Nanoplatforms for biomolecule delivery to the lymph nodes have attracted considerable interest as vectors for immunotherapy. Core-shell iron oxide nanoparticles are particularly appealing because of their potential as theranostic magnetic resonance imaging (MRI)-trackable vehicles for biomolecule delivery. The key challenge for utilizing iron oxide nanoparticles in this capacity is control of their coating shells to produce particles with predictable size. Size determines both the carrier capacity for biomolecule display and the carrier ability to target the lymph nodes. In this study, we develop a novel coating method to produce core-shell iron oxide nanoparticles with controlled size. We utilize lipidlike molecules to stabilize self-assembled lipid shells on the surface of iron oxide nanocrystals, allowing the formation of consistent coatings on nanocrystals of varying size (10-40 nm). We further demonstrate the feasibility of leveraging the ensuing control of nanocarrier size for optimizing the carrier functionalities. Coated nanoparticles with 10 and 30 nm cores supported biomolecule display at 10-fold and 200-fold higher capacities than previously reported iron oxide nanoparticles, while preserving monodisperse sub-100 nm size populations. In addition, accumulation of the coated nanoparticles in the lymph nodes could be tracked by MRI and at 1 h post injection demonstrated significantly enhanced lymph node targeting. Notably, lymph node targeting was 9-40 folds higher than that for previously reported nanocarriers, likely due to the ability of these nanoparticles to robustly maintain their sub-100 nm size in vivo. This approach can be broadly applicable for rational design of theranostic nanoplatforms for image-monitored immunotherapy.

Cross-presentation, the process that facilitates display of exogenous antigens on MHC-I molecules, is a crucial step in the cascade of CD8 T cell activation. Potentiation of cross-presentation therefore represents an essential design criterion for development of subunit vaccines that target the induction of CD8 T cell immunity. Covalent conjugation of CpG-DNA to antigenic proteins has shown the potential to promote cross-presentation and has attracted great interest as a promising approach for vaccine development. However, heterogeneous product mixtures that result from typical conjugation schemes precluded identification of active conjugate species and impeded optimization of cross-presentation activity. In this report, we explore the effect of molecular composition of antigen-CpG conjugates on their cross-presentation activity using model Ovalbumin (OVA)-CpG conjugates. We developed a method to generate antigen-CpG conjugates with defined molecular compositions and leveraged this method to produce a series of OVA-CpG conjugates with one, two, and three CpG molecules linked to OVA. We observed that conjugates containing one CpG per OVA enhanced cross-presentation by 4-fold compared to native OVA, while conjugates with higher contents of CpG provided no cross-presentation enhancement. These differences are likely due to enhanced aggregation propensity observed for conjugates that carry more than one CpG per OVA. Our findings suggest that tuning molecular composition of antigen-CpG conjugates to maintain physical stability may be essential for achieving potent cross-presentation activity. Our method to generate defined conjugates could facilitate such molecular tuning and may be useful for continued development of antigen-CpG vaccines.

Background Canine hemangiosarcoma (HSA) is an aggressive cancer arising from multipotential bone marrow-derived stem cells. Anthracycline chemotherapy drugs have been the mainstay adjuvant chemotherapy following surgery with only modest improvement in survival and an attendant risk for adverse events. Immunotherapy, using a whole cell autologous cancer vaccine adjuvanted with MIM-SIS, may improve outcomes for dogs with HSA with a lower risk for adverse events compared with chemotherapy. Results In cultured DH82 canine monocyte-like cells, autologous cancer vaccines prepared from 13 dogs with HSA increased MHC-II surface expression ranging from 20.0-60.4% on single-stained cells, CD80 surface expression ranging from 23.7–45.9% on single-stained cells, and MHC-II/CD80 surface expression ranging from 7.2–20.1% on double-stained cells. Autologous cancer vaccines were able to, on average, stimulate an up-regulation of MHC-II and CD80 by 48-fold as compared to media only (MHC-II + CD80 + cells: 12.19 ± 3.70% vs. 0.25 ± 0.06%; p < 0.001). The overall median survival time for dogs treated with the autologous cancer vaccine was 142 days (range, 61 to 373 days). Dogs treated with the autologous cancer vaccine or maximum tolerated dose (MTD) chemotherapy had significantly (P < 0.001) longer survival than dogs treated with surgery alone. The 1-year survival rate was 12.5% for dogs treated with the autologous cancer vaccine, and 0% for dogs treated with surgery alone or MTD chemotherapy. No adverse events were observed in the dogs treated with the autologous cancer vaccine. Conclusions The adjuvanted autologous cancer vaccine is capable of up-regulating MHC-II and CD80 in cultured canine monocyte-derived cells, which are important stimulatory molecules in generating an immune response and improves survival time in dogs with metastatic (stage III) HSA when compared to surgical treatment alone. Autologous cancer vaccine-treated dogs had survival similar to those dogs treated with MTD chemotherapy without any observed adverse events. This autologous cancer vaccine represents an effective form of individualized immunotherapy that is an appealing option for dog owners not wanting to pursue adjuvant chemotherapy for HSA.

Although the functionalization of magnetic nanoparticles (MNPs) with biomolecules has been widely explored for various biological applications, achieving efficient bioconjugations with a wide range of biomolecules through a single, universal, and versatile platform remains a challenge, which may significantly impact their applications' outcomes. Here, we report a novel MNP platform composed of Au@Fe core/satellite nanoparticles (CSNPs) for versatile and efficient bioconjugations. The engineering of the CSNPs is facilely formed through the self-assembly of ultrasmall gold nanoparticles (AuNPs, 2−3 nm in diameter) around MNPs with a polysiloxane-containing polymer coating. The formation of the hybrid magnetic nanostructure is revealed by absorption spectroscopy, dynamic light scattering (DLS), transmission electron microscopy (TEM), element analysis using atomic absorption spectroscopy, and vibrating sample magnetometer. The versatility of biomolecule loading to the CSNP is revealed through the bioconjugation of a wide range of relevant biomolecules, including streptavidin, antibodies, peptides, and oligonucleotides. Characterizations including DLS, TEM, lateral flow strip assay, fluorescence assay, giant magnetoresistive nanosensor array, high-performance liquid chromatography, and absorption spectrum are performed to further confirm the efficiency of various bioconjugations to the CSNP. In conclusion, this study demonstrates that the CSNP is a novel MNP-based platform that offers versatile and efficient surface functionalization with various biomolecules.

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