• counting practice plates from last friday in the cell colony counter and doing statistics on them
• setting up the vacuum media aspirator (maybe)

 

June 23, 2016

Clonogenic 0.1 - Colony Counting Protocol

• Open the computer, select Colcount and the password is dnapk*atm
• Once on the desktop screen select colcount from the desktop
• Once the application opens click start, select 50mm UpperPosition (7E2) and then click Load
• Select Cell present and hit browse - use either Nick(good) or small colonies
• Insert the blank with no tip and it will start warming up.
• Optional - Save at desktop and make new folder every day
• Grab the plates with colony and start counting
• Happy Counting

 

Plates	500	1000	2000
1	46	9	22
2	84	1	10
3	63	12	22
4	53	13	8
5	42	7	19
6	43	7	8

 

 

June 27, 2016

Clonogenic 0.2

• Cells were trypsinized and counted using the Bio-Rad counter and plates.

• Well A: 1.17 x 107 cells/ml (76% alive)

• 36 x 1000 cell plates and 36 x 500 cell plates were seeded with their respective amount of cells. 1ml of cell culture was added to 4 ml of McCoy's media with FBS and Pen-Strep.

 

June 29, 2016

HCT116 Clonogenics 1.0

• Our phosphorylated BBI peptide as well as our control scramble peptide came in!
• We tried to seed our 168 plates today (see google docs clonogenics trial 1), but in our cell count, it turns out only 12-14% of our cells are alive??! (even though we redid it 3 times)

• we suspect that the cell counter was acting up as non of the other people's samples were working either. But either way those results are unusable, so we will have to push this off till next Monday to seed as this Friday will be Canada Day.

 

July 4, 2016

HCT116 Clonogenics 1.0

• Used stock cell plate with cells split on June 29th, also tried counting with cells from 2 weeks ago with 1000 seed but decided not to use them because cell survival was 9%
• Cell survival percentage of the stock solution used is average of 53%, decided to go ahead with the trials as we suspect it is our cell counting protocol that needed tweaking as opposed to our cells actually being fifty percent dead

• Seeded 100 plates with 500 cells/plate
• seeded 100 plates with 1000 cells/plate

 

July 5, 2016

HCT116 Clonogenics 1.0

• Plan of our clonogenics trial

	plates #	Volume added (uL)	Radiation(Gy)	Seeding	# Plates	Time drugged	Time irradiated
Control (DMSO+DTT)	1	0	0	500	3	11:20
	2	3	0	500	3	11:21
	3	6	0	500	3	11:25
	4	9	0	500	3	11:28
	5	0	5	1000	3	11:20	5:52
	6	3	5	1000	3	11:21	5:32
	7	6	5	1000	3	11:25	5:42
	8	9	5	1000	3	11:28	5:42
9mer (S)	1	0	0	500	3	11:30
	2	3	0	500	3	11:32
	3	6	0	500	3	11:35
	4	9	0	500	3	11:40
	5	0	5	1000	3	11:30	6:21
	6	3	5	1000	3	11:32	6:02
	7	6	5	1000	3	11:35	6:25
	8	9	5	1000	3	11:40	6:05
KSCI BBI	1	0	0	500	3	11:45
	2	3	0	500	3	11:46
	3	6	0	500	3	11:48
	4	9	0	500	3	11:50
	5	0	5	1000	3	11:45	5:52
	6	3	5	1000	3	11:46	5:36
	7	6	5	1000	3	11:48	6:02
	8	9	5	1000	3	11:50	5:36
Big BBI	1	0	0	500	3	12:00
	2	9	0	500	3	12:02
	3	18	0	500	3	12:05
	4	27	0	500	3	12:08
	5	0	5	1000	3	12:00	5:25
	6	9	5	1000	3	12:02	5:19
	7	18	5	1000	3	12:05	5:25
	8	27	5	1000	3	12:08	5:19
KTI	1	0	0	500	3
	2	12	0	500	3	11:09
	3	24	0	500	3	11:13
	4	36	0	500	3	11:14
	5	0	5	1000	3		6:21
	6	12	5	1000	3	11:23	6:17
	7	24	5	1000	3	11:21	5:05
	8	36	5	1000	3	11:20	6:13
Scramble	1	0	0	500	3
	2	3	0	500	3	11:29
	3	6	0	500	3	11:32
	4	9	0	500	3	11:33
	5	0	5	1000	3		5:56
	6	3	5	1000	3	11:37	6:20
	7	6	5	1000	3	11:40	6:27
	8	9	5	1000	3	11:41	5:47
P-Tyrosine 9mer (S)	1	0	0	500	3
	2	3	0	500	3	11:52
	3	6	0	500	3	11:50
	4	9	0	500	3	11:48
	5	0	5	1000	3		5:56
	6	3	5	1000	3	11:55	6:13
	7	6	5	1000	3	11:57	6:10
	8	9	5	1000	3	11:59	5:47

 

July 12, 2016

HCT116 Clonogenics 1.0

• the cultures with p(y) 9mer looks more yellow than other plates, perhaps indicating more colonies (more to come in 2 weeks)

 

July 15, 2016

HCT116 Clonogenics 1.0

• We had set up a vacuum to suck off the media as well as treat our plates with fix and stain solution. Colonies were counted using ColCount colony counter.

	Concentration (μmol/L)	Radiation(Gy)	Seeding	Colony Count			Treatment Concentration	Surviving Fraction	Trial PE
Control (DMSO+DTT)	0	0	500	250	200	168	0	0.955085482	0.431375
	10	0	500	178	242	215	10	0.98135806
	20	0	500	237	311	220	20	1.186902347
	30	0	500	223	270	250	30	1.148266203
	0	5	1000	16	24	15	0	0.042499759
	10	5	1000	37*	18		10	0.041727036
	20	5	1000	31*	8	16	20	0.027818024
	30	5	1000	20	28	6	30	0.041727036
9mer (S)	0	0	500	208	220		0	0.992176181
	10	0	500	77	191	180	10	0.692359703
	20	0	500	122	129	120	20	0.573360379
	30	0	500	106	117	140	30	0.560996813
	0	5	1000	20	12	9	0	0.031681638
	10	5	1000	17	30	27	10	0.057181493
	20	5	1000	22	7	3	20	0.024727132
	30	5	1000	13	9	26	30	0.037090698
KSCI BBI	0	0	500	194	221	209	0	0.964358157
	10	0	500	212	209	218	10	0.987539844
	20	0	500	247	268	242	20	1.169902444
	30	0	500	227	201	199	30	0.968994494
	0	5	1000	48*	66*	50*	0
	10	5	1000	23	6	13	10	0.032454361
	20	5	1000	5	17		20	0.025499855
	30	5	1000	85*	11		30	0.025499855
Big BBI	0	0	500	184	202	204	0	0.911813001
	10	0	500	250	198	211	10	1.018448759
	20	0	500	256	236		20	1.140538974
	30	0	500	225	290	246	30	1.176084227
	0	5	1000	19	48*	11	0	0.03477253
	10	5	1000	22	15	28	10	0.050226987
	20	5	1000	18	10	23	20	0.039408867
	30	5	1000	50	21	28	30	0.076499565
KTI	0	0	500	247	267	250	0	1.180720564
	10	0	500	251	286	241	10	1.202356805
	20	0	500	222	236	232	20	1.066357578
	30	0	500	198	240	206	30	0.995267072
	0	5	1000	22	43*		0	0.05099971
	10	5	1000	21	79*		10	0.048681542
	20	5	1000	30	83*	33*	20	0.023181686
	30	5	1000	27	30	42	30	0.076499565
Scramble	0	0	500	218	227	243	0	1.063266686
	10	0	500	278	283	335	10	1.384719405
	20	0	500	271	284	252	20	1.247174732
	30	0	500	248	295	240	30	1.210084034
	0	5	1000	17	31	9	0	0.044045204
	10	5	1000	25	33	31	10	0.068772337
	20	5	1000	36	53		20	0.103158505
	30	5	1000	21	13	12	30	0.035545253
P-Tyrosine 9mer (S)	0	0	500	241	216	235	0	1.069448469
	10	0	500	180	226	176	10	0.899449435
	20	0	500	107	126	131	20	0.562542258
	30	0	500	107	108	123	30	0.522360668
	0	5	1000	10	14		0	0.027818024
	10	5	1000	13	19		10	0.037090698
	20	5	1000	51*	51*		20
	30	5	1000	37	8	12	30	0.044045204

>blank spaces indicates the plates were contaminated in the process of obtaining the data, as such data was not obtained and these plates were excluded from counting.

 

HCT116 Clonogenic 1.0 - Graphs

• Graph 1 is of plates seeded with just 500 cells and treated with just variuos peptides and no radiation to see if peptides inherently have toxicity to our cells. Data was created in excel with different treatments overlayed on top of one another. Error bars represent excel calculated standard error for those particular data points.
• Graph 2 is of plates seeded with 1000 cells and treated with various peptides as well as 5Gy radiation to see if our peptides confer radioprotection to our cells. Data was created in excel with different treatments overlayed on top of one another. Error bars represent excel calculated standard error for those particular data points. (note the scale difference between the two graphs)

July 18, 2016

HCT116 Clonogenics 1.0 - Discussion

• None of the peptides we intended to test showed significant radioprotection compared to out control with just DMSO and DTT (the solvent we dissolved our peptide in)
• BBI and KTI were interesting in that at 30μmol/L they seem to be conferring radioprotection to our cell-line compared to the control; but this relationship does not seem linear (no apparent linear correlation between application of the peptide and surviving fraction). Also cannot be sure of the significance of this observation due to the small increase that is apparent (being concious of the scale on the second graph)
• our scramble peptide seemed to increase the surviving fraction of irradiated cells <20μmol/L, but at 30μmol/L seemed to do nothing to help the surviving fraction of our cell-line. Only 2 datapoints were used in the calculation of the surviving fraction of cells treated with 20μmol/L scramble peptide so the error on this observation could be large--> requires more in-depth statistical analysis before conclusions.
• The Serine 9mer as well as the p(Y)9mer seemed to be killing our cells/making them unable to reproduce, suggesting toxicity against HCT116 cells or cancer cells in general; while the rest of our peptides were comparable to the control treatement in terms of toxicity (or lack thereof)
• KTI protein as well as our scramble peptide showed interesting results on cells that did not undergo irradiation, in that those peptides seem to increase the surviving fraction on our cell-line

 

Next steps: Trypan Blue Assay Protocol

• Discussed next steps with our mentors and decided to do a Trypan Blue survivability assay in the interest of time. Trypan Blue Assay should be able to give us some analyzable results in the time-frame of a week

• Grow, treat and irradiate the cell line as needed to test timing as well as different peptides (splitting cells without being concious of the number seeding)
• Aspirate the media and place it in an autoclaved 15mL falcon tube
• wash cells iwth DPBS and Trypsinize to lift cells off the plate, quench, then pipette the solution into the same 15mL falcon tube
• Mix well and stain with trypan blue stain.
• Quickly count total cells and live cell fraction with BioRad 500 cell counter. Record percentage of live cells and tabulate for analysis of survivability.

 

July 20, 2016

HCT116 Trypan Blue 1.1

• attempeted to count cells for trypan blue assay, but after trypsinizing for longer than usual (8min), the cells at the botom of the plate was unwilling to lift off the plate when viewed under a microscope. Seemed like there was a carpet of other smaller material around the cells that is trapping the cells to the bottom of the 60mm plate. Media was normal and was not cloudy (which is why we were unsure about what to think.
• Consulted this observation with our advisor, who announced that it was likely contamination. As a result all plates were discarded (all plates seemed to see the same situation) and the incubator was cleaned out as the water in the incubator was getting gross (which might have been the source of the contamination to begin with)
• Our HCT116 stock cells do not seem to be affected  

August 2, 2016

1BR3 Clonogenic 1.0 – Splitting protocol

• New Materials

• Media was composed of MEM (no extraneous nutrients), 15% FBS, 1% Penstrep, 2mM L-Glutamine (Stock is 200mM or 100x)
• used 0.25% trypsin
• used DPBS with no additives

1. Fibroblasts were looked at under the microscope to ensure over 70% confluency
2. Aseptic technique practiced to a tee (turn on hood 15 min before using, warm up media and trypsin in the 37c water bath, spray and wipe down everything with ethanol, ensure we have sterile/autoclaved 15mL falcon tubes)
3. wash 2 times with DPBS (making sure to pipette PBS on the side of the flask away from our cells)
4. add 3ml trypsin in each flask, incubate at 37c for ~5min
5. bump side of flask to ensure all cells are rounded and deadhered (check under the microscope)
6. add media to quench trypsin effects (around 6mL)
7. pipette media around the flask to "wash" down the surface (making sure declumped and deadhered)
8. remove all media and place in a 15mL falcon tube
9. centrifuge tubes at 2500rpm, 4c, for 3 minutes; make sure there is a pellet (should be visible for a T75 flask)
10. while the tubes are being centrifuged, fill new flasks with 14-18mL of media (T75 flasks)
11. remove the falcon tubes from centrifuge, aspirate the media
12. resuspend cells in 6mL media (for a 1:3 split) or 8mL media (1:4 split)
13. add 2mL into each new flask
14. tilt back and forth for an even spread
15. place back into incubator and do not touch the cells again (to not disturb) until you need to use them next

August 5, 2016

1BR3 Clonogenic 1.1

• Trypsinized/ Lifted a T75 flask of 1BR3 feeder fibroblasts(passage #: 12; 80% confluency) and added to 10mL of media
• Irradiated cells at 35 Gy of radiation (134Cs source, 85 Gy/min)
• Diluted cells to make 110mL of cell solution
• Seeded a layer of feeder cells on 108x60mm plates by pipetting 3mL of media and 1mL of seeder cells
• Placed in 37C, 5% CO2 overnight

August 6, 2016

1BR3 Clonogenic 1.1

• Counted 1BR3 cells from our T25 flask stock

• Well A: 5.98x105 (52% live); Well B: 6.41x105(48% live)

• Dilution and seeding of live 1BR3 cells to feeder plates

August 7, 2016

1BR3 Clonogenic 1.1

• Treatment with various peptides (big BBI, 9mer, p(Y) 9mer, KTI, DTT+DMSO, no treatment control)

• Peptides were added to a final concentration of 20uM

• Irradiation with 3 Gy of radiation according to original plan and seeding
• Placed in Placed in 37C, 5% CO2

August 11, 2016

1BR3 Clonogenic 1.1 – Contamination

• Found our stock flasks are contaminated
• Cleaned out our incubator and streaked reagents (media, DPBS, contaminated plate) on some LB plates to see what is causing the contamination
• Obtained more cells from Dr. Goodarzi – will be able to receive them in the near future

August 12, 2016

1BR3 Colonogenic 1.1 – Contamination results

• Non of our reagents were found to be contaminated through streaking on LB
• Thought about the possibility of our trypsin being contaminated but eliminated the possibility as trypsin is a protease and cell should not grow there
• Contamination most likely due to incubator not being clean, so we thermal cycled our original incubator, and in the meantime we have switched to using a different incubator to avoid further contamination

August 15, 2016

Big Team meeting

• Presented biotarget results to the whole team and all of our advisors
• Dr. Goodarzi, after seeing our presentation, gave us some suggestions on what to do going forward

• Suggested using lower Gy (ex. 0.25, 0.5, 1.0) to test for hyper-radiosensitivity that is observed at low Gy → more applicable to project as typically astronauts receive radiation in 0.25 Gy range (Short S.C., Woodcock M., Marples B., 2003. Joiner M.C. Effects of cell cycle phase on low-dose hyper-radiosensitivity International Journal of Radiation Biology, 79 (2) , pp. 99-105.)
• Suggested altering the concentration of peptide added to our H2AX assay to see the effects of altering concentration on DSB repair
• Suggested looking at more timepoints after radiation for H2AX assay to get a better trend of looking at when BBI works on our cells
• Will be providing more 1BR3 cells for us to work with; Interested in looking at a comparison of how our peptides work in cancer cells vs. primary fibroblasts

August 18,2016

1BR3 Clonogenic 1.2

• Trypsinized/ Lifted a T125 flask of 1BR3 feeder fibroblasts(passage #: 12; 80% confluency) and added to 10mL of media
• Irradiated cells at 35 Gy of radiation (134Cs source, 85 Gy/min)
• Diluted cells to make 225mL of cell solution
• Seeded a layer of feeder cells on 220x60mm plates by pipetting 3mL of media and 1mL of seeder cells
• Placed in 37C, 5% CO2 overnight

August 19, 2016

• Found a patent on a product that is very similar to ours with the inventors Minnie McMillan and Lynn E. Foster (Patent: http://www.google.ch/patents/US20150050250)

1BR3 Clonogenic 1.2

• Counted 1BR3 cells from our T25 flask stock

• Well A: 4.02x105 (100% live); Well B: 2.92x105(100% live)

• Dilution and seeding of live 1BR3 cells to feeder plates

• To make 115 mL cell concentration of 700 cells/mL for our 4 Gy radiation plates

Radiation (Gy)	Seeding concentration (Cells/mL)	Volume for Serial Dilution
		Vol from previous concentration (mL)	Vol Media (mL)	Total Volume (mL)
4	700			115
2	500	75	30	105
1	250	65	60	125
0.5	200	85	21	106
0.25	175	66	9	75
0	150	35	5	40

• Plates previously seeded with feeder layer (August 18) were plated 1mL of cells diluted to the specified concentrations in the table above according to intended radiation treatment
• Plates treated with various types of peptides (big BBI, 9mer, p(Y) 9mer, KTI, DTT+DMSO, control) (4-5PM)

August 20, 2016

1BR3 Clonogenic 1.2

• Cells irradiated with 137Cs source (8.5Gy/min) and designated Gy (0.25/1.7s, 0.5/3.5s, 1/7s, 2/14.1s, 4/28s) at 8AM (total incubation with peptides before radiation: 17hr)

August 22, 2016

1BR3 Clonogenic 1.2 - Contamination

• Plates for both the Clonogenic assay and the H2AX assay were found to be contaminated when looked at under the microscope (40x magnification); both assays are not able to continue
• Contaminated plates were disposed of; kept a few plates for reference to test the source of contamination

August 23, 2016

1BR3 Clonogenic 1.2 - Contamination

• Informed Nick Jette regarding contamination, he suggested we stop working out of the refrigerator as our chassis team, which may be the root of our contamination

• He will contact Dr. Goodarzi to see if we will be able to use some of his lab space so that we do not get any further contamination

• Look for the root of the contamination by plating various reagents on 60mm plates

• New unopened MEM (control plate)
• Old media (5mL)
• DPBS (1mL) + MEM (4mL)
• Trypsin (1mL) + MEM (4mL)
• MEM (5mL) + assortment of mBBI peptides
• MEM pipetted by Nick (to test technique)
• MEM pipetted by Nilesh (to test technique)
• MEM pipetted by Sid (to test technique)
• MEM left open in the incubator (to test bacterial growth in incubator

• Plates were left in the incubator (37C, 5% CO2) overnight

August 24, 2016

1BR3 Clonogenic 1.2 - Contamination Results

• No growth observed in plates containing new or old media, DPBS, media plated by all experimenters
• Bacterial growth found in plates containing trypsin (viewed under 100x)

• More cells (1 x T75 flask (50% confluent as of today)) will be obtained (we will most likely when our trypsin comes in
• Wendy (lab technician) donated 5 x T75 flasks as well as 60 x T25 flasks to us

September 2, 2016

1BR3 Clonogenic 2.0

• Trypsinized/ Lifted a T75 flask of 1BR3 feeder fibroblasts(passage #: 12; 80% confluency) and added to 10mL of media
• Irradiated cells at 35 Gy of radiation (134Cs source, 85 Gy/min)
• Diluted cells to make 110mL of cell solution
• Seeded a layer of feeder cells on 108x60mm plates by pipetting 3mL of media and 1mL of seeder cells
• Placed in 37C, 5% CO2 overnight

September 3, 2016

1BR3 Clonogenic 2.0

• Counted 1BR3 cells from our T25 flask stock

• Well A: 2.56x105 (93% live); Well B: 2.01x105(98% live)

• Dilution and seeding of live 1BR3 cells to feeder plates

• To make 115 mL cell concentration of 700 cells/mL for our 4 Gy radiation plates

Radiation (Gy)	Seeding concentration (Cells/mL)	Volume for Serial Dilution
		Vol from previous concentration (mL)	Vol Media (mL)	Total Volume (mL)
4	700			57
2	500	37	15	52
1	250	32	31	63
0.5	200	43	10	53
0.25	175	33	4	37
0	150	17	3	20

• Plates previously seeded with feeder layer (September 2) were plated 1mL of cells diluted to the specified concentrations in the table above according to intended radiation treatment (4-5 PM)

September 5, 2016

1BR3 Clonogenic 2.0

• Treatment with various peptides (big BBI, 9mer, p(Y) 9mer, KTI, DTT+DMSO, no treatment control)

• Peptides were added to a final concentration of 20uM
• Done at 7 AM

• Irradiation with various Gy of radiation according to original plan and seeding

• 1 PM (for total 6 hr. incubation with peptide)

• Placed in Placed in 37C, 5% CO2

Engagement

Click to learn more about each of our engagements.

Collaboration

Device’s NOTEBOOK

---

About Us

Address: UOFC_CALGARY IGEM 2016

UNIVERSITY OF CALGARY

HM393B - 3330 HOSPITAL DRIVE, NW

CALGARY, AB T2N 4N1

CANADA

CONTRIBUTORS:

Mathematical Modelling/Academics

Noshin Karim

Neliza Mendoza

David Nguyen

Manufacturing/External affairs

David Nguyen

Neliza Mendoza

Visual Modelling/Graphic Designer

Christine Phan

Tiffany Dang

Team Lead/Lab Technologist

Tiffany Dang

Administrative

Tiffany Dang

Noshin Karim

---

Considerations

What we have to determine:

Wednesday, May 4th

• How effectively diffusivity works
• Scaling:

• Model and prototype
• Measurements

• Layers:

• Kill switch in backing layer and bottom layer
• Break controller

• Know how long proteins would stay in the body

 

Friday, May 6th

• Habitation:

• Reservoir/chamber
• Control temperature
• Oxygen permeability
• Specific parts of the body (heat, placement)
• Delivery of nutrients - snapping mechanism?
• Modelling - in different conditions, see the growth rate

• Entire Device:

• Design
• Materials - contaminants
• Eg) rate controlling membranes, diffusion of peptides

• Interface:

• Needle - size, density, material, design
• Compatibility - initiation
• Site of application
• Disinfect prior to use, etc.

Friday, May 20th

• General

• How to apply to body?
• How to discard after use (i.e. when we take it out, won’t the media start to leak?)

• Microneedles/Drug Reservoir

• How to deliver drug through microneedle? (frozen, snap mechanism, isotonic?)
• Hollow needle location → side or straight down?
• What to test? (strain, stress, flow rate, diffusivity, etc.)
• How to construct everything on a small scale?
• Can we build a microneedle based on our own dimensions (i.e. can we customize the microneedle)? Or are the dimensions preset because we are ordering them in?
• Backing layer → is that included in the microneedle or do we need to attach it on?
• Actually getting microneedles
• What is in the drug reservoir? (bacteria, nutrients (LB = nutrients))
• Can we build on a microscopic scale? If so, how?
• What is the media that is in the microneedle?
• How do traditional microneedles work?
• Snapping mechanism

• Size-controlling membrane

• How big should the pores be? (0.1 micrometer or smaller… 0.1 = size before bacteria can go through) → what else other than peptides will go into the body? Will they be harmful?
• What material should it be made of? What else do we need to consider for the membrane (flexibility, etc.)?
• How do we get it? (3M)?
• Can we cut it to get it in a certain size?

Wednesday, May 25th

When researching materials, consider the following:

• cost
• environmentally friendly
• the “biological aspects” (e.g. gas/oxygen permeability, elasticity permeable)
• how can we get the material (local, international)
• think about how can we use the material to assemble the device (e.g. if we need silicon for the device for example, is it easily machined?)

Tuesday, May 31st

****The patch has to be transparent so that the indicator can be noticeable

Monday, June 6th

• How to make the patch long term

• Look at disposable
• Space the package would take
• Shelf life, how long would it take for the patch contents to "expire”

• Think about the worse scenarios that we could get

• Then we can start thinking about alternatives
• 100 mL vs. 10 mL
• Packets being unintentionally popped

• Actually have a plan, but it is also important to know how everything works out

---

Designs

Monday, May 9th

Design of Microneedles:

• Hollow cylindrical microneedle with conical tip
• Enough strength to withstand bending and axial forces
• Pressure uniform in main cavity of needle
• Velocity constant in cavity; increase in outlet
• Flow rate controlled by applied pressure and diameter of hole
• For different materials - strength and deformation compared

Design 1:

• The microneedle - from the machine shop
• Semipermeable membrane - use a filter (different grades of filter paper)
• Bacteria?
• A top layer (to the bacteria)

Design 2:

Part 1: MICRONEEDLE

• The microneedle - from machine shop
• Semipermeable membrane - use a filter (use different grades of filter paper)

Part 2: CAPSULE WITH BACTERIA

• Capsule with bacteria - the capsule can be put in the fridge; after, it would be inserted into the microneedle and then using a snapping mechanism/force to break the capsule which starts the process and peptides go through the semipermeable membrane

Tuesday, May 17th

Device Prototypes:

• Major idea #1: Bacteria, rich media (LB), membrane all in top of drug reservoir        

• Variations of Major idea #1

• Patch on top of microneedle array (patch is directly on top of patch)

• Drug reservoir right on top of needles (i.e. no patch) and size controlling membrane

• Problems: How can we actually put in things in this small drug reservoir?

• Drug reservoir right on top of needles – media is initially frozen and then melted after application to body due to body heat

• Problems: can’t make the media frozen because expensive (buy refrigerator)
• Frozen media will also expand like water, so when it melts it may not come into contact with spores

• 1 packet containing the bacteria in the drug reservoir → snap the packet to release bacteria spore

• Problems: How can we actually put spores in small packets and then put them in the small drug reservoir? How can we break those packets?

• Introduction of a gel (on a “lid”/backing layer that is lowered onto the top of the microneedle array/drug reservoir) OR rate controlling membrane (hence we have a rate and size controlling membrane)

• Problems: How do we put a lid on the top of the microneedle array? How do we stick the gel on the lid? If the gel falls, won’t it block the size controlling membrane? How do we put a rate controlling membrane in the small microneedle array?

• Major idea #2: Leave microneedle as it is; have larger patch that contains 2 chambers to separate LB and spores

• backing material (what the block is made of)
• “valve”
• size controlling membrane
• adhesive layer
• microneedles

Constraints:

• 70 degrees C to activate spores
• Oxygen transmission: available (80-100 cc/m2/24h)

Dr. Dalton’s Microneedles

• Problem 1: Geometry of microneedles

• TIP:

• Insertion force can be independent of the wall thickness
• Fracture force increases with increasing wall thickness and increases with wall angle, also independent of tip radius
• Side opened microneedles instead of standard tip microneedles – prevents tissue clog during insertion
• High needle density can increase fluid flow rate
• Sharper needles require less force for insertion, but has reduce needle tip strength
• Microneedle needs to be 10-15 um but shorter than 50-100 um to avoid pain
• Best option: small tip radius, large wall thickness

• BODY:

• Cylinder: eliminate stress concentrations, stronger needle structure, better self-adhesion

• Problem 2: Material for microneedles

• Single crystalline silicon: high resistance to bending, can be fragile
• Metal – greater strength, but thin metals are soft

• Problem 3: Application/removal

• Application: Elastic nature of skin creates possibility of non-uniform contact of the array with the skin - an issue for correctly metering dosages

• Has to find out which area of the skin where the least strain happens

• Removal: Leakage when microneedle patch is removed

• Development of microvalves within microneedles = passive system

Reference: Ashraf, M., Tayyaba, S., Nisar, A., Afzulpurkar, N., Bodhale, D., & Lomas, T. et al. (2010). Design, Fabrication and Analysis of Silicon Hollow Microneedles for Transdermal Drug Delivery System for Treatment of Hemodynamic Dysfunctions. Cardiovascular Engineering, 10(3), 91-108. http://dx.doi.org/10.1007/s10558-010-9100-5

Monday, May 9th

Future design testing:

• Test the proposed design with skin (animal skin?)
• Change diameter of hole - how much more different is the flow
• Heat of finger - enough to stimulate it?
• Diffusion works?
• Failure load (at what pressure does the needle break?)

STORAGE IDEA 1: Freeze drying

• Order in microneedles > put in freezer > at temperature of freeze dry

• If microneedles survive in the fridge, we should also test if diffusivity still works
• If diffusion did not work, we have to look at pumps

• Membrane/Filter test: does the drug reservoir/peptide actually go through the filter? Does it prevent the bacteria from going through?
• Diffusion test: To test whether diffusion will actually work and what other problems we will face with diffusion (e.g. bacteria secretes peptides which go through the semipermeable membrane… but what about the media that goes through the membrane?
• Making changes to the design

• Is the drug reservoir actually contained properly in the microneedle?

• Bacteria test

Monday, May 16th

Experiment to somehow test the idea:

Objective:

Tested the level of frozen H2O in microneedle as it melts into a cup of H2O (l)

Materials:

• 200 uL pipet tip
• Distilled H2O
• 200 uL pipetter
• Freezer
• 300 uL H2O
• Parafilm/tape
• Black sharpie
• Test tube rack

Procedure:

1. We used a pipet to take in 150 uL of distilled water in a pipet tip and taped the bottom with parafilm/tape. We marked the water level with a black sharpie.
2. Put the pipet tip in freezer and let the H2O freeze (10:50 am - after lunch).
3. Put pipet tip (just the tip area) into test tube of 300 uL (2x the volume of water in the needle). Cap the top with parafilm.
4. Overtime, see whether amount of H2O (l) goes back to original height.

Results/Observation:

After putting the frozen water in the distilled H2O (l) test tube (at normal tap temperature), we put parafilm on top to mimic the microneedle being capped.

• H2O melts extremely fast (this is without adding body heat) - melted within 1-2 minutes
• In actual microneedle, H2O (s) will melt very fast due to body heat. Also, silicon vs. plastic tube
• After melting we noticed H2O (l) level in needle is at a constant level (significantly lower than initial amount). Eg) doesn’t go back to original level
• H2O did expand, but not too significantly from initial observation, we think it won’t affect/damage the needle tip

Monday, May 9th

IDEA 2: Popping Idea

• Instead of pump, have a chamber with cells have a positive pressure already within it - have it sealed
• Capsule - look into the 2014 team
• Thinking about having water in the capsule, keep water in capsule, break it and everything gets hydrated
• Coating the microneedles with palmitic acid, we don’t need to create another compartment
• For medical uses, need only single administration, would we need the bacteria there? Could dissolving microneedles work?

Monday, June 6th

Tentative Materials for Our Patch

Part of the patch	Material	Pros	Cons
Backing Layer Purpose:   provides structural support and protects the middle adhesive layer from the environment	3M CoTran™ 9722 Backing Polyethylene Monolayer Film Alternative: (2nd best out of 3) 3M CoTran™ 9719 Backing Polyethylene Monolayer Film	Out of the three films 3M offers, the qualities that we thought would be beneficial are: Elongation = for movement of patient, has to 600% elongation, highest of the three MVTR = this has the lowest amount out of all 3 Translucent Breathable Printable Can be directly laminated to adhesives Heat Sealable (PE) Designed to resist excipient and drug uptake	We are not sure how much oxygen the bacteria would be using. We are not sure if 6400 cc/m2/day is enough for oxygen transmission. We do not know if this would turn cloudy after a period of time as currently existing patches with clear backing undergo the same issue.
Adhesive Layer Also note that thicker adhesive layer also results in severe cold flow during storage in the pouch, and higher affinity for lint and dirt to adhere to the edge of the patch during wear.	pdf of Duro-Tak Transdermal Adhesives
Release Liner	3M Scotchpak 1022 3M Scotchpak 9741 3M Scotchpak 9742 3M Scotchpak 9744 3M Scotchpak 9755	Good for release with silicon skin contact adhesives, acrylate, PIB and rubber based PSA Excellent chemical stability
Size-controlling membrane	tentative

Important Notes

Monday, June 20th  

• Emailed 3M for products, will talk to them tomorrow if they haven’t replied.

• Got a reply from 3M. Will need to call them later to ask for products.

• Emailed Dow Corning for adhesives
• Emailed Dr. Nezhad, an Electrical Engineering prof whose research is focused on microfluidics, for meeting
• Researched about different assays for material testing

Monday, June 27th

• Contacted 3M again, they are sending us the samples (YES). They should be here by the end of next week.
• Dow Corning will send the adhesives on 29th of June
• Today the device team focused on the math model for diffusion as weird numbers for the initial amount of peptides that needs to be produced have been calculated. Noshin found a paper with a MATLAB code that can be used to calculate the diffusion across a membrane using Fick’s second law of diffusion. An email was sent out to Dr. Nygren to get his opinion on the code and see if we are heading in the right direction
• UPDATE: We meet with Dr. Nygren Wednesday to go over the mathematical model developed. He suggested in his email that the equation is applicable in principle, but the situation is a little different because the membrane and skin surface are probably the main diffusion barriers and the diffusion constant is (very) different at those barriers compared to everywhere else. He also suggest we create a numerical method rather than doing it analytically.

Tuesday, June 28th

• The device group continued to work on both learning Solidworks to build the graphical model as well as the mathematical model of the diffusion of BBI through the patch, skin and into the bloodstream.
• From our results we calculated that we need 2 g of peptide being produced in the patch. This however is not feasible and needs to be reworked.

Wednesday, June 29th

• Protocols Tiff and Dave talked about with Dan

• Test how much media go through and if peptides go through; safety mechanism
• Can we make dye as a qualitative or quantitative aspect of the assay?
• How do we confirm peptides make it through?
• Critical: make sure cells stay where they are, and peptides go through

• There may be a big difference in terms of the diffusion constants depending on the volume, barriers, material
• Dr. Nygren’s suggestions

• DRAW
• Volumes separated by membranes
• Faster production = would reach steady state at some point as too much BBI might stop bacteria from producing
• Work equations out that he derived; make sure units are consistent
• Degradation - might be in the bloodstream, not before the skin
• Diffusion coefficients: testing would be necessary for peptide flow through membrane; for skin, could probably find from literature; or ask yourself, can we just find the diffusion coefficients rather than testing bunch of membranes?
• In our case diffusion is too fast that equilibrium is achievable.
• Make sure to state assumptions and a way to justify it.
• MATLAB: solving ODEs; recall ENGG 407 lecture

Tuesday, July 5th

• The rest of the team continued to work on the diffusion model based on Dr. Nygren’s suggestions. Based on Noshin’s work, there were too many unknowns that we could solve for if we decided to solve the equation as a function of time. Therefore we went back to Fick’s first law where the diffusion was a function of concentration over area. This however had unknowns such as protein solubility that we weren’t sure of
• Adhesive? Gave the green light that the assays are doable

• Backing Layer? Gave a similar opinion that the assays are doable
• Membrane? REASONABLE
• Quantifying about how much peptide go through
• Use of other peptides? (Since we want to quantify diffusion, maybe we could use something that is cheaper)
• TRICKIEST: finding how much went through
• Glucagon, oxytocin (20 - 30 amino acids long)

Wednesday, July 13th

• Contacted Dr. Nezhad about manufacturing patches, asked for resources.
• Contact Dr. Ingalls from Waterloo.
• Update: Waterloo is open for collaboration.
• In terms of modelling:

• Had our equations sent to Brian Ingalls for checking.

• Christine developed the patch using SolidWorks! See below:

Thursday, July 14th

• Meeting with Dr. Sundararaj (UT) next thursday the 21st at CCIT 320 at 1 pm for manufacturing.
• Finished the analytical diffusion model across stratum corneum. See Device folder >> Patch Modelling >> open the only document there >> See Stratum corneum under Layer by Layer heading.
• Tiffany finished her initial models for the bottom layer of the patch

• Optimized the dimensions to hold 10 mL in the main area, and either 0.5 and 1 mL of media in the pockets
• Sent the models into 3D printing services at TFDL <3
• Will hear back in a couple of business days about the progress

Friday, July 15th

Team Meeting

• Nelly has finished the first part of the analytical model for the stratum corneum. She will continue working on the next two parts of the analytical model for the next week
• Dave has been researching ways to manufacture to create the patch. For most industries including 3M they will only manufacture for large scale industry
• As a result Dave is focussing on how to manufacture our patch ourselves or with some professors
• Nelly will help out with Dave with this by researching into the adhesive and how to attach it
• Both Nelly and David have contacted professors for help in manufacturing the patch. We have a meeting with Dr. UT next Thursday at 1:00 pm with David, Nelly and Tiff
• If worse comes to worse and no one can help us, we are visiting the machine shop to see if anyone can help us
• Christine and Tiff have finished their prototypes. Tiff has sent hers to get 3D printed and will hear back in a couple of business days about the progress
• Christine and Tiff will start David’s assay next week and run triplicates throughout the week to see if the patch growth curves matches the ones under optimal conditions already done in the lab
• Discussed with Nishi what needs to be done for mouse trials

• There are three trials being run: 6 mice for a positive and negative control, 6 mice with patches with BBI dissolved in DMSO, and 6 mice with patches with cell culture
• The patch is 1 cm X 1 cm X 0.2 cm
• The patches need to be completed Monday prior the experiments begin the following Monday

• Alina also messaged back Tiff to see how things were going

• She will be escorting an astronaut from the Apollo 11 mission during a science fair and so we can send her questions we can ask the astronauts that will be there
• Tiff will create a document for questions to send to Alina. Focus the questions on either radiation in space or their lifestyles in space
• As well she said she will try to help us with our model. She said to message her as a group if we have questions about the diffusion model. Currently she is working on her own MATLAB project and she said could help us with this aspect specifically

 

Tuesday, July 19th

• This is what Nelly did for the whole day: Check this out. She tried to organize all the models she had on file into a document. Hopefully it is easier to navigate in this format. She also uploaded all the codes, functions, scripts etc. She uploaded it in a folder under Patch Modelling.
• Followed up with Dr. Nezhad because he has not responded for 6 days.
• Finished first draft of the powerpoint about our project. Check *presentation under *device for it.
• Tiffany talked to Dr. Mayi concerning optimizing the diffusion assay

• Instead of an eight hour interval of taking the sample, we will run the project over a seven day period, taking samples at 24 hour period to mimic the patch.
• As well, we are now measuring the the optical densities over a spectrum as no literature points to a wavelength for saline solution/distilled water

Wednesday, July 20th

• Tiffany continued working on the diffusion assay and got initial results to determine the wavelength needed to determine the optical density of saline solution

• The falcon tubes containing LB, B. subtiliis and E.coli were removed from the saline solution
• Saline solution was used as a blank
• Wavelengths of 260 nm, 300 nm, 600 nm, and 700 nm were used to measure the absorbance of the saline solutions contained in the erlenmeyer flask

Wednesday, July 27th

• Went to the machine shop to see if they can help us manufacture patches for prototype testing.

• Although they were not super clear about what we wanted initially they figured out the basics of what we needed from them
• They took care of 3D printing for free for us as we are a student based project and they resolved the issues for us
• Tiffany is in contact with them and will hear back from them in a couple of days
• In addition, we learned that the machine shop cannot manufacture the patch. What they do recommend is the easiest way would be to proceed with something similar to the thermoforming method

• About the thermofolding method

• Suggested we cast a mold that they can help us design using metal. The mold would consist of two parts for the backing layer and the semipermeable membrane
• We would then heat our materials and lay them onto the molds to conform to their shape. From there we place the molds together and inject our media into the mold and heat seal the entire patch together
• Unfortunately they can only help us with the mold. They don’t have equipment we could use

• We’re gonna talk to Dr. Mayi/Nygren before we proceed to see if this is a good idea and how we can proceed about thermoforming.
• We also need to determine experimentally at what temperature we get the two materials to seal together. This has to be determined ourselves
• Tiff will bring hair iron + blow dryer tomorrow to test at what temperature we can heat seal our materials

Thursday, July 28th

• The device team made prototypes today!

• We used the flat iron, iron, and blow dryer. Iron worked best. See pic below!

• Nelly worked on her adhesives. She tested BIO-PSA 7-4101 and 7-4301; however, both tests failed. What she did on her first test was she applied a thin film of adhesive on the release liner and let the heptane evaporate. She waited for 20 minutes, but the adhesive dried completely that it lost its adhesive properties. She tried the second time and she waited for heptane to evaporate for 5 minutes. The adhesives still contained heptane. She would run tests again tomorrow for 10 and 15 minutes.

Friday, July 29th

• Had our weekly meeting. See timeline for what we have to accomplish next week.
• David contacted 3M to ask them to create a crude prototype so we have a better idea what the patch should look like.
• Nelly did rounds of her adhesive testing assays. She found that the BIO-PSA 7-4201 will be the best adhesive for our current purposes. BIO PSA 7-4101 dries up quickly as when it was applied, it lost it adhesiveness. BIO PSA 7-4301, on the other hand, dries up very slowly, which also explained why it is less viscous than the other adhesives. This means it is dissolved in more heptane. In the pic below, Nelly was holding onto the release liner, the other layer was our membrane. The adhesive was successfully stuck to the membrane.

• Recommendations:

• Use of rolling pin for more uniform adhesive distribution.
• Wait 1.5 minutes for the adhesive to dry up once spread as film before attaching the membrane.
• Test on pork skin. Design assays for quantifying amounts of adhesive we need to apply, optimal temperature for drying, etc.
• More prototypes next week!

Tuesday, August 2nd

• David finished a detailed word doc outline of the presentation (with some help from Nelly and Tiff <3). Check under Presentation folder and edit anything you’d like. Now pass on to Christine to create powerpoint. Christine started a rough draft of our presentation.
• In the lab

• Tiff finished the last day of the diffusion assay
• Nelly and David performed some adhesive experiments. They experienced problems and they are as follow:

• The adhesive did not stick to the liner completely
• Uneven distribution of adhesive on liner

Wednesday, August 3rd

• Christine did the data analysis for the diffusion assay

• Nelly and David are testing adhesive. There were different methods used in doing these tests. They are as follow:

1. Rolling pin test

1. An amount of adhesive was applied to the release liner.
2. A cut out of EVA membrane was placed onto the adhesive.
3. Place another layer of the release liner on the membrane to prevent the adhesives from sticking to the rolling pin.
4. Roll the pin on the layer to distribute adhesive.
5. Let it dry.

1. Film application test

1. An amount of adhesive was applied to the release liner. The adhesive must be applied I a straight line.

1. Spread the adhesive using a popsicle stick to form a thin film.
2. Wait for a minute before placing a cut out of EVA membrane onto the release liner to dry

1. Two - release - liner - sandwiched - together test

An amount of adhesive was applied to the release liner. The adhesive must be applied in a spiral manner.

1. Place another layer of the release liner onto the initial liner.
2. Spread the adhesive using a rolling pin, by pressing, etc. until you see a clear film (meaning, no air bubbles, no accumulated glue anywhere, etc.)
3. Wait for 20 minutes to let the adhesives settle for a bit.
4. Peel the liners apart.
5. Place a cutout of the EVA membrane onto the liner.
6. Let it dry.

Results from the adhesive tests:

1. Rolling pin test: Opposite to what was expected, the adhesives were unevenly distributed. Air bubbles and bumps of glue were present. Some had not completely dried up, forming webs when peeled, some had.
2. Film application test: The adhesives were unevenly distributed.
3. Two - release - liner - sandwiched - together test: The adhesives were unevenly distributed, but this method was the most effective one. See pic below!

• Tiffany also picked up the 3D printed models of our prototype from the machine shop

Thursday, August 4th

Team had a meeting with Dr. Nygren

• Updates

• 3D printing

• Patch seemed to be a reasonable size according to Dr. Nygren

• Diffusion assays

• There were bacteria leakage that happened
• Back up plans, solving issues

• Meeting with Dr. UT

• Thermoforming

• Prototyping

• Maybe we could use wax paper or parchment paper. See what happens.
• Machine shop may not be able to help us since they will be closing soon for renovations. They would require us to send the SolidWorks model asap. They can’t do heat sealing for us, but they can make the mould.
• For the mould: instead of making 18 patches at a time, make it simple by making 1 at a time. Easier to manufacture, cheaper
• Adhesive: use of heat

• Manufacturing

• Materials for moulding

• Suggestions he could give us
• Moulding design he could provide

• One at a time

• Contacts for manufacturing

• Companies: nope
• Professors: nope

• Modelling

• Diffusion model Noshin prepared

• MATLAB code
• ODE45 will help us get numerical values instead of having a function as a solution
• C2 degradation
• Flux = moles/(m2. s); production rate = moles/s
• Start with assumed values for production rate and then move forward.

• Diffusion model Nelly prepared

• Will send the document to Dr. Nygren for checking

Friday, August 5th

• Mason completed a SolidWorks model of a previous microneedle prototype design
• Noshin and David continued to manufacture patches in the lab. Following Nelly’s procedure, the made patches that were 1 cm x 1 cm

        

Monday, August 8th

• Tiffany and Christine planned the week to perform the backing layer growth curves. These growth curves will be used to determine if cell growth is affected when gas exchange is limited

• The Plan

• Tuesday start the overnight cultures for the growth curves in the falcon tubes
• Wednesday at 4:00 pm, 12:00 and Thursday at 8:00 am start inoculation for the tubes
• Thursday start overnight cultures for the growth curves in the patch and perform growth curves for falcon tubes
• Perform growth curves in the patch

• Before starting the growth curves in the patch, soak the 3D printed models in 70% ethanol overnight

Tuesday, August 9th

• Tiffany started the overnight cultures for the growth curves in the falcon tubes
• Nelly and David made patch prototypes today. The procedures are as follows:

Materials:

• Strips of release liner ( 5cm x 13.2 cm )
• Strips of EVA membrane ( 5cm x 13.2 cm )
• Square cut outs of backing layer
• BIO PSA adhesive
• Cylindrical metal bar
• Masking tape
• Scissors, marker, ruler
• Flat plastic surface
• Iron
• Syringe + needle

Procedures:

1. Preparation of the adhesive layer

1. Have the materials ready. Perform the process under the fume hood as the adhesives contain heptane. Heptane is flammable and create vapor trails that may cause fire.
2. Note that the coated side of the liner is where the adhesive will be applied. In case you cannot figure which one is the right side, grab a marker and try to write on both sides of the liner. If the ink stayed permanently on the liner, you wrote on the uncoated side. The side where the ink just slipped through would be the coated side. It would be recommended to write which side is which to avoid confusion.
3. Draw a horizontal line on one end of the release liners (1cm from the end) with a marker. This end will be taped to keep the liner in place when the adhesive is being applied.
4. Draw three 3cm x 3cm squares on the release liners. Make sure to leave ample amount of space between the squares. Draw 1cm x 1cm squares inside the initial squares.
5. Take all the materials under the fume hood. Tape the end release liner on the flat plastic surface.
6. Apply the adhesive on the line initially drawn. Apply a constant pea-size amount on the line.
7. Using the metal bar, spread the adhesive onto the liner. Spreading using a metal bar will form a thin film of adhesive on the liner.
8. Wait for about a minute before placing the EVA on the layer.
9. Carefully place the EVA membrane strip on the adhesive layer.
10. Lightly tap the membrane to stick.
11. Wait for the adhesive to completely dry (1 hr - 3 hrs).
12. Cut out the squares.

B. Heat sealing the patch

1. Set the iron to the heat sealing temperature that was previously determined.
2. Place the backing layer on the prepared adhesive-membrane layer.
3. Carefully iron the sides of the layer. Avoid ironing parts of the 1cm x 1cm square  centre drawn. This is where the nutrient rich media and bacteria will be stored.

C. Adding the media in

1. Obtain the required amount/volume of media to be stored in the patch using a syringe.
2. Carefully inject the media into the middle compartments of the prepared patches by poking a hole on one of the corners of the drawn center squares.
3. Heat seal the holes created by the needles.  

***Check the whole document under https://docs.google.com/document/d/18uXLYuw3K3-0EAZaxPTyErA_iBWKPDveNloJpIzlsIU/edit 

Wednesday, August 10th

• The team went to the Foothills machine shop yesterday at around 2pm to consult with Peter Byrne. He was the person Dr. Nygren had referred during our last meeting. We had set the mould specifications for him to follow. He had also given us suggestions to further improve our mould. The mould will be ready for about a week. ($50 per hour of labour by the way ladies and gentlemen).

• The mould will be made with aluminum and brass that may stick to our layers.He then recommended us to get a Teflon coating spray or something the same.

• Nelly finished adding her Powerpoint slides for the presentation on Monday. She also picked up a Dupont Non-stick Lubricant (with Teflon) from Canadian Tire.
• David and Nelly made more adhesive layers before leaving the lab. Some had thicker adhesives applied, some had thinner layers. They will see how this quality would affect adhesion or if it has any effect at all.
• Nelly tried one of the adhesive layer on her wrist. It took her a while to completely stick the layer on because it had thin adhesive coating. It also required applied pressure for it stick better. She had the layer on for 4 hours.

• Result: The layer was somehow painful to remove because some of her hair were stuck on the adhesive. She said the pain felt like a degree lower than a wax sheet slowly being peeled off your skin. There was no redness or irritation that happened on her skin, but itchiness was felt when the layer was on.
• Recommendations: Disinfect the skin area before patch application.

• Christine continued working on the animation for Maya today and edited our presentation for August 15th.
• Tiffany conducted assays in the laboratory today
• Tiffany had also tested in the lab if the 3D models would hold the initially calculated volume of media.

• Results: The pockets exactly held 0.5 mL of liquid, while the content area held exactly 10 mL. Therefore, the 3D model is a good representation of our patch system in terms of holding capacity.

Thursday, August 11th

• Tiffany’s focus today was to conduct the growth curve assays every 40 minutes.
• Meeting with Dr, Nygren

• Potential questions here → Questions for Dr. Nygren document

• Meeting minutes:

• Update:

• Showed Dr. Nygren the full system prototype Nelly and David made
• Continuing assays by Tiffany and Christine
• Christine is working on her video
• Noshin is working on her math model
• Went to machine shop to get the thermoforming mold done

• Modelling:

• Dr. Nygren said Noshin is on the right track. He will email her to clarify some details.

• Prototyping:

• Dr. Nygren suggested talking to Dr. Jenne about the prototype and alter it to better suit the mice.
• Ask Dr. Mayi to buy the heat gun on our own

• Assays:

• Try to make a connection between the assays and math models. Answer the question of why we do the assay, why we have the models, what the connections are. Cohesion is key.

Friday, August 12th

• Christine and Tiffany worked on entering data values of their growth curve assays in a spreadsheet. They created time versus absorption plots.

• Results:

Tube 1

Tube 2

Tube 3

Average of Tube 1, 2 & 3

Monday, August 15th

• Presentation day! What we basically did for today was preparing for our presentation and did run throughs.
• After the presentation: dead. There were challenging questions thrown at us. This means we still have things to be solidified before we can say we’re done.
• We also asked our mentors for comments with regards to our presentation. They are as follows:

• Instead of having a slide with all equations on them, why not just use words that tells our audience what the terms represent.
• Maya animation. Changes to be made could be:

• Making the adhesive liner take up the whole bottom area of the patch.
• We’re not using the indicator system to signal low media, but tells if the bacteria is activated.
• After we have shown how the patch works, we can also extend the story by, say, an astronaut peels off the liner and apply patch, then wears this for this how many hours, and then through our modelling results we’ll figure how long before we pop a packet and then what would happen next and, you know, the story goes on yadi yadi yada.

• Our graphs must be able to speak for themselves, meaning that by the time we look at them, we know right away what it is trying to tell us.
• Explain all the terms that we are talking about.
• CONNECT ALL THINGS TOGETHER.
• Use constants that are from the other teams. The values we get from our model runs must correlate to the models and numbers Chassis and Biotarget get. Both Rai and Dr. Nygren brought this up.
• Our model should also have a flow.

Friday, August 19th

• David and Nelly made more adhesive laminates, preparing for more prototypes

Tuesday, August 23rd

• Tiffany continued doing her diffusion assays. The dialysis membranes came today as well and treated for testing. There were two types of tubing membranes that were used: 20 kD and 628 kD pore size membranes. However, these membranes are not heat sealable which would be a problem in manufacturing. There were other  limitations that were mentioned and these can be found in the email Nilesh sent/cc’d us in. Tiffany will be performing diffusion assays for these membranes tomorrow.

• Must be soaked in room temperature distilled water for 30 minutes instead of being boiled
• “Just soak them- once you have figured out how to glue them together” -- Dr. Volgo

• Nelly made around 80 adhesive laminates.

Wednesday, August 24th

• Tiffany performed her diffusion assays experiment using the dialysis tubing membranes.
• Noshin worked on modelling and sent the codes to Dr. Nygren for checking.

• She also asked Dr. Nygren if he could give us a contact who could help us with cost analysis for our patch.

Friday, August 26th

• Tiffany and Christine picked up the moulds from the Foothills machine shop.
• The results from dialysis membrane diffusion assays came out. There is still diffusion of bacteria through the membrane.
• The team also met with a PhD scholar, Xiaoan Li, whose research project focuses on water filtration using nanoporous membranes. Robert, a student who works in Li’s lab, is also in the meeting.

---

Changing points in our Project

Monday, May 30th

Meeting with Dr. Dalton:

• Contact 3M for hollow microneedles, look at patents
• If he can find his microneedles he will give it to us
• Would have to consider shear stress, what if microneedles break off and go into the body
• Utah array - solid microneedle
• Lab on a chip - chips and tips
• Perhaps make microneedle system separate with valve
• Issues:

• In microfluidic systems are bubbles - bubble will block channels
• LB chamber, what’s stopping the fluid from flowing directly out the microneedles? Perhaps use a mechanical system to control pressure, could also suck the media back in
• Diffusion would be way too slow or not even occur, would need an external syringe pump
• Leaving microneedles in for too long will cause immune response from body, wound infections
• Need to determine how much pressure the membrane can take
• Consider stress on microneedles, fact that skin is very mobile
• Skin elasticity, skin varies by thickness based on ethnicity and age, also have to consider the hairs on the skin

Wednesday, June 22nd

• Meeting with Dr. Amir Nezhad

• About Dr. Amir Nezhad’s Research

• Dr. Sanati-Nezhad' primary research interest involves BioMEMS, Microfluidics, Tissue Engineering, Micro and Nano Technology, and Lab-on-Chip.
• His research group has focus on development of integrated bioinspired microdevices using microfluidics and tissue engineering approaches for disease modeling, biological systems modeling, and drug discovery.
• Another research interest of his group is to develop point-of-care devices for testing infectious diseases, and portable tools for detection of plant and food pathogens.

• On meeting with him, we were invited to his lab where he showed us the process of micro fabricating the labs on the chips and how he uses microfluidics in order to do so
• The fabrication process is difficult. It requires a semi permeable membrane in the middle where the individual cells can be housed.
• Surrounding the permeable membrane is silicone mold to provide structure and handling.
• Although the chip looks simple, actual analysis requires a system of micropumps to transfer media, waste and byproducts across the system. This in turn is connected to a computer or microscope system for analysis.
• In order to work for cells for 30 days, the first 6 - 15 days is incubating the cells and ensuring they are in a happy media. After that, you can start researching on the cells
• He mentioned the importance of finding the optimal flow rate in the chip because anything above or below they flow, will disrupt the cells and kill them
• In concerns with our project

• He thinks the general idea of a patch system releasing various peptides into the body is very interesting and sees potential for collaboration with his lab
• When asked about companies to order from he suggested Dow Corning
• He stressed the importance of drafting up a prototype of our design in AutoCAD so he could better understand what our design will look like (not really? We asked him what he used for visual modelling and he said AutoCAD. However, he did stress making a powerpoint presentation to show people we consult to give them a better idea of what we are doing)

• Gave us access from his lab to purchase AutoCAD

• When asked about fabrication, he volunteered his services or that of the mechanical shop to create a prototype

Thursday, June 23rd

Meeting with Dan and Dr. Mayi

• Contacted Porex for EVA samples - waiting for response
• Also discussed with Dan about assays we can perform for both the semi permeable membrane and backing layer

• For the semi permeable membrane        

• Using a 15 mL falcon tube, we can fill it with overnight cultures and wrap the semi permeable membrane underneath the lid. We then invert it in a larger 50 mL falcon tube with water, salts and at the same pH as the blood and leave it to sit. After hourly intervals, we would take samples of the water and take the OD readings.
• Another assay that we can do is to use a brute force method and apply the same setup as the first assay. However we would centrifuge it down and see at the extremes how well our semi permeable membrane works.

• For the backing layer

• Dr. Mayi suggested creating a small patch with overnight cultures and leave it to sit in conditions similar to those found in the ISS. We take an OD reading initially and then let it sit for eight hours before taking another reading at the end and comparing the growth of our cells. We can also measure the amount of moisture vapour by measuring before and after the mass of the patch.
• Dan also suggested something similar to measure how well our cells grow. Using the small plates, we would aliquot some of our overnight cultures into two plates. We would then cover one with parafilm and the other with our backing layer. We then leave them to shake in the incubator. We take the initial OD and the final OD reading then of both to see how well our cells have grown

Thursday, July 21th

• David replied to 3M, they sent me back an email with useful info. I forwarded to everyone:

• Temperature and pressure depends on system:

• 250F and 40 psi for their small single well sealer - 1 second so the liner doesn’t warp.
• However, multiple well sealer needs 300F, 50 psi
• => May need a few trials to decide the parameters

• They can create a crude prototype to give us some idea of usability
• If making it ourselves, they recommend a sequence of steps:

• coat adhesive onto the liner, laminate the membrane to the liner through a low pressure nip roller and lastly heat seal the backing to the lamination (on the membrane side) by placing the materials to be sealed onto a well type receiving fixture with silicone sealing gaskets and using a platen type seal plate above.

• Our membranes are the most permeable. The main difference is the thickness between the 2. The thicker one (9716) will have a slower transmission rate. If nothing works, they’ll help us pick out another one.

• Meeting with Dr. UT Sundararaj about manufacturing:

• He went over the project with us and said gel media might be safer when being punctured but diffusion might be compromised. However, he said at equilibrium everything should be the same.
• He approved our materials for the patch. He told us to consider the materials’ solubility parameters to make sure they are compatible and nothing will dissolve into each other when heated. EVA and polyethylene are fine. We might want to check hexane/heptane in the silicone adhesive and EVA though, but it should be fine.

• http://cool.conservation-us.org/byauth/burke/solpar/solpar2.html If delta is >+-2 it should be okay.

• He recommended using thermoforming for our mouse patches. The general idea is to make a wood “mold”, heat our backing layer up, put it on, vacuum so it forms a reservoir, pour the media in, put the membrane on top, and then heat seal everything together. A flat iron for hair or something might work. It’s also a good idea to apply heat on both sides.

• Thus, we need to get in touch with the machine shop ASAP

http://www.plasticsmag.com/thermoforming.asp?fIssue=Mar/Apr-03 some reference we pulled out from google today about thermoforming.

Friday, July 22nd

• Dow Corning (finally!) responded to Nelly about the preparation of adhesives.

• Basically, the actual adhesives are dissolved in heptane. This is the reason why handling it without precaution might cause irritation. The adhesive in its pure form is safe to use.
• Easy enough, we just have to let the heptane evaporate so we can use the adhesives. The evaporation rate will depend on the temperature of the environment. In some patent documents she found online, companies dried the same type of adhesive in 100o C oven for 2-3 minutes.
• Note, we must do any adhesive assays under the fume hood.

Wednesday, August 17th

• Meeting with Dr. Jenne (August 17, 2016)
• Mouse orders done on Wed the 24th potentially
• Might take some time to do training, he’s concerned about the timeline since he technically can’t order mice until he got ethics done
• He thinks that the biggest thing for us is mass spectrometry
• Problems we might encounter:

• Peptide not being found in the blood
• If it's found in the blood, what if it’s absorbed by other cells or organs

• The most important thing is what we get out of it, what the results tell us, and our future solutions for it. It’s okay if it fails.
• We have to change the dimensions of our patch to make it narrower and longer.

Monday, August 22nd

• Modelling meeting with Dan

• In our modelling story, this experiment would be in the beginning of the story. This would be use which peptide we are going to use. Once our peptide has gone into the bloodstream, this model would help us determine how fast our peptides would diffuse through the cells to do its functions.
• Different forms of our peptide

• How it would go through our membrane
• Looking at the energy levels

• Applying force to membrane through a bilayer
• Very hydrophobic = low energy in the middle
• Calculate energy

• Size of the system

• Peptides: we want to model the different forms of our peptide and see which diffuse the fastest
• 9-residue monomer - linear (reduced form) and cyclic (oxidized form, ring, forming a disulfide bond = might affect diffusion, should it be reduced or not)
• TD1 tag: compare that to BBI (combination of the 2 may have a better effect)
• KSCI + monomer + F at the end

• Having a negative control

• Like a sodium ion (+ve)
• Hormone that readily diffuses through

• Another important part: bilayer

• Simulate the actual membrane

• One type of human bilayer = have a smoother curve
• Or a hydrocarbon bilayer/disc
• Talk to them if we could have a simple bilayer with true phospholipid
• Explicit water on the outside
• Size of the environment (10nm cube, etc.)

• Ask if there is data already existing

Friday, August 26th

• The team also met with a PhD scholar, Xiaoan Li, whose research project focuses on water filtration using nanoporous membranes. Robert, a student who works in Li’s lab, is also in the meeting. He was a previous member of the winning iGEM team in 201_. Here’s what we talked about:

• Ask Dr. Mayi or Deirdre about other membranes that we could test

• Examples

• centricon: small scale version, 5kD (ask Goodarzi’s godmother): not as big, small scale purification, good for centrifuge works
• filter sterilization membranes (0.22 micron)

• Watch 2011 NASA - video
• figure out what carbon nanotubes we need, they got some. They have PPL, carbon nanotubes. Once we’ve tried everything and still fail, then we should ask them.
• Other ideas

• dissolving O2 on the actual membrane
• We need less than 200 nm of pore sizes
• SEM, AFM: contact Matthias Amrein - does lung cells; Michael (runs SEM)

• use something conductive, Teflon is not conductive
• thin sheet of gold

• figure out what materials we would need at the moment

---

Providers

Companies involved:

3M

• Provided us with all the sample materials used in the team’s assays
• The following materials are as follows:

• 3M CoTran™ 9722 Backing Polyethylene Monolayer Film
• 3M CoTran™ 9719 Backing Polyethylene Monolayer Film
•  3M CoTran™ 9716 Ethylene Vinyl Acetate Membrane
• 3M CoTran™ 9728 Ethylene Vinyl Acetate Membrane
• 3M Scotchpak™ 1022 Release Liner Fluoropolymer Coated Polyester Film

Dow Corning

• Provides us with the adhesive samples used in our patches.
• The following materials are as follows:

• Dow Corning BIO-PSA Silicone-based Adhesive 7-4101
• Dow Corning BIO-PSA Silicone-based Adhesive 7-4201
• Dow Corning BIO-PSA Silicone-based Adhesive 7-4301

---

Collaborations

Thursday, July 7th

• The team continued working on the diffusion model by either researching the equations further to see if there is another perspective we can take on the model or writing a code that can be used in MATLAB
• Nelly was able to get a graph of the result of her MATLAB code [Insert image or comments here about the result Nelly!]
• Meeting with Waterloo

• The modelling team met with the UWaterloo Modelling leads to discuss a possibility for collaboration
• They are working on a project that uses yeast to take advantage of the higher readthrough rates during prion response
• There is not very much commonality between the two projects but one potential would be a collaboration on their protein aggregation model as it would involve transcription rates
• However after discussing, it may not be worth the collaboration between us and Waterloo

iGEM Collaboration UCalgary & UWaterloo MM

07 JULY 2016 / 4:00 PM

ATTENDEES

• Zoë Humphries, Emily Watson, Tiffany Dang, Nelly Mendoza, David Nguyen, Sid Goutam and Nilesh Sharma

BRIEF INTRO

UWaterloo Project Summary

• Creating a system that takes advantage of higher readthrough rates during [PSI+] state of prion response
• Model of iGEM collaboration network from 2015
• Model of plasmid retention - metabolic load of expressing our fusion protein
• Model of protein aggregation - how the prions are distributed through generations

UCalgary Project Summary

• Working a transdermal delivery system to deliver peptides to the body
• Specifically working on the delivery of the Bowman Birk Inhibitor which as shown radioprotective effects
• Modelling the diffusion of BBI into the body to determine the initial concentration needed to be produced
• Model of the required transcription rates to produce the required amount of BBI or what a reasonable amount of BBI can be expected

MORE DETAIL ABOUT WATERLOO MODEL

• Gene Retention Model
• Determine number of copies based on fluorescent protein in plasmids
• Retention is dependent on metabolic load, stability of plasmid,
• Quantify using fluorimetry
• Protein Aggregation Model
• Model how long it takes for aggregation to form (help lab subteam)
• No differential equations as of yet
• Probabilistics important for chance of inheritance in daughter cells
• Quantify aggregation via Western blots
• System should break down prions, will model this disassembly
• Quantify breakdown via GFP (sup35 prion protein begins functioning again)

MORE DETAIL ABOUT CALGARY  MODEL

• Diffusion Model
• Model how long it takes for the diffusion of BBI to reach steady state
• Compartmentalized the diffusion system into three components: the patch to the skin to the blood stream
• No differentials equations at this point but trying to use Fick’s Law in MATLAB to see if we can analytically solve for the steady state
• Also using this model to determine the initial concentration needed in the patch for the desired amount of BBI to be found in the blood stream
• Transcription Rate Model
• Model to determine how fast the peptides are being produced inside the patch and how varying transcription rates will create the necessary concentration of peptide needed in the patch

ACTUAL ITEMS FOR COLLABORATION

UCalgary Assistance

• Help starting with the transcription rate models to determine how fast the peptides are being produced and what is a reasonable initial concentration based on these rates
• Mentoring for the diffusion model. This would be in the form of bouncing questions with Waterloo to see if we are on the right track and if there are any considerations we need to take
• With the gene retention model, UCalgary can help by collaborating on the transcription rates specifically

UWaterloo Assistance

• Gene retention subgroup most likely to benefit from assistance
• Optimising expression of plasmid number over gene copy number in plasmid
• Modelling expression of gene, how quickly the plasmid is lost during normal and prion states ([PSI+] condition)
• Dr. Brian Ingalls works on gene retention in bacteria, is a great resource
• Transcription rates would benefit gene retention and protein aggregation

ACTION ITEMS

• Use Google Drive to maintain collaboration docs
• UWaterloo will send update e-mail to math subteam & CC UCalgary

---

Manufacturing

Wednesday, August 24th

• Dave and Nel worked on manufacturing today. David had figure out a way to properly make our mice testing patches. The protocol is as follows:

        Procedure:

1. Prepare the adhesive laminates following Nelly’s procedure
2. Plug the iron in to preheat.
3. Look through the adhesive laminate under a light source and pick out and most even area. Sketch the 1 x 1 cm square on the liner at the center of the area
4. Trim the laminate to roughly 1.5 x 1.5 cm
5. Lay the laminate membrane-side up on the iron board, and the backing layer on top
6. Iron 1 side of the patch up to the sketch. To make the seal even and bubble free, when sealing, apply more pressure on the side of the iron that’s touching the line, then tilt the iron so that side of the iron lifts up slightly, then press it down again, after each time, move the iron away from the line to the edge of the patch. The seal should look clearer than the center. If not, then the iron is not hot enough, redo using a different side of the iron until it’s clear
7. Seal the other 3 edges. Now the patch should be stuck to the iron board, leave it there. Repeat the process for another 2 patches.
8. Remove the first patch from the iron board. Prevent peeling the patch off the liner by sliding a finger under the patch after you peel off 1 corner when you reach the liner.
9. Hold the patch diagonally, vertically, backing-side facing you. Bend it slightly so the backing folds towards you. The backing at the center (not sealed) should bend and separate from the membrane, creating a thin pocket running diagonally from the top corner to the bottom corner.
10. Fill the syringe with water, use the needle to poke a hole at the top corner of the patch, make sure the needle did not poke anywhere else and the needle is in deeper than half of the patch.
11. Inject the water slowly, keep a bend in the patch so we have a pocket, fill it from the bottom corner, tilt the needle and the patch to get the other 2 corners, then fill it nearly to the top corner. A good volume should be 0.06 - 0.08 mL
12. Remove the needle straight out, a drop of water may leak out, dry it lightly with paper towel, don’t push on it
13. Lay it on the iron board. Make sure there’s no water droplet visible. If there is, dry it gently with a paper towel, then iron on the hole for about 1-2 seconds
14. Press on the patch slightly to see if there’s still any leaks, if yes, dry the droplet of water with paper towel and re-iron the corner until there’s no more leaks.
15. Now the patch is done, trim the sealed sides with scissors to approximately half of the original side dimensions. Round off the sharp corners
16. Cut a bandage in half, horizontally, then cut it vertically to create 2 small rectangles, with the sides approximately to be twice of the sealed sides of the patch
17. To put the patch on a finger, remove the liner, put it on and press all edges lightly but firmly until all edges stay down
18. To reinforce the patch, put the 2 bandage pieces on both sides that go down to the sides of the finger

*Note: A patch with “YES” written on it is on the iron board. It should show you the size of the patch, how clear the seals get, and how I seal the hole. You can ask Tiffany about the process of applying since she had 5 on her fingers today.

*Note 2: This is a pretty reliable method. The seal works great most of the time. Poking more than 1 hole in the patch happens once in awhile. The patch stays on well most of the time. The water will leak when you put on if the seal is not good enough, or if there’s another hole you accidentally poked.

Wednesday, August 31st

• David made some more mice patches for Thursday

Thursday, September 1st

• David put 3 patches of different shapes (square, rectangle, and triangle) on the mice in Dr. Jenne’s lab and reinforced them with tissue glue.

Friday, September 2nd

• All 3 patches from yesterday survived. The rectangle and triangle worked better than the square. Rachelle from Dr. Jenne’s lab said the rectangle looked best.

Tuesday, September 5th

• David created 28 patches for the mouse testing with 9 containing water, 9 containing bacteria and 10 containing pure BBI

 

---

Research

Papers:

http://www.ncbi.nlm.nih.gov/pubmed/21139238

• Hydrophilic large molecular compounds
• Via hollow microneedles

• Loaded into lower epidermis and superficial dermis

• Release rates (Fick’s Law of Diffusion)

• Increase volume of FD-4 injected - faster the FD-4 release rate from skin
• Release rate increases when FD-4 given in multiple injections
• Large molecule = lower release rate from skin

• Silicon - useful material?
• Questions:

• So is the drug being released secreted out of the skin after secretion?

http://www.ncbi.nlm.nih.gov/pubmed/22575858

• Sections 2.4, 3.1,2
• Pressure gradient - greater pressure causes drug to flow through
• Diffusion gradient
• Questions:

• Based on methods of delivery - silicon; have to test and make sure peptide/fluid/bacteria doesn’t react with silicon
• Simulate a large scale prototype of design to test if we can use pressure/diffusion to get the peptide to flow through

• Problem:

• For microneedles that use multiple needles (our suggested form):

• 1 microneedle leaks
• Pressure can’t be equally applied to all needles
• Fluid won’t flow through all microneedles equally

• The small size of microneedles - drugs given on/within microneedles limited to microgram dose

• Applications of microneedles

• Biotherapeutics, drugs (peptides, proteins, DNA,RNA)

• Hollow microneedles - used for insulin delivery in both rats and humans
• Diffusion and active infusion worked, decreased blood-glucose levels after injection
• Humans found it to be:

• Decreased pain
• More preferred (compared to normal needles)
• Increased insulin pharmacokinetics almost 2-fold - may cause better control over post plasma glucose levels
• Had faster insulin absorption and enabled more rapid onset and offset metabolic effect on blood glucose levels than injection

https://www.researchgate.net/publication/228562173_Side-Opening_Hollow_Microneedles_for_Transdermal_Drug_Delivery

• Proposed design - measurements for diameter length of needle, etc.
• pump/syringe for injection

• Need to test diffusion; if diffusion doesn’t work, use hand to press

• Overdose problem

• Reservoir patches give tighter control of delivery rates but can have an initial burst of drug release. If the membrane is damaged, there is also a risk of sudden release of drug into the skin and overdose as potentially a larger area of skin is exposed for drug absorption.
• In a matrix patch, the active ingredient is distributed evenly throughout the patch. One-half of a patch will have half the original surface area and deliver half the original dose per hour. The matrix patch carries less risk of accidental overdose and offers less potential for abuse than the reservoir system.

http://ieeexplore.ieee.org/stamp/stamp.jsp?tp=&arnumber=659727

• The principle of operation is based on the pressure barrier that develops when cross section of capillaries change abruptly in neck and expansion regions. This type of gating device in its normal state can only stop flow, but it can be electrically triggered to re-establish flow when used in combination with two electrodes.
• Mechanics: The capillary stop consists of a region of the tube that is necked down followed by a sharp enlargement. When the liquid as first introduce in the reservoir, it wicks in the necked region and abruptly stops at the neck of the outer edge preventing any outer flow. The pressure barrier provided by the stop can be overcome by external pressure to re-establish a flow.

---

Material Research

Tuesday, May 31st

http://images.alfresco.advanstar.com/alfresco_images/pharma/2014/08/22/ba2e9668-a586-4daf-9179-70f220be6e56/article-18600.pdf

PSA = Pressure Sensitive Adhesives

• Materials that adhere to skin with application of light pressure and do not leave residue upon removal
• See paper for materials used

• Acrylic-, polyisobutylene- & silicone based adhesives commonly used in transdermal patches

• Increasing the polymer content provides a softer and tackier adhesive, whereas higher resin levels result in lower tack but higher adhesion and resistance to cold flow.
• Release liner is peeled off = also has the adhesive properties

Idea to prevent water from leaking out:

• Adding some sort of liquid in pores that will let peptide through and not let media mix with it
• Gel-like fluid for media

Design of patches:

• Polymer membrane partition-controlled TDD systems

• There is a constant release as long as concentration is maintained, but release rapidly decline when device approaches exhaustion
• 

• Reservoir system

• Same concept

• Drug in adhesives

• I don’t think this is applicable for us, but would put it here just for reference

• Micro reservoir type

• Suspension of drug with aqueous solution of water-soluble liquid soluble polymer
• Homogenous dispersion of drug suspension in a lipophilic polymer (silicone elastomer)
• Example: nitroglycerin patches

Competitors:

• TEPI Patch

• Articles:

• http://futurerxdream.com.ng/ibuprofen-patch-heralds-side-effect-free-drug-future/
• http://www.painnewsnetwork.org/stories/2015/12/9/new-skin-patch-delivers-pain-relief-with-ibuprofen

• 24 hour medication
• Constant drug delivery for 24 hours
• The drug is dissolved into the adhesive layer which helped it to release the drug in a steady rate and to take up more drug
• Will be used for pain medication, so this will be applied to the SPECIFIC area where pain is felt
• Will be out in the market for 3 years

Wednesday, June 1st

Adhesive layer research:

• https://geckskin.umass.edu/

• Super-adhesive based on mechanics of gecko feet
• Leaves no residue
• Not sure if we can get our hands on this though…

Friday, June 3rd

http://www.tandfonline.com/doi/pdf/10.1517/17425247.2012.637107

• PSA’s fall into three categories: solvent based, water based and hot-melt

• Solvent based are traditionally used in patch production

• o   Water based and hot-melt are more beneficial for skin irritation, sensitization and environmental contamination risks
• o   Polyisobutylenes are better for allergenicity compared to acrylics and silicone-based
• Patch failures:
• o   Case I: Adhesive failure
• o   Case II: PSA doesn’t adhere to backing layer

o   Case III: matrix has good adhesive strength, poor cohesive strength

o   Case IV: adhesive and cohesive failure

• PIB-based adhesives:

• Disadvantage: easy oxidation and low air and water vapour permeability

• Acrylic-based adhesives:

• Colorless and transparent
• More resistant to oxidation

• Silicon-based adhesives
• When testing in vivo performance:

• Need to find an artificial material that is able to simulate continuous variations of skin humidity - related to critical surface tension, surface roughness and deformability
• Skin deformability is most critical to consider
• Effects of relative adherend humidity on peel adhesion performances can be studied using collagen-coated plates
• When peeling off a patch, need to consider tensile deformation, bending stiffness and substrate deformation
• Stress distribution on skin deformation was measured in vivo by tension, torsion, suction and indentation tests

Some Market Research:

Paper:  Challenges and opportunities in dermal/transdermal delivery

From <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2995530/>

PATCHES that is applicable in our subgroup:

• Clonidine patches

• Catapres TTS® was introduced in 1984 for high blood pressure as the first 7-day patch system
• "The patch should stay in place during showering, bathing, or swimming for a full 7 days." From <http://www.mayoclinic.org/drugs-supplements/clonidine-transdermal-route/proper-use/drg-20073656>
• Clonidine is in a class of medications called centrally acting alpha-agonist hypotensive agents. It works by decreasing your heart rate and relaxing the blood vessels so that blood can flow more easily through the body. From <https://www.nlm.nih.gov/medlineplus/druginfo/meds/a608049.html#precautions>
• How did they solve the problem with loss of adhesion? "If the clonidine patch loosens while wearing it, apply the adhesive cover that comes with the patch. The adhesive cover will help to keep the clonidine patch on until it is time for the patch to be replaced. If the clonidine patch significantly loosens or falls off, replace it with a new one in a different area. Replace the new patch on your next scheduled patch change day."

From <https://www.nlm.nih.gov/medlineplus/druginfo/meds/a608049.html#precautions>

• Climara patches

• Treating conditions due to menopause (eg, hot flashes; vaginal itching, burning, or dryness), treating vulvar and vaginal atrophy, and preventing osteoporosis. It is also used for estrogen replacement therapy after failure of the ovaries and to relieve symptoms of breast cancer. From <http://www.drugs.com/cdi/climara-weekly-patch.html>
• Method of application:  A new patch should be applied to your skin on the same day once a week (i.e., the patch should be changed once every 7 days)

From <http://chealth.canoe.com/Drug/GetDrug/Climara>

Tuesday, June 7th

isosorbide dinitrate (ISDN) Patch

http://www.scielo.br/pdf/bjps/v51n2/1984-8250-bjps-51-02-00373.pdf

***They are dealing with a drug called isosorbide dinitrate (ISDN)  which is used as  vasodilator for angina, congestive heart failure, and esophageal spasms.

What they are trying to create:

• Acrylate polymers = they used this as the rate controlling membrane, as this polymer has not been extensively used in patches before

• Why use this? keep drug release for at least 48 hours at constant rate

Here's how their patch looks like: poor mouse :’(

• Patch was stored in a sealed aluminium pouch =  minimise the loss of solvent

Part of the Patch	Brand or what material was used	Why was it chosen
Backing layer		Same material as what is existing	Temporary liner and the backing layer were then heat-sealed and cut to the appropriate sizes
Temporary Liner	3M, Scotchpak 1022	Packaging purposes, protects adhesive	Adhesive solution was coated onto the temporary liner (3M, Scotchpak 1022), allowed to dry completely
Rate controlling membrane	Polyacrylate membrane (made from scratch), synthesised by 2-hydroxy-3-phenoxypropylacrylate, 4-hydroxybutyl acrylate and diethyl maleate	To keep drug release for at least 48 hours at constant rate Better permeation, does not involve degradation, erosion or dissolution of the polymer	These are non-degradable polymers Thickness: 14 microns Pore sizes are fabricated randomly by polymer chains
Reservoir layer	75% PVA, 10% ISDN and 5% urea	Showed better permeation Permeation rate is increased by 25.4 fold, a 31.1-fold cumulative release after 24 h, and a 30.8-fold cumulative ratio of release at 24 h compared to EC	Also has the penetration enhancers
Adhesive layer	PSA (pressure sensitive adhesives): 5% ISDN dispersed in the mixture of PVP K90, PEG400 and gelatine	This whole combination helps enhance drug permeation
THEIR CONCLUSION	This presented a longer release time at a sustained release rate Would promote patient satisfaction Sustained release rate, owing to the rate-controlling membrane, A higher loading-drug amount, owing to the separated drug reservoir layer Easier to tune release rate and release time to achieve the prediction

Patch 2:

Contraceptive patch http://www.google.com/patents/WO2013112806A2?cl=en

Dosage: 20 mg per day, $14 for a full month of medication

Pros about this patch	Cons about this patch
Entire patch is flexible enough to effectively and comfortably adhere to contoured sites of the body Used destrogel mixed with carriers
Part of the patch	Material used	Why did they use it
Backing layer
Release liner
Adhesives	Duro Tak® 87-4098 by Henkel Corporation., Bridgewater, N.J. Comprises a certain percentage of vinyl acetate co-monomer PIB adhesives such as 0.1 to 30 wt% PVP (i.e., povidone) or a PVP co-polymer such as PVP/VA (i.e., copovidone) as a humectant and plasticizer	PVPs are very hydrophilic as compared to PIBs, which are hydrophobic, has an ability to absorb moisture. The use of PVP copolymers, such as PVP/VA, can improve compatibility with other polymers and modulate the water absorption.

Recommendations:	WHY
use of water soluble polymers is generally less preferred	Would cause dissolution or erosion of the matrix Would affect the release rate of the desogestrel Would affect capability of the dosage unit to remain in place on the skin
Incorporate cross-linking monomeric units or sites	Would solve problems with polymers having glass transition temperatures below room temperature which are used to form a polymer matrix as the transdermal desogestrel-containing composition
In development of suitable polyisobutylene PSAs, one consideration is that PIBs are not crosslinked so they flow slightly.	Within a patch, that slight flow can cause an unsightly ring around the patch when it is worn for several days.
A higher content of high molecular weight PIB in the PSA formulation. **Polybutene in certain PIB formulations, such as the Oppanol B-12 functions as a plasticizer to allow for incorporation of more high molecular weight PIB. Mineral oil can be used as a plasticizer for the same purpose.

Wednesday, June 8th

http://www.technicaljournalsonline.com/ijpsr/VOL%20II/IJPSR%20VOL%20II%20ISSUE%20I%20JANUARY%20MARCH%202011/IJPSR%20VOL%20II%20ISSUE%20I%20Article%2019.pdf

Polymer requirements:

• Biocompatible + Chemically compatible -> with both drug and body

Different companies use different polymer systems:

• Alza Corporation: EVA (Ethylene Vinyl Acetate) or microporous polypropylene
• Searle Pharmacia: Silicone rubber

Backing Layer: Most common is Polyester-polyethylene composite

Rate controlling membrane: 

• EVA: The percentage of VA can be manipulated. Higher VA -> higher permeability and higher polarity. Maximum VA is 60% by weight (or else glass transition temperature will increase)
•  Silicone rubber: Biocompatible, ease of fabrication, high permeability (especially steroids), free rotation around silicone rubber backbone -> Low microscopic viscosity within polymer

        Adhesive: (also referred to as PSA) We talked about PIB today

        Release liners: Fluoropolymers

Monday, June 13th  

Media Release Material

Water Soluble Materials

• Aquasol uses a mixture of sodium carboxy cellulose and wooden pulp to create the water soluble material
• Aquasol specifically designs their packaging to biodegrade over time or with the introduction of water at any various temperature
• Monosol is another company which specializes in the use of water soluble packaging and dispersible materials. The water soluble film is made from PVOH (Poly Vinyl Alcohol)
• Like Aquasol, the material is water soluble at all temperatures. At higher temperatures, the material is more soluble. That is why they suggest moderate temperatures of 10-20 degrees celsius with relative humidity of 30-60%
• With the material being quickly degraded, water soluble materials are not the best materials to use for the media release. The material however does not harm the environment as the bacteria naturally found in wastewater can break it down into harmless components
• As well, there are no specifics about their material as you have to customize it to your needs

Polyethylene and Bubble Wrap

• An alternative would be to use a single layer of polyethylene used for bubble wrap. By filling the media bubble with media and air, it’ll form a pouch that can be popped.
• Polycell makes a bubble wrap called Oxo-B Eco Bubble which incorporates their Reverte Oxo Biodegradable into their polyethylene resins. After discard, through the use of substantial UV light, oxygen and/or heat it will break down in smaller pieces. These smaller pieces are then broken down further by the ingestion of bacteria and through respiration will degrade the plastic into carbon dioxide and water.  
• Will look into plastics that degrade over time

Membrane: 

• Contacted Dr. Mintchev for a meeting about transdermal patch + materials.
• Reading this paper: page 13

https://www.ualberta.ca/~csps/JPPS8(1)/N.Udupa/glibenclamide.htm

• This paper tested EVA 2%, 9% and 19% both in vitro and in vivo. The trend held true that the higher the % of EVA, the more drug diffuses.
• They also tested ethyl cellulose, Eudragit RS-100 and Eudragit RL-100 but only in vitro.
• The drug was glibenclamide to treat diabetes.

Reading this atm:

http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2014/12/WC500179071.pdf

Release Liner:

• Needs to be chemically inert with drug penetration, penetration enhancer and water
• 3M Scotchpak 1022
• 3M Scotchpak 9741
• 3M Scotchpak 9742
• 3M Scotchpak 9744
• 3M Scotchpak 9755

• Fluoropolymer Coated Polyester Film
• Good for release with silicon skin contact adhesives, acrylate, PIB and rubber based PSA
• Excellent chemical stability

Adhesives:

Links:

1. https://label.averydennison.co.za/content/dam/averydennison/lpm/na/en/doc/home/resource%20center/Adhesive%20Overview(1).pdf
2. https://www.researchgate.net/publication/51881833_Adhesive_properties_a_critical_issue_in_transdermal_patch_development_Expert_Opin_Drug_Deliv_933-45

The typical adhesive properties include:

• Initial Tack - The immediate holding power of the label upon contact with the substrate. A label with high initial tack will grab the substrate quickly. A label with low initial tack will exhibit a low level of adhesion when first applied and may remove cleanly.
• Ultimate Adhesion - The ultimate or maximum holding power that the label will achieve as the adhesive penetrates into the substrate. The time required to obtain ultimate adhesion may depend on the stiffness (shear) of the adhesive, the roughness of the substrate and the temperature of the environment.
• Shear Resistance - A measure of the internal cohesive strength of the adhesive. The shear of the adhesive is an indication of how soft an adhesive is. A low-shear adhesive (soft) has more of a tendency to flow (resulting in higher initial tack), and has a higher chance that the adhesive will split apart if put under stress. A high-shear adhesive (firm) is less likely to split under stress because of its good internal cohesive strength, and will be less likely to flow (possibly lower initial tack).

• Solvent-based

• Traditionally used in patch production
• Good for extended wear
• Provide tighter hold
• Stingy

• Silicone-based

• High oxygen/gas permeability
• Low pain upon removal to sensitive skin
• Can be customized to improve chemical compatibility and stability with cationic drugs
• Increased diffusivity
• Tendency to cause drug crystallization

• DOW CORNING® BIO-PSA 7-4101 SILICONE ADHESIVE, DOW CORNING® BIO-PSA 7-4201 SILICONE ADHESIVE and DOW CORNING® BIO-PSA 7-4301 SILICONE ADHESIVE that way you can compare the different levels of tack. They are all amine compatible with heptane as the carrier solvent.”

---

Math Research

Diffusion:

Factors affecting diffusivity:

Search up: Chapter 2: Overview of Controlled Release Mechanisms by Ronald A. Siegel and Michael J. Rathbone

• Depends on size of molecule and medium, as well as the membrane that we’re using
• For a hard spherical molecule diffusing through:

Equation: D = kT/(6*pi*a*n)

• a = molecule's radius
• T = absolute temperature (K)
• n = solvent viscosity
• k = Boltzmann's constant  (this accounts for intensity of thermal agitation)

• In terms of the medium: in free volume theory, each drug, solvent, and polymer molecule contains an impenetrable core that is surrounded by nanovoids, called free volume

• Thermal motions cause the size of voids to fluctuate. Occasionally, a void becomes large enough for a diffusing molecule to move into or through it

• Free volume of a matrix depends on the its composition
• Free volume can also be increased substantially by sorption of small molecules, such as water. (sorption is the physical and chemical process by which one substance becomes attached to another)

• For a molecule diffusing through a water-swollen hydrogel, diffusivity of drug is affected by the viscosity of the water space and also by obstructions placed in the drug molecule’s path by the hydrogel chains

Monday, June 6th

What we need to model:

• Oxygen diffusion through the backing layer (outside to inside and vice versa)
• The amount of peptides that go through the rate membrane
• How much force would we apply to the pouches for them to break
• Mixing of media in patch and in pouches
• Start modelling the system

• Play around with the sizes of the packets
• Do math around, having some idea about how something works would be necessary, at least we have an idea how it works
• Determine what we’re actually trying to solve
• MATLAB = useful way we could deal with our model - trying different values for a parameter we don’t know about and see how it changed, identify what it represents, it’s a framework of understanding which parameters are important

Friday, June 10th

Rate of release of therapeutic agents from reservoir transdermal systems:

• https://www.researchgate.net/file.PostFileLoader.html?id=55f196f8614325b8798b456f&assetKey=AS%3A272146475778050%401441896184273 
• Assuming mechanism of drug delivery involves these steps:

• Drug dissolution within the reservoir matrix
• Drug diffusion and partitioning into the membrane
• Drug diffusion within the membrane and partitioning into the adhesive layer
• Drug diffusion within the adhesive and partitioning into the stratum corneum

• Rate controlling membrane controls drug diffusion into the adjacent adhesive layer and therefore is the rate-limiting step in the diffusion process

https://www.quora.com/Why-arent-patches-used-as-drug-delivery-systems-more-often

http://ceaccp.oxfordjournals.org/content/7/5/171.full

• Reservoir = drug concentration is established, drug moves further into the skin, into the capillaries, and then into the circulation
• There is a time to reach steady state of plasma concentration

Parameters that we have to consider:

• Diffusing peptide must not affect the adhesive and vice versa
• Skin compatibility, chemical compatibility
• Tack, peel adhesion, skin adhesion and cohesive strength
• Hydration of skin

• Tissue swells when skin is saturated with water and its permeability increases = this would be an important factor to increase penetration

• Temperature
• Diffusion coefficient

• Diffusion speed of molecules depend on the state of matter in the medium

• Drug concentration

• Drug permeation usually follows the Fick’s law. The flux of solute is proportional to the concentration gradient across the entire barrier phase

• Partition coefficient

• Important in establishing flux of drug through stratum corneum

• Molecular size

iGEM

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