Particles engineered to engage and interact with cell surface ligands and to modulate cells can be harnessed to explore basic biological questions as well as to devise cellular therapies. Biology has inspired the design of these particles, such as artificial antigen-presenting cells (aAPCs) for use in immunotherapy. While much has been learned about mimicking antigen presenting cell biology, as we decrease the size of aAPCs to the nanometer scale, we need to extend biomimetic design to include considerations of T cell biology-including T-cell receptor (TCR) organization. Here we describe the first quantitative analysis of particle size effect on aAPCs with both Signals 1 and 2 based on T cell biology. We show that aAPCs, larger than 300 nm, activate T cells more efficiently than smaller aAPCs, 50 nm. The 50 nm aAPCs require saturating doses or require artificial magnetic clustering to activate T cells. Increasing ligand density alone on the 50 nm aAPCs did not increase their ability to stimulate CD8+ T cells, confirming the size-dependent phenomenon. These data support the need for multireceptor ligation and activation of T-cell receptor (TCR) nanoclusters of similar sizes to 300 nm aAPCs. Quantitative analysis and modeling of a nanoparticle system provides insight into engineering constraints of aAPCs for T cell immunotherapy applications and offers a case study for other cell-modulating particles.

Development of nontoxic, tumor-targetable, and potent in vivo RNA delivery systems remains an arduous challenge for clinical application of RNAi therapeutics. Herein, we report a versatile RNAi nanoplatform based on tumor-targeted and pH-responsive nanoformulas (NFs). The NF was engineered by combination of an artificial RNA receptor, Zn(II)-DPA, with a tumor-targetable and drug-loadable hyaluronic acid nanoparticle, which was further modified with a calcium phosphate (CaP) coating by in situ mineralization. The NF can encapsulate small-molecule drugs within its hydrophobic inner core and strongly secure various RNA molecules (siRNAs, miRNAs, and oligonucleotides) by utilizing Zn(II)-DPA and a robust CaP coating. We substantiated the versatility of the RNAi nanoplatform by demonstrating effective delivery of siRNA and miRNA for gene silencing or miRNA replacement into different human types of cancer cells in vitro and into tumor-bearing mice in vivo by intravenous administration. The therapeutic potential of NFs coloaded with an anticancer drug doxorubicin (Dox) and multidrug resistance 1 gene target siRNA (siMDR) was also demonstrated in this study. NFs loaded with Dox and siMDR could successfully sensitize drug-resistant OVCAR8/ADR cells to Dox and suppress OVCAR8/ADR tumor cell proliferation in vitro and tumor growth in vivo. This gene/drug delivery system appears to be a highly effective nonviral method to deliver chemo- and RNAi therapeutics into host cells.

No abstract

Polyelectrolyte complex (PEC) nanoparticles assembled from plasmid DNA (pDNA) and polycations such as linear polyethylenimine (lPEI) represent a major nonviral delivery vehicle for gene therapy tested thus far. Efforts to control the size, shape, and surface properties of pDNA/polycation nanoparticles have been primarily focused on fine-tuning the molecular structures of the polycationic carriers and on assembly conditions such as medium polarity, pH, and temperature. However, reproducible production of these nanoparticles hinges on the ability to control the assembly kinetics, given the nonequilibrium nature of the assembly process and nanoparticle composition. Here we adopt a kinetically controlled mixing process, termed flash nanocomplexation (FNC), that accelerates the mixing of pDNA solution with polycation lPEI solution to match the PEC assembly kinetics through turbulent mixing in a microchamber. This achieves explicit control of the kinetic conditions for pDNA/lPEI nanoparticle assembly, as demonstrated by the tunability of nanoparticle size, composition, and pDNA payload. Through a combined experimental and simulation approach, we prepared pDNA/lPEI nanoparticles having an average of 1.3 to 21.8 copies of pDNA per nanoparticle and average size of 35 to 130 nm in a more uniform and scalable manner than bulk mixing methods. Using these nanoparticles with defined compositions and sizes, we showed the correlation of pDNA payload and nanoparticle formulation composition with the transfection efficiencies and toxicity in vivo. These nanoparticles exhibited long-term stability at −20 °C for at least 9 months in a lyophilized formulation, validating scalable manufacture of an off-the-shelf nanoparticle product with well-defined characteristics as a gene medicine.