- Akshay ChaudhariApril 15th 2009

Overview of multi scale modeling of a Paclitaxel Drug Delivery system

Why image? Why cryo-TEM?

To corroborate structure of the modeled PGGP molecule and its true physiochemical structure

Methods1. Mounting2. Glow Discharging3. Super cooling the PGGP4. Transfer to EM

Feedback?

The premise of this study is to utilize a multi scale modeling procedure in order to create the most effective drug delivery system.

Paclitaxel, a mitotic inhibitor, is loaded onto Poly (Glutamyl – Glutamate) backbone.

Using coarse graining and the MARTINI force field, the size and shape of a single PGGP molecule can be elucidated.

Coarse graining of PGGP Mesoscopic model of PGGP micelle

The potential energy of bond interactions can be calculated using the MARTINI force field, creating an atomistic model, which can be further used to construct a mesoscopic model of PGGP.

The physiochemical structures that are predicted through modeling cannot hold much value unless they are corroborated by physical evidence.

Transmission Electron Microscopy can be used to image the PGGP molecules to find the similarities amongst the predicted model and the physical model.

• Cryogenic Transmission Electron Microscopy (cryo-EM) is one of the most pragmatic ways to image particles such as PGGP, due to its ability to capture PGGP in its native environment.

• High resolution image of Egg Phosphatidylcholine Liposomes visible at range of 100nm

•The following is a JEOL 2010 TEM, which is capable of High Resolution TEM, sample tilting to 45 degrees, along with the ability to hold cooled samples (1).

X-Ray Crystallography:

•Although widely used to image biological molecules, XRC is more developed for the imaging of macromolecules rather than “small molecules” due to low resolution imaging (3).

•(4) Structure of Photosystem I determined using X-ray crystallography in Petra Fromme's lab at ASU.

Nuclear Magnetic Resonance Spectroscopy:

• NMR utilizes a very powerful magnetic field to image particles. Motion of molecules, however, results in low resolution images(6).

Fragment of an enteropathogenic e.coli (5)

Ability to minimize conformational changes.

Low dosage of radiation applied. In addition, the process of cryo – TEM has

been established to image amphiphilic molecules (7).

Temperature lowered to 60 – 77 Kelvin and put through same procedure as that for EM.

1) Mount onto surface(8)(8)

The PGGP molecule is mounted onto a holey carbon film, in either a water or a buffered solution.

Clean the mount by H2O2 beforehand. The shape prevents the formation of ice crystals on the surface of the mount surface. Increased power spectrum. Ability to make surface hydrophilic.

Quanticoil R 2/2 Holey Carbon Film (9)

• Proper mounting techniques are quintessential for the return of good images.

Glow discharging is achieved by placing an inert gas (sometimes an non-reactive gas) between two electrodes, to produce a hydrophilic mount.

A potential difference of 100V – a few kV is applied to the electrodes and the pressure of this system is varied from around a few mTorr to atmospheric pressure. (10)

Apply 3-5 μL of a PGGP solution onto the holey carbon film. To create a thin layer containing the aqueous suspensions of

PGGP, that is up to 100nm thick. Set the mount into a cryo holder. Plunge the sample rapidly into ethane, until it has reached a

temperature of liquid nitrogen. Around 70 K.

Gatan 70 Cryo transfer holder, workstation and cover, cryo gloves.

Pre cool the workstation along with any tools that might be used in the process.

(Gatan 70 Cryo transfer holder, workstation and cover, cryo gloves.

Pre cool the workstation along with any tools that might be used in the process.

The frozen grids are transferred to the workstation using pre cooled tweezers.

Attach the cryo holder dewar to the workstation. This is will constantly keeping the grids at liquid nitrogen temperature.

Fill the dewar with liquid nitrogen while being careful not to spill it or bring it around any inflammable materials

Transfer the workstation and cryo holder to the EM. Top off the anti contaminator to minimize contamination of the vicinity of the sample

• Insert the holder into the airlock• Rotate the pre tilted holder so that the dewar can be fit in to the high vacuum column of the EM• Top off the dewar with more ethane• Wait for 15-45 minutes for the holder to thermally equilibrate.

Image PGGP to corroborate predicted model to the physiochemical structure.

Use cryo – TEM since it is the most effective form of imaging for PGGP.

Minimize any chance for conformational changes. Super cool the PGGP to bring it to its native

environment. Compare the predicted model to the real structure.

Questions? Comments?

(1) Picture of a Transmission Electron Microscope:Penn Regional Nanotechnology Facility http://www.seas.upenn.edu/nanotechfacility/em/jeol2010.html

(2)http://www.mardre.com/homepage/mic/tem/samples/colloid/pc_samples/egg_pc_liposome_dispersion_cryo_tem2.html

(3)X-Ray Crystallography: William Clegg, School of Natural Sciences (Chemistry), University of Newcastle, Newcastle upon Tyne, UK, Comments on Inorganic Chemistry, Volume 26, Issue 3 & 4 May 2005 , pages 165 - 182 http://www.informaworld.com/smpp/section?content=a725618401&fulltext=713240928

(4)X-Ray Crystallography image of Photosynthesis 1: Diane Boudreau, “Catching some rays: Harnessing the power of photosynthesis”http://researchmag.asu.edu/2007/03/catching_some_rays.html

(5)Kelly, G., Prasannan, S., Daniell, S., Flemming, K., Frankel, G., Dougan, G., Connerton, I. and Matthews, S. ‘The Solution Structure of the 30.1 kDa Cell-Adhesion Fragment of Intimin from Enteropathogenic E. coli’  Nature Struct. Biol.  6, 313-318 (1999) – [IP13.6]

(6)Hrabe J, Kaur G, Guilfoyle DN. Principles and limitations of NMR diffusion measurements. J Med Phys [serial online] 2007 [cited 2009 Apr 9];32:34-42. Available from: http://www.jmp.org.in/text.asp?2007/32/1/34/31148

(7)Viveka Alfredsso. Cryo-TEM studies of DNA and DNA–lipid structures. Current Opinion in Colloid & Interface ScienceVolume 10, Issues 5-6, December 2005, Pages 269-273

(8)(8) Linda Melanson Gatan Inc. Cryo –TEM workshop, Baylor College of Medicine. Linda Melanson Gatan Inc. Cryo –TEM workshop, Baylor College of Medicine. http://ncmi.bcm.tmc.edu/ncmi/events/workshops/workshops_64/proceeding/Cryo%20TEM%20fromhttp://ncmi.bcm.tmc.edu/ncmi/events/workshops/workshops_64/proceeding/Cryo%20TEM%20from%20specimen%20prep%20to%20the%20microscope_Gatan%20Presentation_LAM1.pdf%20specimen%20prep%20to%20the%20microscope_Gatan%20Presentation_LAM1.pdf

(9) Description of the product Quantifoil, product website. http://www.emsdiasum.com/microscopy/products/grids/quantifoil.aspx

(10) What is a glow discharge and what is it used for ? http://webh01.ua.ac.be/plasma/pages/glow-discharge.html