The overall goal of this procedure is to deposit biomaterial vaccine particles into lymph nodes using a direct injection technique. First, synthesize the lipid stabilized polymer particles using a double emulsion method. Then wash the particles and measure the material properties such as size, cargo, loading or stability.
Next, administer a tracer dye at the tail base of the mouse to allow visualization after drainage of the dye to the lymph node. The final step is to identify the D labeled lymph node and inject a small volume of polymer particles at this location. Histology, immunofluorescence and confocal microscopy can be used to confirm the presence and distribution of particles in the inguinal lymph nodes.
Combining the direct injection of lymph nodes with biomaterials for vaccination enables tight control over the combinations and doses of vaccine components in the lymph node microenvironment, and allows for controlled release of cargo in these tissues. All vaccines must reach lymph nodes to be effective. So defining how biomaterials and incorporated immune cues impact local lymph node signaling is important to link these events to systemic immune response.
This knowledge will help us better understand how biomaterial vaccines administer along traditional routes function. Although intra lymph node delivery of biomaterials serves as a tool to study the effects of biomaterials on the organization of lymph nodes, this platform also provides an applied opportunity to develop new therapeutic vaccines and immunotherapies aimed at cancer and autoimmune disorders. For microparticles sonicate, the organic phase containing the polymer lipid and other water insoluble cargo on ice at 12 watts.
To create the water and oil emulsion, add 500 microliters of distilled H2O or H2O containing one milligram of peptide protein or other water-soluble cargo. Continue fornicating for 30 seconds. Gently rocking the vial up and down side to side around the ator tip to ensure complete emulsification.
Now prepare the water and oil in water emulsion by pouring the water oil emulsion into 40 milliliters of H2O homogenized for three minutes at 16, 000 RPM. Next, add a magnetic stir bar and stir the water and oil in water emulsion overnight to remove the excess solvent after overnight removal of solvent. Pour the emulsion through a 40 micron nylon mesh cell strainer into a 50 milliliter conical tube centrifuge for five minutes, decant the supernatant, resuspend the pellet of particles in one milliliter of water and transfer the suspended particles to a 1.5 milliliter micro centrifuge tube.
Collect the particles by a five minute centrifugation. Measure particle size by laser diffraction, or light scattering. She the volume of water added to the fraction cell is at a sufficient level for alignment and blanking Pipette 10 microliters of particle suspension into the fraction cell.
Close the particle size analyzer compartment door. Then measure particle size for PLGA. Use a refractive index of 1.60.
Use the software interface to calculate particle diameter using a number basis one day prior to injection. Anesthetize the mouse according to an iacuc approved animal protocol. Evaluate the depth of anesthesia with a toe.
Pinch reflex tests and monitor breathing to ensure a respiratory rate of 100 to 120 breaths per minute. Shave the hair at the base of the tail and the hind quarter. Remove the hair from the ventral side of the animal and laterally around to the dorsal side just above the joint of the hind leg.
For each dye injection, use a micro pipette to transfer 10 microliters of dye solution into a microcenter fuge tube and aspirate the entire 10 microliters through a 31 gauge needle into a one milliliter insulin syringe. Now inject 10 microliters of dye solution subcutaneously on each side of the tail base for loading in between injections. Apply a mild depilatory cream to remove the remaining hair.
Be sure to coat the area in between the hind leg and the abdomen. After three minutes, use a wet gloved hand with warm H two oh and gently rub the depilatory cream into the skin. Repeat immediately to remove the excess depilatory.
Next, wet a soft cloth or paper towel with warm water and in a single motion wipe the lower portion of the mouse. Place the mouse under a heat lamp to recover and then return to holding for at least 12 hours. Examine the anesthetized mouse to confirm drainage of trace or dye into each inguinal lymph node.
The lymph node should be visible as a dark spot near the hind thigh and abdomen. Now resus resuspend the particles in distilled water at desired injection concentration for each injection. Use a micro pipette to transfer 10 microliters of particle solution into a micro centrifuge tube.
Aspirate the entire 10 microliters into a 31 gauge insulin needle attached to a one milliliter syringe. Pull the skin taut around the dyed lymph node with the needle at a 90 degree angle to the skin. Penetrate the skin to a depth of one millimeter.
Slowly inject the entire volume monitoring for visible lymph node enlargement. Allow the mouse to recover under a heat lamp and then return to holding or conduct additional testing. First, confirm the particle synthesis and size distribution.
The emulsion solvent evaporation synthesis protocol can be qualitatively assessed by visual inspection of the final emulsions.Generated. Emulsions should be a homogenous suspension free of visible aggregates. Part particle size distribution can be confirmed by laser diffraction or dynamic light scattering particle samples should exhibit a mono modal distribution.
Further qualitative assessment of the particle synthesis can be achieved through modification of the protocol to incorporate one or more fluorescent cargoes, such as a fluorescent peptide or lipophilic dye. Dye can be used to locate and target the lymph node for particle injection During training, mice may be euthanized and necropsy following dye injection to gain familiarity with lymph node locations. Particle distribution within the t and b cell zones of the infected lymph node is confirmed by confocal microscopy of sectioned and stained lymph nodes.
Differences in particle properties such as size can be observed in lymph nodes following injection and imaging by fluorescence microscopy Once mastered, this technique can be completed over the span of two days. Particle synthesis and animal preparation occur on the first day. Particle washing characterization and intra lymph node injection are performed on the second day.
After watching this video, you should have a good understanding of how a trace or dye can be used to visualize and inject the inguinal lymph nodes of mice. This technique enables non-surgical delivery of biomaterial vaccine carriers directly to the lymph node with the level of control that was not previously possible. Direct lymph node delivery of biomaterials will allow scientists and engineers and immunology and vaccine development to explore the fundamental interactions of biomaterials vaccines and immune signals with the lymph node shedding new light on the mechanisms by which these materials stimulate and shape immunity.
