Sustained antigen availability during germinal center initiation enhances antibody responses to vaccination.

Natural infections expose the immune system to escalating antigen and inflammation over days to weeks, whereas nonlive vaccines are single bolus events. We explored whether the immune system responds optimally to antigen kinetics most similar to replicating infections, rather than a bolus dose. Using HIV antigens, we found that administering a given total dose of antigen and adjuvant over 1-2 wk through repeated injections or osmotic pumps enhanced humoral responses, with exponentially increasing (exp-inc) dosing profiles eliciting >10-fold increases in antibody production relative to bolus vaccination post prime. Computational modeling of the germinal center response suggested that antigen availability as higher-affinity antibodies evolve enhances antigen capture in lymph nodes. Consistent with these predictions, we found that exp-inc dosing led to prolonged antigen retention in lymph nodes and increased Tfh cell and germinal center B-cell numbers. Thus, regulating the antigen and adjuvant kinetics may enable increased vaccine potency.

Dendrimer-Inspired Nanomaterials for the in Vivo Delivery of siRNA to Lung Vasculature.

Targeted RNA delivery to lung endothelial cells has the potential to treat conditions that involve inflammation, such as chronic asthma and obstructive pulmonary disease. To this end, chemically modified dendrimer nanomaterials were synthesized and optimized for targeted small interfering RNA (siRNA) delivery to lung vasculature. Using a combinatorial approach, the free amines on multigenerational poly(amido amine) and poly(propylenimine) dendrimers were substituted with alkyl chains of increasing length. The top performing materials from in vivo screens were found to primarily target Tie2-expressing lung endothelial cells. At high doses, the dendrimer-lipid derivatives did not cause chronic increases in proinflammatory cytokines, and animals did not suffer weight loss due to toxicity. We believe these materials have potential as agents for the pulmonary delivery of RNA therapeutics.

Ionizable amphiphilic dendrimer-based nanomaterials with alkyl-chain-substituted amines for tunable siRNA delivery to the liver endothelium in vivo.

A library of dendrimers was synthesized and optimized for targeted small interfering RNA (siRNA) delivery to different cell subpopulations within the liver. Using a combinatorial approach, a library of these nanoparticle-forming materials was produced wherein the free amines on multigenerational poly(amido amine) and poly(propylenimine) dendrimers were substituted with alkyl chains of increasing length, and evaluated for their ability to deliver siRNA to liver cell subpopulations. Interestingly, two lead delivery materials could be formulated in a manner to alter their tissue tropism within the liver-with formulations from the same material capable of preferentially delivering siRNA to 1) endothelial cells, 2) endothelial cells and hepatocytes, or 3) endothelial cells, hepatocytes, and tumor cells in vivo. The ability to broaden or narrow the cellular destination of siRNA within the liver may provide a useful tool to address a range of liver diseases.

A combinatorial library of bi-functional polymeric vectors for siRNA delivery in vitro.

PURPOSE: To apply a combinatorial chemistry approach toward the design of polymeric vectors, and to evaluate their effectiveness as siRNA delivery systems in vitro. METHODS: Poly(acrylic acid) (pAA) was synthesized via RAFT polymerization with well-controlled molecular weights (M (n): 3 kDa, 5 kDa, 10 kDa and 21 kDa). A polymer library was generated from the pAA precursors by conjugating two distinct moieties, agmatine (Agm) and D-(+)-galactosamine (Gal), at various ratios. Biophysical and cellular characterization was evaluated in vitro for these polymeric vectors using MDA-MB-231-luc+ cells. RESULTS: A critical balance between Agm/Gal content and polymer molecular weight must be attained to achieve favorable transfection efficacies. From the library of 22 polymers, only a few had knockdown efficiencies commensurate with effective siRNA delivery, particularly those with polymer precursor M (n) of 5 kDa and 10 kDa. Highest protein knockdown of 84% was achieved by a polymer conjugate with a 5 kDa pAA backbone with a side chain composition of 55% Agm and 17% Gal. CONCLUSIONS: Effective delivery of siRNA was found to be highly dependent on the molecular structure of the polymeric vector. The combinatorial approach employed provided the tools to identify optimal structural properties leading to efficient siRNA delivery for this class of vector.

An in-depth analysis of polymer-analogous conjugation using DMTMM.

Combinatorial libraries have become increasingly popular in the field of functional biomaterials. One approach for creating diverse polymer libraries is polymer-analogous conjugation of functional groups to polymer scaffolds. In this study, we show that a water-soluble condensing agent, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM), can be employed to conjugate two disparate model ligands, d-(+)-galactosamine (Gal) and agmatine (Agm), to the side chains of either poly(methacrylic acid) (pMAA) or poly(acrylic acid) (pAA) at various substitution ratios. The degree of substitution was found to be directly influenced by media pH, polymer concentration, structure of ligands, and polymer precursor. A nearly 2-fold increase in conjugation efficiencies for both ligands to pAA was achieved as compared to pMAA under identical conditions reaching up to 56% and 78% of Gal and Agm of total content, respectively. These two structurally similar polymers showed remarkably different performances, which reveals that the selection of a polymer precursor is crucial for the optimal design of polymeric libraries, particularly when complex structural ligands are involved. The approach employed provides a basis from which larger and more diverse combinatorial libraries of functionalized polymers with multiple moieties can be generated.

Nucleic acid-mediated intracellular protein delivery by lipid-like nanoparticles.

Intracellular protein delivery has potential biotechnological and therapeutic application, but remains technically challenging. In contrast, a plethora of nucleic acid carriers have been developed, with lipid-based nanoparticles (LNPs) among the most clinically advanced reagents for oligonucleotide delivery. Here, we validate the hypothesis that oligonucleotides can serve as packaging materials to facilitate protein entrapment within and intracellular delivery by LNPs. Using two distinct model proteins, horseradish peroxidase and NeutrAvidin, we demonstrate that LNPs can yield efficient intracellular protein delivery in vitro when one or more oligonucleotides have been conjugated to the protein cargo. Moreover, in experiments with NeutrAvidin in vivo, we show that oligonucleotide conjugation significantly enhances LNP-mediated protein uptake within various spleen cell populations, suggesting that this approach may be particularly suitable for improved delivery of protein-based vaccines to antigen-presenting cells.

Lipid-like nanomaterials for simultaneous gene expression and silencing in vivo.

New lipid-like nanomaterials are developed to simultaneously regulate expression of multiple genes. Self-assembled nanoparticles are capable of efficiently encapsulating pDNA and siRNA. These nanoparticles are shown to induce simultaneous gene expression and silencing both in vitro and in vivo.

Rational design of a biomimetic cell penetrating peptide library.

Cell penetrating peptides have demonstrated potential to facilitate the cellular delivery of therapeutic molecules. Here we develop a set of 50 cell penetrating peptide based formulations with potential to deliver small interfering RNAs intercellularly. The transfection efficacy of siRNA containing lipid-like nanoparticles decorated with different peptides was evaluated both in vitro and in vivo and correlated with the peptide physical and chemical properties. In vitro, these particles were internalized primarily through macropinocytosis. When the peptides were presented to bone marrow-derived dendritic cells, they induce low immunoactivation relative to control cell penetrating peptides including the antennapedia homeodomain and TAT, as quantified by the expression of activation specific surface proteins like CD80, CD86, and major histocompatibility complex class II. In vivo, peptide decorated nanoparticles primarily accumulated in the lungs and the liver. Three human peptides derived from surfactant protein B (a lung surfactant protein), orexin (a neuropeptide hormone, and lactoferricin (a globular glycoprotein) that exist in many physiological fluids facilitated the in vivo delivery of siRNA and induce significant knock down (90%) of a hepatocyte expressed protein, coagulation Factor VII.

Lipid-modified aminoglycoside derivatives for in vivo siRNA delivery.

Rationally designed siRNA delivery materials that are enabled by lipid-modified aminoglycosides are demonstrated. Leading materials identified are able to self-assemble with siRNA into well-defined nanoparticles and induce efficient gene knockdown both in vitro and in vivo. Histology studies and liver function tests reveal that no apparent toxicity is caused by these nanoparticles at doses over two orders of magnitude.

Modular 'click-in-emulsion' bone-targeted nanogels.

A new class of nanogel demonstrates modular biodistribution and affinity for bone. Nanogels, ∼70 nm in diameter and synthesized via an astoichiometric click-chemistry in-emulsion method, controllably display residual, free clickable functional groups. Functionalization with a bisphosphonate ligand results in significant binding to bone on the inner walls of marrow cavities, liver avoidance, and anti-osteoporotic effects.