Purpose: Artificial antigen-presenting cells (aAPCs) are promising for immune modulation, and anisotropic (ellipsoidal) particles have shown improved T-cell stimulation over spherical particles. However, widely used methods for generating anisotropic particles, such as thin-film stretching, can be difficult to implement reproducibly. A standardized, scalable protocol is needed for fabricating biodegradable anisotropic aAPCs with tunable size, shape, and signal presentation.
Hypothesis: If biodegradable polymeric particles are fabricated into anisotropic ellipsoidal shapes and surface-conjugated with T-cell stimulatory proteins, then they will activate and expand CD8+ T cells more effectively than spherical aAPCs, due to increased surface contact area, enhanced binding, reduced nonspecific uptake, and improved pharmacokinetic properties.
Aims: Provide a rapid, standardized protocol for fabricating anisotropic biodegradable polymeric aAPCs with tunable size, shape, and signal presentation. - Describe synthesis of PLGA micro- and nanoparticles and deformation into prolate or oblate ellipsoidal shapes via thin-film stretching. - Detail surface conjugation of signal 1 (MHC-Ig dimer/anti-CD3) and signal 2 (anti-CD28) proteins via EDC/NHS chemistry. - Outline characterization of particle size, morphology, aspect ratio, and surface protein content. - Assess functionality by in vitro CD8+ T-cell proliferation and expansion.
Delivery system:
Component: Particle core; Details: Biodegradable PLGA, optionally hybrid PLGA/poly(beta-amino ester) (PBAE)
Component: Particle types; Details: Micro- and nanoparticles
Component: Shapes; Details: Spherical; prolate ellipsoidal (1D stretch); oblate ellipsoidal (2D stretch)
Component: Fabrication; Details: Single emulsion for spherical particles; thin-film stretching at 90°C for anisotropic shapes
Component: Surface conjugation; Details: EDC/NHS coupling of primary amines on proteins to carboxyl groups on particle surface
Component: Signal proteins; Details: Signal 1: peptide-loaded MHC IgG dimer or anti-CD3; Signal 2: anti-mouse CD28
Component: Targeting ligand; Details: None
Component: Payload; Details: No genetic/drug payload; surface protein signals only
Component: Key feature; Details: Anisotropic shape and biodegradable polymeric core for aAPC function
Approach: In vitro protocol. No in vivo studies. - Cell source: CD8+ T cells isolated from Black 6 mouse splenocytes. - T-cell activation: CFSE-labeled CD8+ T cells incubated with spherical or prolate ellipsoidal micro/nano-aAPC at doses of 0.01, 0.1, and 1 mg for 3 and 7 days. - Controls: Non-cognate controls, spherical aAPC, untreated cells. - Disease context: Cancer immunotherapy / immunoengineering. - Group structure: Triplicate replicates; error bars represent standard error of the mean.
Key methods: Size and morphology: Scanning electron microscopy (SEM) for microparticles; transmission electron microscopy (TEM) for nanoparticles; dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA) for nanoparticle size. - Aspect ratio: Image analysis of long axis divided by short axis. - Protein conjugation efficiency: Fluorescently labeled signal proteins and fluorescence plate reader. - T-cell proliferation: Flow cytometry for CFSE dilution after 3 days. - T-cell expansion: Manual counting with hemocytometer after 7 days; fold expansion normalized to initial count.
Key results: Particle size: Spherical nanoparticles = 237.3 ± 4.0 nm by DLS and 224 nm by NTA; spherical microparticles = 3 ± 1 µm. - Aspect ratios: Spherical = 1.05 ± 0.04; 1D-stretched prolate ellipsoidal = 3.6 ± 0.8; 2D-stretched oblate ellipsoidal = 1.2 ± 0.2. - Protein conjugation efficiency: 15–20% for EDC/NHS coupling. - T-cell activation: Prolate ellipsoidal aAPC induced higher levels of T-cell proliferation at sub-saturating doses than spherical aAPC, with best separation at 0.01 mg dose. - Expansion: After 7 days, prolate ellipsoidal aAPC more effectively stimulated T cells than spherical counterparts at both micro- and nanoscale; dose-dependent T-cell expansion was observed.
Interpretation: The authors claim that thin-film stretching is scalable, highly reproducible, and inexpensive for generating anisotropic polymeric particles. Anisotropic ellipsoidal aAPCs are more effective than spherical aAPCs at stimulating CD8+ T-cell proliferation and expansion. This modular platform is suitable for “off-the-shelf” immunotherapies and can be adapted to other drug delivery applications by conjugating different proteins of interest.
Limitations: Protocol paper: Describes methods; no new in vivo therapeutic efficacy data. - In vitro only: T-cell activation assessed in vitro; no in vivo T-cell expansion or antitumor efficacy. - Mouse model only: Uses mouse CD8+ T cells and mouse-specific antibodies; human translation not demonstrated. - No targeting ligand: aAPC relies on nonspecific particle–cell interactions. - No encapsulated cytokines or genetic payload: Only surface protein signals. - Specialized equipment: Requires automated thin-film stretcher or manual stretching device. - No long-term toxicity or safety data. - No direct comparison to clinical-grade aAPC or other anisotropic fabrication methods beyond discussion.
