The overall goal of this procedure is to study in vivo tumor targeting and biodistribution of fluorescently labeled viral nanoparticles. In mouse tumor models first propagate and purify the virus, then conjugate the dyes and peg to the particles. After characterizing the functionalized particles proceed to inject them into mice with tumor xenografts.
Results from maestro imaging, fluorescence intensity, measurements of tissue homogenous and immunofluorescence show localization of the viral nanoparticles in the tumor and various organs throughout the body.Platform. Nanoparticles or BMPs are promising platforms for applications in biomedicine. Here we describe the procedures for PLA VMP, propagation, purification characterization, and bowel conjugation.
Finally, we show the application of V mps for tumor homing and imaging using a malino graft model and fluorescence imaging. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. The main advantages of using bio nanoparticles over synthetic materials such as metal or polymeric nano materials is that BMPs from plants are monitor dispersed and their structures are known to atomic resolution.
Plant viruses can be produced in large scale through molecular farming and are amendable to both genetic and chemical engineering. In this demonstration, we show the chemical modification of viral nanoparticles with Fluor fours and peg. The Fluor fours will be used for imaging enhancement and the PEG will be used to improve the pharmacokinetics.
Chemical modifications can be extended to load viral nanoparticles with therapies and contrast agents for clinical imaging such as MRI. Here we demonstrated biodistribution study of ated viral nanoparticles like other nanoparticles. Tumor humming of V NPS is based on enhanced permeability and retention, which is described as passive tumor homemaking.
We recently found that the biodistribution profiles of different plant viruses are distinct. For this demonstration, we'll use copy mosa virus, which is considered the gold standard for viral N technology. We have a particular interest to develop, engineer and study the nano materials from plant viruses.
My lab focuses on gaining a fundamental understanding of how Ed cow p mosaic virus bro mosaic virus, rock shaped bacca mosaic virus and filamentous potato virus X interact with the mammalian system. Specifically, we like to learn how the shape and the flexibility impacts and EVO properties and how to best tailor them for applications such as tissue specific imaging and drug delivery. This manuscript details the preparation of kalp mosaic virus, potato virus X, brown mosaic virus and tobacco mosaic virus.
From Vic, Gil and Nicotiana, we describe their functionalization and application in a preclinical mouse model. In this demonstration, we'll use the cow P mosaic virus In an indoor incubation chamber. Plant three cow P seeds per pot.
On day 10, infect each leaf with 0.1 micrograms per microliter of CPMV by mechanical inoculation using a light dusting of carum followed by a light rubbing of the leaves. After 10 days, harvest the leaves and store at negative 80 degrees Celsius. In a standard blender homogenize 100 grams of frozen leaves in two volumes of cold 0.1 molar potassium phosphate buffer pH seven, filter the homogenate through three layers of cheesecloth and centrifuge at 5, 500 G for 20 minutes.
Collect the supernatant, then perform an organic extraction of the plant material with 0.7 volumes of one-to-one chloroform to one butanol, stirring the mixture for at least 30 minutes. After centrifuging the solution at 5, 500 G for 20 minutes. Collect the upper aqueous phase and ultracentrifuge.
Add 160, 000 G for three hours. Next, resuspend the pellet in five milliliters of phosphate buffer and incubate the sample at four degrees Celsius overnight. Also, prepare a 10 to 40%sucrose gradient using an equal volume of 10, 20, 30, and 40%sucrose in buffer.
Placing the heaviest first equilibrate the gradient overnight at room temperature. Layer the RESUSPENDED palette over the sucrose gradient and centrifuge at 100, 000 G for two hours. Collect the light scattering band and dialyze against buffer in order to conjugate the dyes and peg to surface lysine residues at 2, 500 molar equivalents of Fluor and 4, 500 equivalents of NHS PEG to CPMV.
After purifying the reaction, measure the absorbance spectrum of two microliters of sample using the nano drop, noting the values at 260, 280 and 651 nanometers. Proceed to analyze the particles by size, exclusion, fast protein liquid chromatography for a super oh six size exclusion column, and the ACTA Explorer load 50 to 100 micrograms of the VNP sample. In 200 microliters of 0.1 molar potassium phosphate buffer.
pH 7.0 set the detectors to 260 nanometers for nucleic acids and 280 nanometers for proteins. Also set the absorbance wavelength of any dyes attached. Run the separation at a flow rate of 0.5 milliliters per minute for 72 minutes.
The Ellucian profile in the 2 62 80 ratio indicates whether the reaction is pure and that the V NPS are fully assembled and intact. Next, prepare a denaturing SDS gel electrophoresis to analyze conjugation to individual coat proteins. For each sample, add three microliters of four XLDS sample buffer to 10 micrograms of the particles in nine microliters of potassium phosphate buffer denature in a heat block for five minutes at 100 degrees Celsius.
Load the samples onto an SDS gel and run at 200 volts for one hour. In one X mops running buffer document the gel under white light. After staining, These viral nanoparticles are now ready for tumor targeting and imaging using a xenograft mouse model.
Typically, we use a tail vein injection to administer these particles and intravenously To evaluate tumor homing of VN ps. Analyze the tissues with the maestro fluorescence imaging instrument. Use yellow excitation and emission filters with 800 milliseconds exposure to detect the presence of fluorescent signals derived from the A 6 47 label conjugated to the VN PS for fluorescence quantification.
Homogenize the tissues for two to three minutes in PBS. Then transfer the homogenous to fuge tubes for each PBS and VNP formulation per time point transfer 100 microliters of sample Senna into a 384 well black UV plate. Measure the fluorescence intensity using excitation and emission wavelength of six and 665 nanometers.
Successful VNP propagation can be determined by the appearance of mosaic symptoms on the leaves about a week after mechanical inoculation. UV visible spectroscopy can be used to assess the purity of the particles. Absorbance at 260 nanometers is proportional to the nucleic acid concentration and absorbance at 280 nanometers is proportional to the protein concentration.
These size exclusion elution profiles of VN PS were obtained by using a Superos six column and ACTA purifier. As with the UV visible spectra, the A two 60 to a two 80 ratio can be used to assess purity of VNP preparations. In addition, the elucian profile can be used to determine purity of the particles.
SDS gel electrophoresis separates the denatured coat protein subunits. The CPMV particles are formed by two proteins, a large 42 kilodalton protein, and a small 24 kilodalton protein.Note. The smaller protein has two electrophoretic forms in these TEM images of virus particles.
The TEM grids were negatively stained with 2%urinal acetate, so the particles appear light on a dark background. The CPMV particles are sized at 30 nanometers. The V NPS can then be functionalized via bio conjugate chemistries.
For instance, CPMV have lysines that can be used to react with functionalized NHS Esters to confirm functionalization of the V NPS characterization using the same methods as previously for the purified particles is performed. These are representative data of fluorescently labeled and pegylated VN ps. The functionalized particles can then be utilized for downstream applications such as the investigation of biodistribution and tumor homing with pegylated and thigh labeled VN ps.
Here, biodistribution of CPMV is evaluated in nude mice implanted with human HT 29 xenografts biodistribution and tumor homing is evaluated using maestro imaging of tissues. Ex vivo no fluorescence was observed for the PBS control. While for CPMV conjugated with a 6 47 and peg, there was prominent fluorescence in the liver and some in the spleen due to clearing by the reticuloendothelial system.
Prominent fluorescence in the tumor indicates tumor homing via the enhanced permeability and retention effect. These quantitative data were obtained via fluorescence measurement of homogenized tissues normalized by tissue weight. As expected data indicate clearance of CPMV by liver and spleen.
CPMV is also detected in the tumor. Further intratumoral localization using immuno staining of cryo sectioned tissues show the intratumoral localization of CPMV. CPMV is shown in red.
The blue is the nuclei, and green is a blood vessel stained with anti CD 31. The CPMV was mainly found near the vasculature of the tumor. The described techniques have brought applicability to a variety of bio nanoparticle systems.
The Stein lab studies VMP, such as cow mosaic virus, bro mosaic virus, potato virus X, and tobacco mosaic virus. The methodologies however, could be applied to any other viral nanoparticle or protein nano structures such as heat shock proteins. Following this procedure, additional methods such as R-T-P-C-R or Western Block can be performed as independent methods.
Cryosectioning and immunohistochemistry can be used to gain additional information on the intratumoral location of the v ps and potential colocalization with tumor associated macrophages or other organelles. After watching this video, you should have a good understanding of how to propagate purify functionalize, and characterize viral nanoparticles and how to study the in vivo by distribution using a tumor mouse model.
