NDs before and after mixing with OVA were analyzed for their sizes and zeta potentials. Transmission electron microscopy (TEM) of bare NDs after strong oxidative acid washes showed that the particles were irregular in shape and varied considerably in size (inset in Figure 1A). Dynamic light scattering measurements of these acid-treated NDs in distilled deionized water (DDW) revealed a mean hydrodynamic diameter of ~100 nm with a polydispersity index of 0.12. The average diameter of the particles after mixing with OVA in DDW increased by about 20 nm, an indication that the NDs have been successfully coated with OVA by physical adsorption. The typical zeta potential of the bare ND particles was –45 mV (Figure 1A). As the isoelectric point of OVA is 4.5 , which means that the protein molecules are negatively charged in DDW, the change of the zeta potential from –45 mV of NDs to –23 mV of OVA-ND implied that forces (such as hydrophobic forces) other than electrostatic interaction were involved in the protein adsorption processes.
As a member of nanocarbon family, the surface of NDs can be conveniently modified with a variety of oxygen-containing groups such as –COOH, –COH, –COOC–, etc. by extensive washes in strong oxidative acids. Uniquely, the acid-washed NDs exhibit an exceptionally high affinity for a variety of protein molecules, including bovine serum albumin (BSA), myoglobin, cytochrome c, lysozyme, and luciferase [30–32]. Moreover, the structural integrity of these proteins is retained, as demonstrated by the catalytic activities of lysozyme and luciferase after adsorption onto NDs [31, 32]. Chicken OVA is a phosphorylated glycoprotein consisting of 385 amino acid residues with a molecular weight of 42.7 kDa or a total molecular weight of 45 kDa (including the carbohydrate and phosphate portions) . To evaluate the amount of OVA that could be loaded on the acid-washed NDs, we measured the changes in absorbance of free OVA at 280 nm before and after mixing with the nanoparticles, as shown in Figure 1B. For OVA adsorbed on 100-nm NDs, we determined a protein loading capacity of 0.12 g/g at saturation. Assuming a spherical shape of the adsorbent, this loading capacity suggests that each 100-nm ND (weight of ~1.8 fg/particle) can accommodate more than 3000 OVA molecules on surface.
Prior to the in vivo experiments, we investigated whether or not the OVA-ND conjugates could be readily taken up by APCs in vitro. To address this issue, we chose to use RAW264.7 macrophages as the model cell line [25, 27], which was established from tumors induced by Abelson murine leukemia virus in BALB/c mice . Additionally, in order to observe the cellular uptake in real time, we replaced NDs by FNDs that contain a dense ensemble of nitrogen-vacancy centers (~10 ppm) as fluorophores to facilitate their detection by fluorescence imaging . When excited by a green laser, the FNDs produce far-red emission at ~700 nm where the level of cell autofluorescence is low. Moreover, the nitrogen-vacancy centers in the diamond matrix are magneto-optical with unique spin properties, which enable them to be detected with high sensitivity and exceptional selectivity by magnetic modulation . It should be emphasized here that the surface properties of these two types of particles (NDs and FNDs) are essentially identical to each other. They are surface-modified in the same manner (air oxidation and oxidative acid washes), except that the diamond powders used to prepare FNDs are pretreated with electron irradiation and vacuum annealing to create the color centers .
Figure 2 presents the confocal fluorescence images of RAW264.7 macrophages after incubation with OVA-FNDs at a particle concentration of 100 µg/mL for 4 h. The images were acquired after removal of the excessive amount of the particles by repeated washes with phosphate-buffered saline (PBS), followed by incubation of the cells in culture medium for more than 3 days. As revealed by the fluorescence images, the OVA-FND conjugates could be spontaneously internalized by the cells in culture medium. Moreover, the cells stayed healthy over 72 h of the incubation, despite that a large quantity of FNDs were still present inside the cells. The results are in line with that reported by Eidi, et al. , who made a comparative study on the cytotoxicity of surface-modified FNDs and alum particles. Cell viability assays by the authors using the NSC-34 neuron-like cell line showed that FNDs were non-toxic at the doses of up to 110 µg/mL, whereas alum displayed a distinct toxic or severely toxic effect at all doses used over 1 – 50 µg/mL. The high biocompatibility and photostability of FND makes it useful as a tag for tracking and tracing adjuvant particles in vivo.
Aside from the fluorescence imaging, we further quantified the amount of OVA-FNDs taken up by the macrophages. This was achieved by measuring the fluorescence intensities of the cells dispersed in DDW using the magnetic modulation technique detailed in ref. . It was found that the number of the internalized FNDs scaled nearly linearly with the OVA-FND concentration but gradually leveled off at the particle concentration above 100 µg/mL (Figure 3). For cells incubated with the nanoparticle bioconjugates at 100 µg/mL, the average number of the internalized OVA-FNDs was 208. Since each 100-nm FND can carry more than 3000 OVA molecules as that of 100-nm NDs, this result suggests that the total number of OVA molecules taken up by the individual RAW264.7 macrophages can exceed 6 × 105. These antigens have a high likelihood to be presented on the surface of the murine macrophages and possibly other APCs as well.
The in vivo experiments were started by mixing 5 µL OVA solution (1 mg/mL) with 30 µL ND suspension (2 mg/mL), followed by dispersion of the mixtures in CFA or IFA. The corresponding control experiments consisted of 5 µg OVA only without NDs. Prior to subcutaneous injection of the ND-based adjuvants into BALB/C mice, it is crucial to know the fraction of OVA attached NDs, which could act as a depot of the antigens, thus promoting their cellular uptake by APCs. UV-Vis spectrophotometric analysis of the OVA-ND mixture showed that about 40% of the OVA molecules were attached to NDs and the rest of them were free in solution. They are in dynamic equilibrium with each other. Comparing these values with the protein loading capacity of 0.12 g/g as determined earlier suggests a surface coverage of 27% for OVA on NDs.
Depicted in Figure 4A is the timeline of immunization and blood collection in this experiment. The water-in-oil emulsions formed small nodules and appeared as soft capsules at the injection sites upon immunization. We evaluated the OVA-specific IgG antibody responses with the sera of the immunized mice by using enzyme-linked immunosorbent assays (ELISA) after the second and third immunizations with OVA and OVA/ND in CFA. As shown in Figure 4B and 4C, the OVA/ND/CFA treatments induced a significantly higher amount of OVA-specific IgG antibodies in the mouse sera than OVA/CFA alone (3.5 versus 1.6 folds) after the second and third immunizations. It is demonstrated that the addition of NDs in CFA is able to elicit efficient and protective immune responses against OVA in the mouse body, in good agreement with a previous report with NDs showing enhanced immune responses against recombinant HA/H7N9 in mice .
Next, we investigated the dose dependence of the immune response by employing OVA/CFA and OVA/ND/CFA containing 25 µg OVA each. The amount of NDs used in these assays increased accordingly to 300 µg. Indeed, a 2-fold increase of the OVA-specific IgG antibody production was found in the OVA/CFA treatment (Figure 4A). However, the response did not exceed that of the 5-µg treatment with OVA/ND/CFA. Notably, further increase of the OVA dose failed to boost the immune response in the OVA/ND/CFA treatment. The result pointed toward a saturation effect, where no higher levels of anti-OVA could be reached irrespective of the doses of OVA applied. An important implication of this finding is that the use of NDs as additives in CFA can help reduce the consumption of antigens in producing the antibodies of interest, which is a valuable feature for industrial production of antibodies and vaccines.
We explored further if the same level of immune response by OVA/ND/CFA could be maintained without the need of allergic components such as inactivated mycobacteria in CFA. The dose groups of 5 µg OVA were employed in this experiment. As shown in Figure 5, we found no significant differences in the results between the OVA/ND/IFA and OVA/ND/CFA treatments in these groups, indicating that the substitution of dead mycobacteria by NDs as additives in the mineral oil not only can improve the safety but also can maintain the efficacy of the vaccine adjuvant. This new combination of substances is expected to work properly also as immune drug delivery vehicles to promote directed antitumor activities with minimal systemic toxicity .
The new formulation of NDs in oil emulsions is applicable as antitumor therapeutic agents as well. We demonstrated the applications by using the mouse lymphoma cell lines, EL4 and E.G7-OVA. The latter cells were derived from the C57BL/6 mouse lymphoma cell line (EL4) transfected with pAc-neo-OVA plasmids . They are able to express OVA and have been widely used in cancer immunotherapy studies. Depicted in Figure 6A is the timeline for the subcutaneous injection of OVA/ND/IFA first, followed by inoculation of EL4 and E.G7-OVA cells in C57BL/6 mice. The doses of OVA used in both groups were 5 µg. By referring to the unvaccinated groups, we found that the treatment with OVA/ND/IFA in the EL4 model was unable to delay the tumor growth (Figure 6B). In contrast, the OVA/ND/IFA treatment could effectively inhibit the tumor progression in the E.G7 model over 3 weeks post inoculation of the cells (Figure 6C). Notably, half of the mice (4 out of 7 mice) in the E.G7 model maintained their tumor-free status for more than 15 days after cell inoculation (Figure 6D) and survived up to 35 days post tumor cell challenges (Figure 6E). In Figure 6F, we show photographs of the tumors isolated on day 24 from vaccinated and non-vaccinated mice. The difference in tumor size between these two groups (in triplicate) of mice is substantially, about 10 times in total volume.
We next verified the hypothesis that NDs played an important role in the antitumor therapeutic treatment. The verification was made by replacing NDs with FNDs in the adjuvants and searching for the particles in sacrificed mice by fluorescence detection. In this experiment, we followed the same procedures described above for the OVA/ND/IFA treatment and collected the spleen tissues of the mice inoculated with EL4 and E.G7-OVA cells on day 24 after the vaccination with OVA/FND/IFA. With the aid of magnetic modulation to achieve selective detection of FNDs in the tissue digests without any pre-separation [34, 38], we were able to clearly identify the presence of these particles in mouse spleens by measuring the intensities of the far-red fluorescence from nitrogen-vacancy centers at wavelengths longer than 750 nm. The amount of FNDs found inside the spleens was 0.13 µg, obtained after subtraction of the backgrounds from the signals between these two groups (Figure 6G). Given a total weight of 60 µg for the FNDs used in vaccination, the recovery rate was estimated to be 0.2%. Taken together, the data displayed in Figure 6 strongly corroborate the suggestion that the presently developed nanovaccines with ND/IFA as adjuvants are promising agents for cancer immunotherapy.
To further assess the therapeutic potential of OVA/ND/IFA, we investigated the in vivo immunostimulatory activity of the agent with just one dose in each mouse. Single-dose therapy has several advantages over multiple-dose therapy, including greater patient compliance, less risk of side effects, and lower costs . In particular, in the efforts of protecting livestock (such as cattle, sheep, pigs, and goats) from infectious diseases, single-dose veterinary vaccine renders it easier for suppliers to streamline the production process and distribution of the agents to rural areas . Furthermore, knowing the effectiveness of the single-dose COVID-19 vaccines composed of either whole viruses, protein subunits, viral vectors, or nucleic acids is crucial in the prevention and control of SARS-CoV-2 infections today .
In this single-shot experiment, mice were first administrated with OVA/ND/IFA via subcutaneous injection and then examined by measuring the production of anti-OVA IgG in the mouse sera on a weekly basis. We found that the OVA/ND/IFA treatment could dramatically induce the production of OVA-specific IgG antibodies on day 28 and day 35 after the administration (Figure 7). Compared with the OVA/ND and OVA/IFA groups using the same amount of antigens, the OVA/ND/IFA treatment boosted the levels of anti-OVA IgG by 432 and 6 times on day 28, respectively. The enhancement factor increased to 1717 and 19 times on day 35. It is demonstrated that the addition of NDs can substantially improve the effectiveness of IFA as a single-dose vaccine adjuvant, which is capable of sustaining its immunostimulatory activities over an extended period of time.
Finally, we explored whether or not the addition of NDs in IFA altered the mechanism of the immune response elicited by IFA alone, which is known to proceed predominantly through the Th2 pathway (i.e. humoral immune response) [17, 42]. We addressed the question by performing ELISA assays for cytokines in the sera of C57BL/6 mice after subcutaneous injection with OVA/ND/IFA. As shown in Figure 8, only a small difference in the interleukin 2 (IL-2) level was found between the control and treatment groups, whereas a marked elevation of the interleukin 4 (IL-4) concentration in the vaccinated group was detected. Together with the results obtained with FNDs as described in the previous sections, we were led to a possible predominant mechanism for the initiation of the immune response by the ND/IFA-based vaccine as follows: (i) formation of nodules with loose structure in mouse tissues after subcutaneous injection of the antigen-loaded ND/IFA emulsion, in which the adjuvants act as a depot; (ii) sustained release of the antigens from NDs in the water phase of the emulsions; (iii) active and continuous recruitment of immature immune cells to the depot; (iv) uptake of the antigen-loaded NDs by the immune cells through endocytosis; and (v) promotion of Th2 response, where helper T cells bind with the antigen presenting cells and activate the development of B cells into antibody-producing plasma cells in spleens. The proposed mechanism is depicted in Figure 9.