Fabrication of Anisotropic Polymeric Artificial Antigen Presenting Cells for CD8+ T Cell Activation

This method can help answer key questions in the amino-engineering field. Such as, what is the role of particle physical properties on biomaterial immune cell interactions and activation. The main advantage of this technique is that it enables an independent control of the size and shape of biomimetic particles.

The implications of this technique extend toward the therapy of cancer as it is an off the shelf particle therapy that can cause in vivo cell mediated immune responses. Though this method can provide insight into cancer amino-therapy, it can also be applied to other systems. Such as, infectious disease or Type One diabetes.

For micro-particle synthesis, begin by weighing out 100 milligrams of poly(lactic-co-glycolic acid)or PLGA, into a scintillation vial and dissolving the PLGA in five milliliters of dichloromethane by vortexing. Place a homogenizer in a beaker containing 50 milliliters of 1%polyvinyl alcohol, or PVA solution, so that the homogenizer is as close to the bottom of the beaker as possible without touching it. Turn on the homogenizer to the appropriate speed and add the PLGA solution to the beaker for one minute of homogenization.

At the end of the homogenization, pour the polyvinyl alcohol PLGA micro-particle solution into a beaker containing 100 milliliters of 5%PVA solution on a stir plate in a chemical hood and stir the solution for at least four hours. When the solvent has evaporated, pour the particle solution into 50 milliliter conical tubes and collect the particles by centrifugation. Replace the supernatant in each tube with 20 milliliters of de-ionized water and re-suspend the particles by vortexing.

Then bring the final volume in each tube up to 50 milliliters with fresh de-ionized water and wash the particles two more times as just demonstrated. For nano-particle synthesis, dissolve 200 milligrams of PLGA in five milliliters of dichloromethane and place a sonicator probe in a beaker containing 50 milliliters of 1%PVA solution on ice without touching the bottom of the beaker. Begin the sonication at 12 watts and immediately add the PLGA solution to the beaker for a two minute sonication to generate nano-particles with an approximate 200 nanometer diameter.

After sonication, pour the 1%polyvinyl alcohol PLGA nano-particle solution into a beaker containing 100 milliliters of 5 PVA solution on a stir plate for four hours of solvent evaporation in a chemical fume hood. When all of the solvent is evaporated, pour the particle solution into 50 milliliter conical tubes for centrifugation to remove any micro-particles and remove the supernatants. Then transfer the nano-particles into high speed centrifuge tubes for three washes in de-ionized water as demonstrated.

For polymeric particle fabrication, after the last wash, re-suspend the particles in approximately one milliliter of fresh de-ionized water and add film casting solution to the particles to a final concentration of 2.5 milligrams per milliliter particles. Transfer the particle suspension in 10 milliliter aliquots into 75 by 50 millimeter rectangular Petri dishes for one dimensional stretching. Or in 15 milliliter aliquots into 100 by 100 millimeter square Petri dishes for two dimensional stretching.

After overnight drying in a chemical hood, use tweezers to remove the films and trim the edges with scissors. For 1D stretching, place the two short edges of one film between two pieces of neoprene rubber for mounting onto the aluminum blocks of one axis of an automated thin film stretching device. Then use an Allen wrench to screw the metal grips on top of the rubber to hold the film in place.

For 2D stretching, mount all four edges onto four aluminum blocks to stretch the film on both axes measure and record the length of film in between the aluminum blocks on one axis for one D stretching or both axes for 2D stretching and calculate the distance required to stretch the film in one or two directions based on the desired fold stretch. Then place the film loaded stretching device in an oven at 90 degrees Celsius next to a larger beaker of a small amount of water to bring the film to temperature over 10 minutes. When the stretching is complete, let the film cool to room temperature for 20 minutes.

For 1D stretching, cut the film out of the stretching device at the edges. For 2D stretching, cut out and save the center square of film that is uniformly stretched on both axes. Place the films in 50 milliliter conical tubes with no more than two films per tube and add approximately 25 milliliters of de-ionized water to each tube for vortexing.

When the films have dissolved, wash the particles three times in de-ionized water as demonstrated. Re-suspending the particles in approximately one milliliter of fresh de-ionized water per tube after the last wash. Then freeze the particles at minus 80 degrees Celsius for one hour or overnight lyophilization.

The next morning, re-suspend the lyophilized micro or nano-particles at 20 milligrams per milliliter in freshly prepared MES buffer with vortexing. Add 100 microliters of the particle solution to a polypropylene micro centrifuge tube containing 900 microliters of MES buffer and add 100 microliters of freshly prepared EDC NHS solution. Mix the particles by vortexing and incubate the particles on an inverter at room temperature for 30 minutes.

At the end of the incubation, collect the particles by centrifugation and re-suspend the micro-particle pellets in one milliliter of PBS by vortexing or re-suspend the nano-particles in one milliliter of PBS by five seconds of sonication at two to three watts. Next, add eight micrograms of a suitable signal one protein and 10 micrograms of anti mouse CD28 antibody to the micro-particles, or 16 micrograms of the signal one protein and 20 micrograms of anti mouse CD28 to the nano-particles. Bring the final volume in each tube to 1.1 milliliter with PBS and incubate the particles overnight on an inverter at four degrees Celsius.

The next day, wash the particles three times in de-ionized water as demonstrated. These PLGA nano and micro-particles were synthesized using the single emulsion techniques as demonstrated and imaged using transmission and scanning electron microscopy. After homogenization, the micro-particles demonstrated an average diameter of about three micrometers.

The spherical micro-particles had an aspect ration of about one, while the 1D stretched prolate ellipsoidal particles had a larger aspect ratio of about three and a half and the 2D stretched oblate ellipsoidal particles had an aspect ratio of about 1.2, roughly maintaining an aspect ratio of one. Conjugation efficiency results reveal the similar amounts of protein on the surface of spherical and ellipsoidal micro-artificial antigen presenting cells, or AAPC and nano-AAPC, demonstrating that protein coupling during AAPC synthesis occurs in a concentration dependent manner. Prolate ellipsoidal AAPC were found to induce higher levels of T cell proliferation at sub-saturating doses than spherical AAPC with the best separation achieved at a 01 milligram dose.

After seven days, manual counting of the T cells revealed that prolate ellipsoidal AAPC more effectively stimulate T cells compared to their spherical counterparts at the micro and nano scale in a dose dependent manner. Following this procedure, AAPC can be administrated in vivo to gauge their therapeutic efficacy and pharmacokinetics. After its development, this technique helped pave the way for researchers in the field of immuno-engineering to explore the effect of anisotropy on AAPC efficacy.