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Meeting Notes

5/14/2013

Drugs Update

Doxorubicin

TNF Alpha – using nanoparticles to localize

Which type of cancer are we targeting?

Already have leukemia cells (different types) available to us

Direction for continued research : Pick 3 (maybe 5) top candidates of drugs for testing that have already been tested in leukemia

Will test in vitro for now, maybe in vivo down the road

Shapes Update

Shape involvement seems to be related to loading ability

Ideas: sphere, dense vs. non-dense, surface area

Use shape for delivery, ligand for targeting

Nanoparticle or liposomal delivery

Is uptake efficiency affected by shape?

Direction for continued research : Limit to 3 designs, tube as starting point, then change aspect ratio or number of layers (density)

Already have 6-helix bundle that could be used

Maximize intercalation efficiency and cell uptake efficiency

Targeting Update

Ideas:

folate targeting – overexpressed in tumor site Liposomes, pei, peg (folate receptors). Size is an issue for penetrating membrane.
magnetic nanoparticles – induced magnetic field at tumor site ferrofluids mixed with drug
peptide ligand targeting – peptides on outside of tumor capillary growth hormones

Direction for continued research: surface bound receptor (as opposed to secreted)

Goal may be B-cell targeting, even if we can’t target malignant B-cell > proof of principal

CD 20 receptor

Design Direction: Focus on leukemia

· Easier to test by delivering into blood stream, easily reaches cancer

Leukemia cells available

· Drugs (daunorubicin, idarubicin) already tested in leukemia

Plans & Targets

More focused literature search on drugs and targeting
Look for ligands

What is overexpressed on leukemia cell surface?
What is commercially available?

Begin designing and drawing cross-sections for shape ideas

Look at degradation – If shape lasts longer, does it get closer to nucleus, etc
Focus on targeting, effects of size/shape on uptake
Potential mechanisms to explain difference in results between tube and triangle
How did they measure loading efficiency?
What shapes do we already have?
Preliminary tests to check hypothesis for loading efficiency if no data is available
Does intercalation affect folding?
Hypothesis – dense would have lower loading efficiency because layers are inaccessible to solution
Incubation period?

5/21/2013

Drugs Update

Intercalating agents:

Daunorubicin (doxorubicin is a derivative of this)

Mitoxantrone

Used in combination with vincristine, inhibits mytosis

Anthrapyrazols

Rituximab commonly used for CLL but not an intercalating agent

Direction: research drug mechanisms

Targeting Update

ROR-1 is overexpressed in CLL

Monoclonal antibodies used to target

How do we attach antibodies to structure?

Direction : research how to target ROR-1

Shapes Update

Plan: use structures already available in the lab for prelimary tests

Tube (6 helix bundle) as control, adjust diameter (6, 12, 18 helix bundles), extended cylinder, density, layers (bennett linkage)

From test results, develop 3 structures to test

Measure intercalation loading efficiency by intensity

Direction: investigate DNA structures in the lab that would be useful, research how loading efficiency was measured in doxorubicin study

5/23/2013

Drugs Update

Drug possibilities:

Daunorubicin

Epirubicin

Mitoxantrone

All intercalating agents, none approved for treatment of CLL, mostly in combination with other drugs, few studies about efficacies on their own

Possibility of using a combination of drugs?

Targeting Update

New research at OSU regarding dual-ligand immunoliposome using CD20 and CD37

In combination even more selective than ROR-1

Anti CD20 is commonly known as rituximab and is widely used for CLL treatment

ROR-1 in 2012 study was found on healthy frozen reactive lymph nodes, but overall effective for targeting

Will need biotinylated antibodies if we can get them

Candidates: CD37 and ROR-1

Will attempt to contact Robert Lee’s lab for dissertation on dual ligand liposome

Shapes Update

Selected 5 or 6 structures with varying densities, surface areas

Examples: 18 helix bundle of 2 different lengths, Bennett linkage (64 helices thick, can be held in open or closed configuration), soak Bennett linkage in open position, and then close it as a possibility

5/28/2013

Drugs Update

Direction: Which of the three drugs is most readily used in CLL? Is it commercially available and what is the cost? Will have selection for next meeting on Thursday May 30

Targeting Update

Group has selected ROR-1 for targeting

It is commercially available but not biotinylated

This is pending information from the OSU dual-ligand liposome study

Per Chris, our lab is already working with them and collaboration is possible

Direction: Read dissertation on CD20 & CD37 once available

Shapes Update

Presented important follow-up questions regarding time in blood stream and renal filtering

Selected structures per presentation

Direction: will wait for drug selection to begin loading efficiency testing

Need to verify that measurement process from doxorubicin study will work for selected drug

5/30/2013

Targeting Update

ROR1 would require characterizations of the cells

o Can do with antibodies

o Cannot find biotinylated ROR1 so the other option would consist of attaching streptavidin on the origami if we cannot find biotinylated ROR1.

Shapes Update

Shapes depend on loading efficiency – keeping in mind the concentration of drug.

o The concentration of drug that will be intercalated within the origami.

o The goal is the control the concentration of the drug to be less cytotoxic.

Drugs Update

Daunorubicin has been selected as the drug of choice between Mitoxantrone and Epirubicin. Daunorubisin has the most trials (around 146 in CLL) and epirubisin had around 11. Mioxantrone was ruled out because it attacks T-cells while CLL is in B-cells

Other:

We will be using MEC1 and OSUCLL cells

Questions that we want answered:

- Does the shape depend on uptake?

- How long does Daunorubicin take to kill cells?

Plans for upcoming weeks:

- Tuesday: Doug and Emily will be presenting article about Doxorubicin with DNA origami.

- Other days: Start thinking about WetLab, Design team, Analysis, and Modeling groups.

6/4/2013

Drugs Update

Daunorubicin has been ordered, should arrive any day

Direction: Start experimentation of cell viability with/without drugs

Design:

Can start viability assays without drugs immediately now as controls

Options:

M(13) and cells
M(13) and dauno and cells (single stranded and double stranded)
Daunorubicin and cells
6-helix and cells
Bennett linkage and cells
6-helix and dauno and cells
bennett and dauno and cells

culture cells in presence of these conditions for 24 hours and then take read-outs for every 4 hours (6, 8, etc)

What can we expect to see as results for these tests? Cell viability should decrease with increasing drug concentration but should not be affected by dna strand

Resistant cells? Don’t have that cell available to us at this point

Intercalation loading efficiency with structures

How can we control loading?

6/6/2013

LLS Proposal – Dr Castro is further revising, will request letter of support from Dr.

Byrd to send to LLS along with our proposal

Presentation on Doxorubicin Study: Doug

Powerpoint will be uploaded to google drive

Supporting figures: tested many of the same controls we suggested

Discussed in depth how concentrations of drug were calculated (i.e. once intercalated into origami structures)

Groups

Wet Lab - will be soaking and preparing the different combinations we want to test, performing cytotoxicity assays

Analysis/Microscope – will be looking at results under microscope, imaging

Design – design origami structures, models, experiment protocol

Structures are ready for testing

Need to determine how to measure final concentration of supernatant

Doug will write up simple beginning protocol for control testing

6/18/2013

WetLab Progress:

Made solution out of daunorubicin
Purified Bennett linkage structures

Next Steps:

· Once structures are purified, they will be soaked and incubated with drug solution for 24+ hours

· May look at the effect of daunorubicin on the DNA after extended incubation times

· May have difficulties measuring concentration of dense structures, will estimate if necessary

· Will discuss amounts and specific protocol for incubation at meeting tonight (June 18)

Direction for Microscope Group:

will meet to research imaging done in Bing study

· training on microscope with Molly – available every day at 5pm

Direction for Design Group:

create logo for OHIOmod
maybe go through Union for t-shirts and designs
design protocol for making resistant CLL cells

6/20/2013

Upcoming BIOmod deadlines

Abstract due in September

WetLab Progress:

· So far, closed Bennett linkage is showing most promising results

24, and 36 hour batches have been completed

Microscope Progress:

Doug and Josh have started training with Molly
Will start next week

Direction for Design Group:

· Dr Byrd has protocol on resistant cell line, Paul will contact him or Molly

· Dr Byrd published paper with good results regarding CLL and ibrutinib – will need to research

· The reason DNA origami MDR-1 receptor, transmembrane protein, stops daunorubicin and doxorubicin, DNA may be shielding the drug from this protein – this will be an important mechanism to research, especially for wiki and presentation

6/27/2013

WetLab Update:

Have tested CBL, OBL, hinge, and 18 HB
Need to test 12 and 6 HB

· Need a baseline for PCR: absorbance reading after dilution is different than expected

· Test absorbance with different buffers: water, FOB, PBS

· Sodium levels may be dropping with dilution in water and may be causing different results

· Alex already has curve from buffer excited at 488

Possible reaction from PEG, odor

· Paul got liposomes from Dr Lee’s lab that can be used

Microscope Update:

· Will meet with Chris today regarding cell lines and imaging ideas

Chris recommends 7-AAD cell viability assays

7/2/2013

Design Updates:

Wiki is set-up, will continue to add info – Mike will send link

Spreadsheet on google drive for member bio information that will go on the website

Video – vote on top ideas in meeting

Scope Updates :

Next week: Start to look at Daunorubicin (free drug) on both cell lines (cell viability) at multiple incubation times (24, 36, 48 or 24, 48, 72)

Need baseline daunorubicin concentration for comparison

After: Unloaded origami structures and cell lines

Loaded origami and cell lines

In the meantime, start resistant cell line

Will try to schedule in the morning to avoid conflicts

WetLab Updates:

Gel baseline results should be in tomorrow
TEM – start next week

Video blog – under 60 seconds each, What is OhioMod? (Pat), What is our project? (Vidhi), What is DNA origami? (Tyler)

7/9/2013

Scope Updates :

Started free drug experiments this week

· Incubating with 5 different concentrations of daunorubicin and cells only, two different incubation times (24 and 48)

· At 24 hours, most of the cells are dying (MEC1 and OSU CLL cells lines)

· Next will be origami controls (plain origami and cells)

· After, drug-loaded structures by the end of next week, still need to establish baseline concentrations

Explore multiple ways to test cell viability
Need to order more daunorubicin

WetLab Updates:

Met with Dr. Lucas from Dr. Byrd’s lab

· Already have resistant line that should be resistant to daunorubicin, developing drug resistant cell lines can take months

· Work extensively with Silvestrol – may try to attach to origami (covalently, or intercalation)

They are on board with our project

· TEM – view loaded structures tomorrow from 12-2pm

· Friday – loading efficiency results, Bennett linkages are loading more than helix bundles on average

Video Blog:

Paul - why cll?

Mike (biochem) - how big is nano?

7/11/2013

Chris ordered more daunorubicin

Design Updates:

· Plain double stranded dna for controls: Need to lay out scaffold, staples that follow path of scaffold, no crossovers, 42 bases long, will have to force the staple crossovers to be at the same place as the scaffold crossovers

Scope Updates :

Running free daunorubicin on controls
Changes: may add 12 hour incubation time
Structure controls planned for next week
Need people available later morning

WetLab Updates:

· Gel results: 400 micromolar – 900 micromolar (what was absorbed by structure) found using plate reader

TEM – reviewed images of loaded structures

· No sure if shapes are due to crystallization or the actual structure

Flat 18 helix bundles stuck together a lot
unexplained blobs
images on the google drive

· Need to determine if structures are breaking down after being stored in PBS

· Will try with additional salt (Carlos suggested adding 10 millimolar mgcl)

Will try varying concentration of daunorubicin

· Image structures by themselves before for comparison

7/16/2013

Design Updates:

Went over movie ideas
Limited to 4 minutes

Scope Updates :

Completed cell viability with structures and CLL
18 helix bundles killed some CLL cells

WetLab Updates:

· 3 ready for imaging on Friday, other lab may be able to do additional imaging

· making changes – structures seemed to have degreaded in the PBS by hour 40

TIRF images this week

7/18/2013

Design Updates:

Will start working on new structure

· Pat’s idea is something more square shaped and densely packed

· Square lattice is more densely packed than honeycomb

Scope Updates :

· Resistant cells now growing – 5 different cell lines (HL60 – doxo resistant and other strains that are silvestrol resistant)

· Showed images from cell viability with MEC 1 and daunorubicin at different concentrations at 24 and 48 hours

Now attempting a 12 hour incubation and culture
Also ran 2 cell only controls
Will complete counts next week

· Will need to consider concentration of drug delivery clinically and how our test concentrations compare

WetLab Updates:

have more 6 and 12 helix bundles to retest
Chris has updates to make process easier

7/25/2013

Scope Updates :

· When imaging resistant cells, they spotted something unexpected, possibly contamination of the cell line

· HL60 cells are supposed to be resistant at 2-2.5 micromolar, going to adjust concentrations around this range

WetLab Updates:

Scheduling additional TEM time

8/1/2013

Design Updates:

Copyright meeting this week
Cando – maya toolkit for animation

Scope Updates :

· When imaging resistant cells, they spotted something unexpected, possibly contamination of the cell line

· HL60 cells are supposed to be resistant at 2-2.5 micromolar, going to adjust concentrations around this range

WetLab Updates:

· If people are available and want to work in the lab/learn protocols before school starts, contact Pat

8/15/2013

Design Updates:

· Script is mostly complete with movie clip/animations/etc in place

· Copyright meeting last week – doesn’t seem to be a problem

· Structure is mostly complete (5x6 square lattice cross-section with 4 holes)

Scope Updates :

· OTT (cell viability) assay yesterday, free daunorubicin at 5 different concentrations with and without structures

· Bennett linkage appeared to have an impact on results relative to free daunorubicin at same concentration (10-15% difference)

None of the other cells had that affect

· Removing .1 micromolar and adding 50 micromolar for next trial, Dr Castro suggests factors of 10 (bigger change is less senstie to variability)

WetLab Updates:

Currently incubating more structures for imaging
TEM tomorrow

8/20/2013

Design Updates:

· Adding staple overhangs to structure and then will be ready to order

Need lab notes, pictures for website

Scope Updates :

· MTT – will re run with different concentrations and without 12hb

· MTT cell viability results are different than TIRF results with same concentrations

WetLab Updates:

New folded structures have polymer
Will wait for new structure

8/27/2013

Design Updates:

Need lab notes, pictures for website

Scope Updates :

Running TIRF on Bennett linkages now

· Want to run MTT in the next week with more Bennett linkages

WetLab Updates:

488 and 480 absorbance and fluorescence
6 helix bundle had the highest concentration
inconsistent results for drug loading efficiency

· may continue to use the 6 helix bundle for comparison to original paper

control double stranded scaffold is folded

Carl gave scaffold presentation

9/5/2013

General:

Abstract due Sept 13, first draft is finished

Design Updates:

Website – split lab notes into two sections

TEAM GOALS

a. More structures (Bennett linkage, double stranded, checkered structure, filled structure)

i. Rachel will design second structure ASAP

b. Evidence that shows circumvention of drug resistance

i. MTT

ii. TIRF

c. Comparing shapes

i. Plate reader

d. Attach antibodies

e. Proposed Mechanism (MDR I, or concentration)

i. Site literature (Mike Haupt)

f. Research DNA structures in vivo - Molly

Alex gave folding presentation

9/12/2013

Plan for next week’s meeting: compile all results and decide next steps

Week of 10/7/2013

Ohio MOD October 2013

Weeks of 10/7 10/14

M

T

W

T

F

S

7

Gel on BL Structures (setup 11 AM – Needs cut 4PM)

8

Seed cell plate for TIRF imaging (10:30 P.M.-12:00 A.M.)
Gel
Plate Reader Concentrations
Structure incubation

9

Wash cells with PBS (11:30-1 P.M.)

10

Meeting 8:00AM
End Incubation
New Video Day 4PM-???

· Seed Cell plate for TIRF on double stranded DNA and daunorubicin(~10:30 P.M. -12)

11

· 11:30-12:45 Wash cells from Thursday night with PBS and put back in cell plate

12:30-3 PM TIRF image cells from loaded structures

12

13

· 11:30 A.M.-2 P.M. TIRF image of double stranded DNA and daunorubicin controls

10/17/2013

Lab Work Day: 10/20/2013

Goals:

Last folding batch

Running 3 TIRF and analysis

Finalizing Presentation outline

Editing Website

Wet Lab Update:

Pat finalized loading control experiment

Higher daunorubicin concentration (10 mM) – loaded 3 times standard

Thermal ramp from 37 C down – loaded double of standard incubation

Room temperature (25 C)

Refrigerated (4 C)

Scope Update:

Because of high loading efficiency, once structures are diluted to match free drug concentrations, there are not enough structures in solution to get consistent results

Will test undiluted loaded structures

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Biomod/2013/OSU/meetingnotes

Contents

Meeting Notes

5/14/2013

5/21/2013

5/23/2013

5/28/2013

5/30/2013

6/4/2013

6/6/2013

6/20/2013

6/27/2013

7/2/2013

7/11/2013

7/16/2013

7/18/2013

7/25/2013

8/1/2013

8/15/2013

8/20/2013

8/27/2013

9/5/2013

9/12/2013

Week of 10/7/2013

10/17/2013

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