The success of clinically relevant immunotherapies requires reversing tumor-induced immunosuppression.Here we demonstrated that linear polyethylenimine-based (PEI-based) nanoparticles encapsulating siRNA were preferentially and avidly engulfed by regulatory DCs expressing CD11c and programmed cell death 1-ligand 1 (PD-L1) at ovarian cancer locations in mice. PEI-siRNA uptake transformed these DCs from immunosuppressive cells to efficient antigen-presenting cells that activated tumor-reactive lymphocytes and exerted direct tumoricidal activity, both in vivo and in situ. PEI triggered robust and selective TLR5 activation in vitro and elicited the production of hallmark TLR5-inducible cytokines in WT mice, but not in Tlr5 -/-littermates. Thus, PEI is a TLR5 agonist that, to our knowledge, was not previously recognized. In addition, PEI-complexed nontargeting siRNA oligonucleotides stimulated TLR3 and TLR7. The nonspecific activation of multiple TLRs (specifically, TLR5 and TLR7) reversed the tolerogenic phenotype of human and mouse ovarian tumor-associated DCs. In ovarian carcinoma-bearing mice, this induced T cell-mediated tumor regression and prolonged survival in a manner dependent upon myeloid differentiation primary response gene 88 (MyD88; i.e., independent of TLR3). Furthermore, gene-specific siRNA-PEI nanocomplexes that silenced immunosuppressive molecules on mouse tumor-associated DCs elicited discernibly superior antitumor immunity and enhanced therapeutic effects compared with nontargeting siRNA-PEI nanocomplexes. Our results demonstrate that the intrinsic TLR5 and TLR7 stimulation of siRNA-PEI nanoparticles synergizes with the gene-specific silencing activity of siRNA to transform tumor-infiltrating regulatory DCs into DCs capable of promoting therapeutic antitumor immunity.

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SYNOPSISA new algorithm is presented for the prediction of equilibrium morphology in latex particles.Thermodynamic equilibrium calculations of the interfacial free energies associated with a large array of possible morphologies serve to create a continuous free-energy surface upon which the preferred morphology is located. When the computations are performed on a 486DX33 PC, the calculations are essentially instantaneous. The graphical output can be refreshed on the monitor screen in less than 2 s. The algorithm is applied to simulate the conversion-dependent morphology for several latex systems composed of polystyrene and poly(methy1 methacrylate) and two very different surfactants.

Cross-linking of the seed latex polymer introduces elastic forces into the thermodynamic analysis of the morphology of composite particles. By determining the elastic storage energy necessary to maintain a deformation within the seed latex particle (as in an occlusion of second-stage polymer), it can be combined with the interfacial energies internal to the particle and at its aqueous phase boundary to compute the total free energy of a specific particle morphology. At low levels of cross-linking it is found that the elastic and interfacial energies are of the same order of magnitude, with the final morphology determined by the balance between them. Elastic energies are dependent upon the state of deformation within the particle, and this makes the thermodynamic equilibrium morphology analysis dependent upon cross-link level, seed latex particle size, stage ratio (second polymer/seed polymer), and the interfacial tensions at the polymer/polymer and polymer/water interfaces. Computational results are presented which show the effects of each of these variables on the predicted morphology.