Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

The overall goal of chemical functionalization of the surface of HEV nanoparticles is to provide a repeatable and consistent approach for conjugation of targeting immunogenic, therapeutic and diagnostic molecules to the surface of HEV nanoparticles for diagnosis and treatment purposes. This method can help answer key questions in the nanomedicine field such as how enhanced targeting of cancer and distribution of nano therapeutics can be monitored. The main advantage of this technique is that it's highly reproducible, efficient and much easier to achieve when compared to generate genetic engineering.

Demonstrating the procedures will be Michelle Nguyen and Ivan Ruiz, research assistants from our lab. Begin this protocol with hepatitis E virus nano particle production and purification as described in the text protocol. Dilute the HEV nanoparticles samples to between 0.5 and two milligrams per milliliter with 10 millimolar MES, pH 6.2 for a TEM imaging.

Ionize the carbon coated grids with 40 milliamp glow discharge for 30 seconds to produce a hydrophilic carbon surface. The hydrophilic carbon surface of the grids can only last for 30 minutes after glow discharge treatment. Following ionization, hold the grid and tweezers and add two microliters of the HEV nanoparticle sample to the grid.

After 15 to 30 seconds, blot the grid with filter paper. Then immediately wash the grid with double distilled water and blot again with filter paper. Immediately add two microliters of two percent uranyl acetate to the grid.

After 15 seconds, blot the grid with filter paper. Dry the sample grids by putting them in an electronic dehumidifier dry cabinet overnight. Depending on the sample, cryogenic electron microscopy or cryo-EM may be necessary for structure visualization and characterization.

Transfer the grid into a TEM and image at 10 to 80 K magnification. HEV nanoparticles appear in TEM as empty icosahedral proteins that are approximately 27 nanometers in diameter due to the absence of viral RNA. To perform the one-step conjugation of HEV nanoparticles and maleimide-linked biotin, first apply the HEV nanoparticles to mini dialysis units.

Dialyze the nanoparticles against 0.01 molar PBS pH 7.4 at room temperature for one hour according to the manufacturer's protocol. Following dialysis, transfer the HEV nanoparticles to 1.5 milliliter tubes and measure the protein concentration at 280 nanometers using a spectrophotometer. Mix the nanoparticles at one milligram per milliliter with an equal amount of 100 micromolar maleimide biotin and 0.01 molar PBS pH 7.4 to make a one to five molar ratio.

After reacting overnight at four degrees celsius, remove unbound maleimide biotin with a spin desalting column procedure according to the manufacturer's protocol. Analyze the samples through a standard reducing SDS page. Using the standard procedures, prepare a chemiluminescent Western blot using HRP-linked streptavidin and capture the chemiluminescent signal by x-ray film.

To perform two-step conjugation of the breast cancer cell specific ligand, LXY30, to surface exposed cysteine on HEV nanoparticles, first apply the nanoparticles to mini dialysis units and dialyze against 0.01 molar PBS pH 7.4 at room temperature for one hour. Then transfer the HEV nanoparticles to 1.5 milliliter tubes and measure protein concentration at 280 nanometers using a spectrophotometer. Next, combine 650 micromolar maleimide azide and 650 micromolar alkyne LXY30 with 200 micromolar copper sulfate and one millimolar ascorbic acid.

This forms maleimide-linked LXY 30 at 650 micromolar. Incubate the mixture at four degrees celsius overnight. The next day, mix the HEV nanoparticles to one milligram per milliliter with about 10%volume of maleimide-linked LXY 30 to make a one to three molar ratio.

React overnight at four degrees celsius. Due to the relatively high concentration of maleimide LXY 30, the final concentrations of the reactants such as copper sulfate are reduced about 10 times after mixing. To avoid their damage to the hepatitis E virus nanoparticles another option is the copper free contribution method.

Remove unbound maleimide click LXY 30 with a spin desalting column according to the manufacturer's protocol. Keep the LXY 30-linked HEV nanoparticles at four degrees celsius. To perform one-step conjugation of the LXY 30 HEV nanoparticles and Cy5.5 fluorescent tag, first mix the LXY 30-linked nanoparticles to one milligram per milliliter with an equal volume of Cy5.5 fluorescent tag to make a one to five molar ratio.

After reacting the mixture overnight at four degrees celsius, remove the unbound Cy5.5 NHS with a spin desalting column procedure according to the manufacturer's protocol. Keep the LXY 30 Cy5.5-linked HEV nanoparticles at four degrees celsius. Two methods were attempted for the LXY 30 functionalization of HEV nanoparticles for tumor cell targeting.

When HEV 573C nanoparticles were bound to maleimide azide first, followed by a click chemistry reaction to LXY 30 alkyne, the HEV 573C nanoparticles appeared to disassemble under TEM observation. However, an initial click chemistry reaction between LXY 30 alkyne and maleimide azide to form maleimide-linked LXY 30 followed by conjugation to assist modified HEV nanoparticles did not affect its structure and the HEV 573C nanoparticles remained intact. LXY 30 and Cy5.5-linked HEV nanoparticles were applied to cultured breast cancer cells and observed by confocal microscopy.

The near infrared fluorescence images by confocal microscopy indicate that LXY 30 Cy5.5-linked HEV nanoparticles had higher binding affinity for an increased internalization in MDA MB 231 breast cancer cells as compared with Cy5.5-linked HEV nanoparticles. The implications of this technique extend toward examination of multimodal targeting and therapeutic molecules which allow for accurate targeting and diagnosis of tumor without causing damage to normal cells. Though this method can provide insight into cancer and tumor targeting through chemical surface modulization, it can be applied to early cancer diagnosis, screening and imaging guided treatment.