Vaccines able to stimulate CD8+T cells are crucial in controlling a broad range of infectious diseases and tumors. To induce effective CD8+T cell responses, exogenous antigen has to be cross-presented onto major histocompatibility complex (MHC) class I molecules by dendritic cells. Although particle size has been recognized as a critical factor of vaccine design, it is unclear how the size of vaccine carriers impacts the intracellular processing of exogenous antigen and cross-presentation onto MHC class I molecules. In this study, by using polystyrene beads with narrowly defined sizes as model antigen carriers, we demonstrate that particle size mediates the efficiency of cross-presentation of exogenous antigens. By examining the intracellular trafficking, kinetics of phagosomal pH and degradation of antigens bounded to beads, we illustrate the possible mechanisms attributed to the profound effect of particle size on the efficiency of cross-presentation. Antigen bounded to 50 nm beads was shuttled rapidly to an acidic environment within half an hour post-exposure to cells, leading to its rapid and unregulated degradation and inefficient cross-presentation. In contrast, antigen bounded to 500 nm and 3 μm beads remained in a more neutral environment, which preserved the majority of antigens, leaving it available for the generation of peptides to be loaded onto MHC class I molecules. We conclude that the size of antigen carriers plays a critical role in directing antigen to the class I antigen presentation pathway. Our results, together with previous in vivo studies on the effect of particle size on CD8+T cell responses, provide insight into the rational design of vaccines for the stimulation of cell-mediated immunity.

Delivering genes to mediate functions of cells is a crucial technology for both basic science and clinical applications. Though numerous non-viral gene delivery systems have been developed, the diversity of mammalian cells poses a great challenge to the material design. Here, we demonstrate that surface-induced mineralization represents a promising approach to systematically customize DNA delivery with respect to the characteristics of cells. We initially examined gene transfer in nine cell types derived from different tissues and organisms by surface-induced DNA/calcium carbonate nanocomposites derived from a library of mineral solutions. Subsequently, we correlated gene transfer efficiency with cellular uptake, pH responsiveness of nanocomposites, and phagosomal pH of individual cell types. Based on the correlation, we were able to optimize the DNA delivery to the cell types of interest. Surface-induced mineralization possesses great potential for customizing gene transfer in realizing gene-and cell-based therapy and probing functions of genes.

The efficiency of the RNA-guided AsCas12a nuclease of Acidaminococcus sp. was compared with SpCas9 from Streptococcus pyogenes, for functional genomics in Schistosoma mansoni. We deployed optimized conditions for the ratio of guide RNAs to the nuclease, donor templates, and electroporation parameters, to target a key schistosome enzyme termed omega-1. Programmed cleavages catalyzed by Cas12a and Cas9 resulted in staggered- and blunt-ended strand breaks, respectively. AsCas12a was more efficient than SpCas9 for gene knockout, as determined by TIDE analysis. CRISPResso2 analysis confirmed that most mutations were deletions. Knockout efficiency of both nucleases markedly increased in the presence of single-stranded oligodeoxynucleotide (ssODN) template. With AsCas12a, ssODNs representative of both the non-CRISPR target (NT) and target (T) strands were tested, resulting in KO efficiencies of 15.67, 28.71, and 21.43% in the SpCas9 plus ssODN, AsCas12a plus NT-ssODN, and AsCas12a plus T-ssODN groups, respectively. Trans-cleavage against the ssODNs by activated AsCas12a was not apparent in vitro. SpCas9 catalyzed more precise transgene insertion, with knock-in efficiencies of 17.07% for the KI_Cas9 group, 14.58% for KI_Cas12a-NT-ssODN, and 12.37% for KI_Cas12a-T-ssODN. Although AsCas12a induced fewer mutations per genome than SpCas9, the phenotypic impact on transcription and expression of omega-1 was similar for both nucleases.

Lymphatic trafficking of particles to the secondary lymphoid organs, such as lymph nodes, and the cell types that particles access are critical factors that control the quality and quantity of immune responses. In this study, we evaluated the effect of PEGylation on the lymphatic trafficking and accumulation of particles in draining lymph nodes (dLNs) as well as the cell types that internalized particles. As a model system, 200 nm polystyrene (PS) particles were modified with different densities of poly(ethylene glycol) (PEG) and administered subcutaneously to mice. PEGylation enhanced the efficiency of particle drainage away from the injection site as well as the access of particles to dendritic cells (DCs). The accumulation of particles in dLNs was dependent on the PEG density. PEGylation also enhanced uptake by DCs while reducing internalization by B cells at the single cell level. Our results indicate that PEGylation facilitated the trafficking of particles to dLNs either through enhanced trafficking in lymphatic vessels or by enhanced internalization by migratory DCs. This study provides insight into utilizing PEGylated particles for the development of synthetic vaccines.

Biomaterials interface with toll-like receptor (TLR) 9-mediated innate immunity in a wide range of medical applications, such as tissue implants and drug delivery systems. The stimulation of TLR9 can lead to two different signaling pathways, resulting in the generation of proinflammatory cytokines (i.e. IL-6) and/or type I interferons (IFNs, i.e. IFN-α). These two categories of cytokines differentially influence both innate and adaptive immunity. Although particle size is known to be a critical parameter of biomaterials, its role in TLR9-mediated cytokine profiles is not clear. Here, we examined how the size of biomaterials impacted cytokine profiles by using polystyrene particles of defined sizes as model carriers for TLR9 agonists (CpG oligonucleotides (CpG ODNs)). CpG ODNs bound to nano- to submicro- particles stimulated the production of both IL-6 and IFN-α, while those bound to microparticles resulted in IL-6 secretions only. The differential TLR9-mediated cytokine profiles were attributed to the pH of endosomes that particles trafficked to. The magnitude of IFN-α production was highly sensitive to the change in endosomal pH in comparison to that of IL-6. Our results define two critical design variables, size and the ability to modulate endosomal pH, for the engineering of biomaterials that potentially interface with TLR9-mediated innate immunity. The fine control of these two variables will allow us to fully exploit the beneficial facets of TLR9-mediated innate immunity while minimizing undesirable side effects.