Quantifying nanodiamonds biodistribution in whole cells with correlative iono-nanoscopy.

Correlative imaging and quantification of intracellular nanoparticles with the underlying ultrastructure is crucial for understanding cell-nanoparticle interactions in biological research. However, correlative nanoscale imaging of whole cells still remains a daunting challenge. Here, we report a straightforward nanoscopic approach for whole-cell correlative imaging, by simultaneous ionoluminescence and ultrastructure mapping implemented with a highly focused beam of alpha particles. We demonstrate that fluorescent nanodiamonds exhibit fast, ultrabright and stable emission upon excitation by alpha particles. Thus, by using fluorescent nanodiamonds as imaging probes, our approach enables quantification and correlative localization of single nanodiamonds within a whole cell at sub-30 nm resolution. As an application example, we show that our approach, together with Monte Carlo simulations and radiobiological experiments, can be employed to provide unique insights into the mechanisms of nanodiamond radiosensitization at the single whole-cell level. These findings may benefit clinical studies of radio-enhancement effects by nanoparticles in charged-particle cancer therapy.

Recent advances and applications of synthetic diamonds in solid-phase extraction and high-performance liquid chromatography.

Since the advent of diamond-based adsorbents in the late 1960s, the interest in their use for solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) has steadily increased. This is primarily due to their unique properties, such as extreme chemical and thermal stability, high mechanical strength and biocompatibility, and complex mixed-mode retention mechanisms. Currently, the most commonly used synthetic diamonds in SPE and HPLC are detonation nanodiamonds (DND), high-pressure high-temperature (HPHT) diamonds, and chemical vapour deposition (CVD) diamonds. These diamonds have been either used as individual particles (in both modified and unmodified forms), or for surface modification, or entrapped within composites and core-shell particles to develop new diamond-based adsorbents. These diamond-based adsorbents have been used for a variety of applications, including streamlined proteome analysis; extraction of anions, cations, actinides, uranium, lanthanides, alkaline earth metals, transition metals, and post-transition metals; and development of reversed-phase, normal phase, hydrophilic interaction, ion chromatography, and mixed-mode liquid chromatography columns, to name but a few. These varied applications of different types of diamonds are typically governed by their specific properties. This review discusses the various surface and bulk properties of DND, HPHT diamonds, and CVD diamonds that facilitate or limit their use in different SPE and HPLC based applications.

General top-down strategy for generating single-digit nanodiamonds for bioimaging.

A variety of techniques exist for synthesizing nanodiamonds. However, it is challenging to produce nanoparticles with a size smaller than 4 nm without aggregation and large volumes of colloidal solutions containing single-digit nanodiamonds. In this study, we demonstrate a facile top-down strategy for the fabrication of monodisperse colloidal fluorescent nanodiamonds with a mean size of 3.6 nm from a suspension of commercial high-pressure and high-temperature diamond microcrystals (raw industrial materials) in an ambient environment using laser ablation in liquids. The formation of colloidal nanodiamonds is ascribed to a mechanism in which diamond microcrystals are first converted into disordered carbon nanoparticles through laser ablation. Subsequently, the amorphous carbon nanoparticles as an intermediate phase are converted into the final nanodiamonds under laser irradiation. Through the in situ covalent linking of ester and ketone groups on the surfaces of the nanodiamonds, tunable, high-performance fluorescence bioimaging can be achieved. The results suggest that single-digit fluorescent nanodiamonds can be generated from colloidal solutions.

Toxicity of nanodiamonds to white rot fungi Phanerochaete chrysosporium through oxidative stress.

Nanodiamonds (NDs) are produced with large scale and applied in many areas, thus the environmental impacts and hazards of NDs should be systematically investigated. In this study, we evaluated the interaction between detonation NDs and white rot fungus Phanerochaete chrysosporium and the impact on the fungus decompositions activities. NDs did not influence the biomass gain of P. chrysosporium and the culture medium pH values. The mycelia of P. chrysosporium were destroyed upon the direct contact with NDs, while the rest retained the fibrous structure. Ultrastructural observations suggested that small aggregates of NDs seldom entered the fungus cells, but the break of cell wall and the loss of cytoplasm were induced by NDs. Under both optical and electron microscopes, the aggregation of colloidal ND particles was observed, which was the possible reason of low toxicity. High concentrations of NDs inhibited the laccase activity and manganese peroxidase activity of P. chrysosporium, which led to the decrease of decomposition activity for pollutants. Colloidal ND particles were not well dispersed in sawdust degradation evaluations, so no inhibitive effect was observed for wood degradation. The toxicological mechanism of NDs was assigned to oxidative stress. The results collectively suggested that NDs had low toxicity to white rot fungi and could be applied safely. The colloid dispersion/aggregation of nanoparticles in biological systems should be carefully considered during the design of safe nanomaterials.

Nanodiamond-supported silver nanoparticles as potent and safe antibacterial agents.

Since its discovery nearly a century ago, antibiotics has been one of the most effective methods in treating infectious diseases and limiting pathogen spread. However, pathogens often build up antibiotic resistance over time, leading to serious failure of the treatment. Silver nanoparticle (AgNP) is an appealing alternative, but successful treatment of the bacterial infection requires a plentiful supply of AgNP, which can negatively impact human health if people are excessively exposed to the particles. Here, we present a method to overcome this challenge by synthesizing nanodiamond-supported AgNP noncovalently conjugated with albumin molecules to achieve enhanced antibacterial activity and strengthened biocompatibility. Using Escherichia coli as a model bacterium, we found that the albumin-conjugated silver-diamond nanohybrids showed a long-term bactericidal effect after 36 days of the treatment at the AgNP concentration of 250 µg mL-1. Moreover, the toxicity of the nanohybrids to human cells (including human fibroblasts, lung adenocarcinoma epithelial cells, and breast adenocarcinoma cells) is low even at the particle concentration of 500 µg mL-1. The method provides a general and practical solution to the concerns of bacterial resistance against AgNP and issues associated with the size, shape, aggregation, and toxicity of AgNP are largely resolved. Finally, we demonstrate that the nanohybrids can be readily incorporated into natural polysaccharides (such as guar gum) to form three-in-one hydrogels, showing promising applications in nanomedicine.

Unraveling In Vivo Brain Transport of Protein-Coated Fluorescent Nanodiamonds.

Nanotheranostics, combining diagnostics and therapy, has the potential to revolutionize treatment of neurological disorders. But one of the major obstacles for treating central nervous system diseases is the blood-brain barrier (BBB) preventing systemic delivery of drugs and optical probes into the brain. To overcome these limitations, nanodiamonds (NDs) are investigated in this study as they are a powerful sensing and imaging platform for various biological applications and possess outstanding stable far-red fluorescence, do not photobleach, and are highly biocompatible. Herein, fluorescent NDs encapsulated by a customized human serum albumin-based biopolymer (polyethylene glycol) coating (dcHSA-PEG) are taken up by target brain cells. In vitro BBB models reveal transcytosis and an additional direct cell-cell transport via tunneling nanotubes. Systemic application of dcHSA-NDs confirms their ability to cross the BBB in a mouse model. Tracking of dcHSA-NDs is possible at the single cell level and reveals their uptake into neurons and astrocytes in vivo. This study shows for the first time systemic NDs brain delivery and suggests transport mechanisms across the BBB and direct cell-cell transport. Fluorescent NDs are envisioned as traceable transporters for in vivo brain imaging, sensing, and drug delivery.

Preferential Tumor Accumulation of Polyglycerol Functionalized Nanodiamond Conjugated with Cyanine Dye Leading to Near-Infrared Fluorescence In Vivo Tumor Imaging.

Preferential accumulation of nanoparticles in a tumor is realized commonly by combined effects of active and passive targeting. However, passive targeting based on an enhanced permeation and retention (EPR) effect is not sufficient to observe clear tumor fluorescence images in most of the in vivo experiments using tumor-bearing mice. Herein, polyglycerol-functionalized nanodiamonds (ND-PG) conjugated with cyanine dye (Cy7) are synthesized and it is found that the resulting ND-PG-Cy7 is preferentially accumulated in the tumor, giving clear fluorescence in in vivo and ex vivo fluorescence images. One of the plausible reasons is the longer in vivo blood circulation time of ND-PG-Cy7 (half-life: 58 h determined by the pharmacokinetic analysis) than that of other nanoparticles (half-life: <20 h in most of the previous reports). In a typical example, the fluorescence intensity of tumors increases due to continuous tumor accumulation of ND-PG-Cy7, even more than one week postinjection. This may be owing to the stealth effect of PG that was reported previously, avoiding recognition and excretion by reticuloendothelial cells, which are abundant in liver and spleen. In fact, the fluorescence intensities from the liver and spleen is similar to those from other organs, while the tumor exhibits much stronger fluorescence in the ex vivo image.

Multiwall and bamboo-like carbon nanotubes from the Allende chondrite: A probable source of asymmetry.

This study presents multiwall and bamboo-like carbon nanotubes found in samples from the Allende carbonaceous chondrite using high-resolution transmission electron microscopy (HRTEM). A highly disordered lattice observed in this material suggests the presence of chiral domains in it. Our results also show amorphous and poorly-graphitized carbon, nanodiamonds, and onion-like fullerenes. The presence of multiwall and bamboo-like carbon nanotubes have important implications for hypotheses that explain how a probable source of asymmetry in carbonaceous chondrites might have contributed to the enantiomeric excess in soluble organics under extraterrestrial scenarios. This is the first study proving the existence of carbon nanotubes in carbonaceous chondrites.

Polyaniline-grafted nanodiamonds for efficient photothermal tumor therapy.

Polyaniline-grafted nanodiamond (PAN-ND) nanoparticles were fabricated by polymerizing aniline at the surface of amine-modified NDs for efficient photothermal therapy (PTT). A series of PAN from different aniline concentrations were also prepared to compare the properties and the efficiency of PTT. The polymerization rate of aniline was faster in the presence of NDs than that of aniline alone. Compared to PAN nanoparticles, PAN-ND has a spherical shape, smaller size, and ultimately higher cellular uptake efficiency. The temperature of aqueous PAN-ND dispersion increased to 44.4 °C after laser irradiation for 5 min. In addition, the UV absorbance intensity of PAN-ND increased at the lower pH at the near infrared (NIR) region, resulting in an enhanced photothermal effect at a tumor site. Notably, the viability of HeLa cells treated with PAN-ND decreased by less than 20%, suggesting the high efficiency of PTT. The PAN-ND can be a potential candidate for efficient photothermal tumor therapy.

Amphipathic Nanodiamond Supraparticles for Anticancer Drug Loading and Delivery.

Nanodiamonds (NDs) have been attracting considerable attention due to their outstanding chemical, physical, and physiological properties. Additional functionalization of NDs can be carried out by the self-assembly technique. This study reports a straightforward chemical route for self-assembled supraparticles (SPs) based on ND (ND-SPs) using alkyl carboxylic acids with different aliphatic alkyl chain lengths by carbodiimide chemistry and sonication. Poly(ethylene glycol) (PEG)-modified ND-SPs are synthesized successfully for effective nanodrug formulations with the hydrophobic anticancer drug paclitaxel (PTX). The properties of these ND-SP nanomedicines are investigated thoroughly by complementary analytical, spectroscopic, and microscopic techniques. This simple methodology permitted the application of PEG-modified ND-SP-encapsulating PTX as a potent drug carrier, achieving greater efficacy than commercial Abraxane. Results revealed that the morphology, particle size, and water dispersibility of the prepared ND-SP nanoclusters affect the drug efficacy. These PEG-modified ND-SP nanoclusters serve as novel nanomedicine for a passive drug delivery system as well as anticancer chemotherapy.

Nanodiamond/poly-ε-caprolactone nanofibrous scaffold for wound management.

We describe preparation, characterization and cytocompatibility of nanodiamond (ND) dispersed in poly (ε-caprolactone) (PCL) based nanofibrous scaffold. The results show that this unique scaffold potentially provides essential properties for wound healing by enhancing proliferation of epithelial cells, in addition to restricting the microbial activities. Electrospinning technique was used to fabricate and develop PCL-NDs nanocomposite scaffold. The developed nanocomposites were characterized for morphology, thermal, surface and biological properties. The incorporation of ND into the PCL matrix resulted in better moisture management and higher thermal stability. Transmission electron microscopy images and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy showed existence of ND particles on the surface of the nanofibers. The aggregation of ND particles increased with the increase in their concentration in nanofiber. The developed scaffolds showed no cytotoxicity and, due to improved hydrophilicity, better cellular activities with Chinese hamster ovarian (CHO) cells, 43%, 38% and 22% more cell proliferation for PCL-5% ND for 1, 3- and 7-days incubations in compare with PCL. Furthermore, Staphylococcus aureus (S. aureus) showed significantly less affinity to the scaffold surface with the increase in ND concentration, ~56% less for PCL-5% ND in compare with PCL, indicating that such ND dispersed nanofibrous scaffold maybe asuitable choice for complex wound management.

Tumor selective uptake of drug-nanodiamond complexes improves therapeutic outcome in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer-related deaths and novel treatment approaches are urgently needed. Here we show that poly(ethylene glycol)-functionalized nanodiamonds loaded with doxorubicin (ND-PEG-DOX) afforded a considerable improvement over free drug in an orthotopic pancreatic xenograft model. ND-PEG-DOX complexes were also superior to free DOX in 3-dimensional (3D) tumor spheroids of PDAC. ND-PEG showed no cytotoxicity towards macrophages, and histopathological analysis showed no abnormalities of major organs upon in vivo administration of ND-PEG-DOX. These results provide evidence that ND-mediated drug delivery may serve as a means of improving the therapeutic outcome in PDAC.

High-Contrast Imaging of Nanodiamonds in Cells by Energy Filtered and Correlative Light-Electron Microscopy: Toward a Quantitative Nanoparticle-Cell Analysis.

Fluorescent nanodiamonds (fNDs) represent an emerging class of nanomaterials offering great opportunities for ultrahigh resolution imaging, sensing and drug delivery applications. Their biocompatibility, exceptional chemical and consistent photostability renders them particularly attractive for correlative light-electron microscopy studies providing unique insights into nanoparticle-cell interactions. Herein, we demonstrate a stringent procedure to image and quantify fNDs with a high contrast down to the single particle level in cells. Individual fNDs were directly visualized by energy-filtered transmission electron microscopy, that is, inside newly forming, early endosomal vesicles during their cellular uptake processes as well as inside cellular organelles such as a mitochondrion. Furthermore, we demonstrate the unequivocal identification, localization, and quantification of individual fNDs in larger fND clusters inside intracellular vesicles. Our studies are of great relevance to obtain quantitative information on nanoparticle trafficking and their various interactions with cells, membranes, and organelles, which will be crucial to design-improved sensors, imaging probes, and nanotherapeutics based on quantitative data.

Doxorubicin-polyglycerol-nanodiamond composites stimulate glioblastoma cell immunogenicity through activation of autophagy.

Immunosuppression is a salient feature of GBM associated with the disease's grim prognosis and the limited success of anti-GBM immunotherapy. Stimulating immunogenicity of the GBM cells (GC) is a promising approach to subverting the GBM-associated immunosuppression. We had previously devised a drug composite based on polyglycerol-functionalized nanodiamonds bearing doxorubicin (Nano-DOX) and demonstrated that Nano-DOX effectively modulated GBM's immunosuppressive microenvironment through stimulating the immunogenicity of GC and initiated anti-GBM immune responses. The present study now explored the mechanism of Nano-DOX's immunostimulatory action. Nano-DOX was found to induce autophagy rather than apoptosis in GC and stimulated GC to emit antigens and damage-associated molecular patterns (DAMPs) that are potent adjuvants, which resulted in enhanced activation of dendritic cells (DC). Heightened autophagosome release was observed in Nano-DOX-treated GC but was shown not to be a major channel of antigen donation. Blocking autophagy in GC not only reduced Nano-DOX-stimulated GC antigen donation and DAMPs emission, but also efficiently attenuated DC activation stimulated by Nano-DOX-treated GC. Taken together, these findings suggest that activation of autophagy is a central mechanism whereby Nano-DOX stimulates GC's immunogenicity. Our work provides new insight on how nanotechnology can be applied to therapeutically modulate the GBM immune microenvironment by harnessing autophagy in the cancer cells. STATEMENT OF SIGNIFICANCE: Immunosuppression is a salient feature of GBM associated with the grim prognosis of the disease and the limited success of anti-GBM immunotherapy. We demonstrated that Doxorubicin-polyglycerol-nanodiamond composites could activate autophagy in GBM cells and thereby stimulate the immunogenecity of GBM cells. This discovery 1, sheds new light on how nanotechnology could be applied to therapeutically modulate the tumor immune microenvironment, and 2, provides a powerful tool for subverting the GBM's immunosuppressive microenvironment, which has great therapeutic potential for the treatment of GBM.

Recognition protein C1q of innate immunity agglutinates nanodiamonds without activating complement.

Nanodiamonds are promising nanomedicines for diagnostic and therapeutic applications. As nanodiamonds are mainly administered intravenously, it is critical to understand the humoral immune response upon exposure to nanodiamonds. Here, we report the interactions of pristine, oxidized, and PEG-functionalized nanodiamonds with human complement, an important part of our humoral innate immunity. In particular, we report the nanodiamond binding properties of the recognition protein of the classical complement pathway: C1q, which also takes part in many other physiological and pathological processes. Our results show similar trends in the effects of C1q on the three types of nanodiamonds. Complement activation assays using human serum show that the nanodiamonds trigger slight activities via the alternative pathway and no response via the classical pathway. Nevertheless, surface plasmon resonance shows that C1q binds the nanodiamonds and transmission electron microscopy reveals their agglutination. Studies with macrophages further show that C1q attachment affects their phagocytosis and cytokine response.

Pilot study on biocompatibility of fluorescent nanodiamond-(NV)-Z~800 particles in rats: safety, pharmacokinetics, and bio-distribution (part III).

INTRODUCTION: We hereby report on studies aimed to characterize safety, pharmacokinetics, and bio-distribution of fluorescent nanodiamond particles (NV)-Z~800 (FNDP-(NV)) administered to rats by intravenous infusion in a single high dose. METHODS: Broad scale biological variables were monitored following acute (90 minutes) and subacute (5 or 14 days) exposure to FNDP-(NV). Primary endpoints included morbidity and mortality, while secondary endpoints focused on hematology and clinical biochemistry biomarkers. Particle distribution (liver, spleen, lung, heart, and kidney) was assessed by whole organ near infrared imaging using an in vivo imaging system. This was validated by the quantification of particles extracted from the same organs and visualized by fluorescent and scanning electron microscopy. FNDP-(NV)-treated rats showed no change in morbidity or mortality and preserved normal motor and sensory function, as assessed by six different tests. RESULTS: Blood cell counts and plasma biochemistry remained normal. The particles were principally distributed in the liver and spleen. The liver particle load accounted for 51%, 24%, and 18% at 90 minutes, 5 days, and 14 days, respectively. A pilot study of particle clearance from blood indicated 50% clearance 33 minutes following the end of particle infusion. CONCLUSION: We concluded that systemic exposure of rats to a single high dose of FDNP-(NV)-Z~800 (60 mg/kg) appeared to be safe and well tolerated over at least 2 weeks. These data suggest that FNDP-(NV) should proceed to preclinical development in the near future.

Designing of membrane-active nano-antimicrobials based on cationic copolymer functionalized nanodiamond: Influence of hydrophilic segment on antimicrobial activity and selectivity.

Designing cationic nano-antimicrobial is a promising solution for combating drug resistant microbes. In this work, hydrophilic cationic copolymer was applied for the surface functionalization of nanodiamonds (NDs) aiming at developing a highly membrane-active nano-antibacterial agent with satisfactory selectivity. As a result, after functionalization, the increased repulsive forces within NDs and interaction with solvent molecular network made the heavily aggregated pristine NDs break down into tiny nanoparticles with particle size ranging from 10 to 100 nm. The improved hydrophilicity and enlarged surface area endowed QND-H5 and QND-H10 a powerful bactericidal capability toward both of Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus). In the further bactericidal assessment, it was also demonstrated that the formation of hydrogen bonding between the 2-hydroxyethyl methacrylate (HEMA) side chains and lipid head groups of bacterial membrane also contributed to the enhanced bactericidal ability. Field emission scanning electron microscopy analysis confirmed that as-prepared nano-hybrid acted bactericidal ability via physical nature of outer membrane and cytoplasmic membrane-separating destruction mechanism toward E. coli, which may derive from the hydrogen bonding ability, making them more effective toward bacterial. More importantly, it was found that with just 10% of HEMA, QND-H10 displayed good selectivity toward bacteria over mammalian cells as shown by the high HC50 values with relatively low MIC values, suggesting the great potential application in medical fields. These results indicate that hydrogen bonding is an important element to achieve the desired high antibacterial activity and selectivity, particularly when cationic nano-antibacterial agents are required for medical application.

Preparation of water dispersible and biocompatible nanodiamond-poly(amino acid) composites through the ring-opening polymerization.

Nanodiamond (ND) is one of the most fascinating carbon materials that have been extensively investigated for biomedical applications owing to its small size, high specific surface areas, chemical inert and desirable biocompatibility. It has been reported that surface modification of ND with polymers could not only improve the dispersibility of final ND based composites but also endow them novel functions to fulfill the requirement for biomedical applications. Although some strategies have been developed previously, surface modification of ND with poly(amino acid)s has not been reported previously. In this work, poly(amino acid)s functionalized ND composites were fabricated through a ring-opening polymerization of α-amino acid N-carboxyanhydrides (NCAs), which was synthesized by conjugation of hydrophilic ethylene glycol with glutamic acid. The successful preparation of ND-GluEG composites was confirmed by a series of characterization techniques. The results suggest that the water dispersibility of final ND-GluEG composites is obviously improved. Moreover, ND-GluEG composites show low toxicity and are of great potential for biomedical applications.

Fluorescent Nanodiamonds in Biomedical Applications.

Nanoparticles have an extended surface and a large surface area, which is the ratio of the size of the surface area to the volume. A functionalized surface can give rise to more modifications and therefore allows this nanomaterial to have new properties. Fluorescent molecules contain fluorophore, which is capable of being excited via the absorption of light energy at a specific wavelength and subsequently emitting radiation energy of a longer wavelength. A chemically modified surface of nanodiamond (ND; by carboxylation) demonstrated biocompatibility with DNA, cytochrome C, and antigens. In turn, fluorescent nanodiamonds (FNDs) belong to a group of new nanomaterials. Their surface can be modified by joining functional groups such as carboxyl, hydroxyl, or amino, after which they can be employed as a fluorescence agent. Their fluorescent properties result from defects in the crystal lattice. FNDs reach dimensions of 4-100 nm, have attributes such as photostability, long fluorescence lifetimes (10 ns), and fluorescence emission between 600 and 700 nm. They are also nontoxic, chemically inert, biocompatible, and environmentally harmless. The main purpose of this article was to present the medical applications of various types of modified NDs.

Nanodiamond enhances immune responses in mice against recombinant HA/H7N9 protein.

BACKGROUND: The continuing spread of the newly emerged H7N9 virus among poultry in China, as well as the possibility of human-to-human transmission, has attracted numerous efforts to develop an effective vaccine against H7N9. The use of nanoparticles in vaccinology is inspired by the fact that most pathogens have a dimension within the nano-size range and therefore can be processed efficiently by the immune system, which leads to a potent immune response. Herein, we report a facile approach to increase antigen size to achieve not only fast but also effective responses against the recombinant HA/H7N9 protein via a simple conjugation of the protein onto the surface of nanodiamond particles. RESULTS: In this study, trimeric Haemagglutinin (H7) that is transiently expressed in N. benthamiana was purified using affinity chromatography, and its trimeric state was revealed successfully by the cross-linking reaction. The trimeric H7 solution was subsequently mixed with a nanodiamond suspension in different ratios. The successful conjugation of the trimeric H7 onto the surface of nanodiamond particles was demonstrated by the changes in size and Zeta-potential of the particles before and after protein coating, Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and Western-blot analysis. Next, biofunction of the protein-nanodiamond conjugates was screened using a haemagglutination assay. A mixture containing 5 µg of trimeric H7 and 60 µg of nanodiamond corresponds to a ratio of 1:12 (w/w) of agglutinated chicken red blood cells at HA titer of 1024, which is 512-fold higher than the HA titer of free trimeric H7. After the 2nd and 3rd immunization in mice, ELISA and Western blot analyses demonstrated that the physical mixture of trimeric H7 protein and nanodiamond (1:12, w/w) elicited statistically significant stronger H7-specific-IgG response demonstrated by higher amounts of H7N9-specific IgG (over 15.4-fold with P < 0.05 after the second immunization). CONCLUSIONS: These results indicated a potential effect inherent to nanodiamond towards modulating immune systems, which should be further evaluated and broadly applied in nanovaccine development.