Immunoactive dna-tethered nanocomplexes of antigen-cpg and iron-oxide nanoparticles as potential cancer nanovaccines

摘要

Introduction: Cancer vaccines hold promising potential for cancer imunotherapy due to their systemic action, high specificity, limited toxicity and treatment durability. However, several critical challenges impede realization of cancer vaccines in the clinic. These challenges include inadequate immunological priming and inefficient in-vivo delivery to the lymph nodes. To address these challenges, we set out to develop a new two-component nanovaccine. This nanovaccine is composed of CpG-modified model antigen ovalbumin and anti-CpG-modified iron-oxide nanoparticles; components that are tethered through complementary single stranded CpG and anti-CpG DNA. We hypothesized that the covalent modification of the model antigen ovalbumin (OVA) with the immunostimulatory adjuvant CpG would improve immunological priming, while tethering to iron-oxide nanoparticles (NP) would serve as a lymph node delivery vehicle. Herein, we present evidence for the formation and in-vitro functionality of OVA-CpG/anti-CpG-NP nanocomplexes and initial proof-of-concept delivery in-vivo. Materials and Methods: The model nanovaccine was formulated in three stages as depicted in Figure 1. OVA-CpG conjugates were analyzed with anion exchange chromatography and SYBR Gold and BCA quantitative spectrophotometric assays for single stranded DNA (ssDNA) and protein, respectively. Complexation between conjugates and iron-oxide nanoparticles was verified with agarose gel electrophoresis and analysis of zeta-potential. The functionality of the nanovaccine was analyzed with the B3Z T-cell hybridoma assay, a colorimetric assay for ovalbumin-specific T-cell activation in-vitro. Delivery to lymph nodes was performed by subcutaneous injections into the footpad of C57BL/6 mice. Nanoparticles were quantified in excised lymph nodes by Electron Paramagnetic Resonance (EPR) spectroscopy. Results and Discussion: The successful formation of OVA-CpG conjugates was confirmed with anion exchange chromatography and spectrophotometric analysis. The elution of OVA-CpG from the High Q anion exchange column required significantly higher concentrations of NaCl as compared to free OVA (p = 0.001). Furthermore, the SYBR Gold assay specific for ssDNA revealed a significantly higher fluorescent signal for OVA-CpG conjugates as compared to free OVA (p = 0.03). Quantification of ssDNA and protein in Ova-CpG conjugates further confirmed that the conjugates could be formed with defined degree of CpG modification (～1,2 and 3 mote CpG/mole OVA). Nanoparticle functionalization with anti-CpG and subsequent complexation with OVA-CpG to form OVA-CpG/anti-CpG-NP nanocomplexes was confirmed by agarose gel electrophoresis and zeta-potential (-1,8±0.5 mV, -27±1 mV and -40 ±2 mV for NP, anti-CpG-NP, and OVA-CpG/anti-CpG-NP, respectively, p＜0.001). Functional analysis revealed that the OVA-CpG conjugates (1 mole CpG/mole OVA) and OVA-CpG/anti-CpG-NP nanocomplexes enhanced OVA-specific T cell activation by 2.5-fold (23 ± 1 mU, p＜0.001) and 4-fold (37±5 mU, p＜0.001), respectively, as compared to free ovalbumin (9±1 mU). Additionally, EPR quantification revealed delivery of approximately 25% of a subcutaneously injected dose of nanoparticles into axillary lymph nodes in mice 24 hours post-injection. Conclusion: Results presented confirm successful preparation of OVA-CpG/anti-CpG-NP immunoactive DNA-tethered nanocomplexes. In addition, functional in vitro analysis and initial delivery results suggest that these nanocomplexes may combine two highly desirable cancer vaccine properties - enhanced immunological priming and enhanced lymph node delivery. The reported nanocomplexation method could pave the way to development of new potent cancer nanovaccines and warrants further investigation.