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One-Shot Immunomodulatory Nanodiamond Agents for
Cancer Immunotherapy
Yu Zhang, Zhifen Cui, Huating Kong, Kai Xia, Liang Pan, Jiang Li, Yanhong Sun,
Jiye Shi, Lihua Wang, Ying Zhu,* and Chunhai Fan*
Therapeutic nucleic acids hold great promise in treating a
wide range of diseases. Nevertheless, it remains highly desir-
able to develop new delivery systems to improve their trans-
fection efficiency and in vivo stability for sustained biological
activities. Here, we report on a type of functional nanodiamond
(fND)-based agent for facile assembly and efficient delivery of
immunostimulatory cytosinephosphateguanine (CpG) oli-
gonucleotides (ODNs). We found that complexation of CpG
ODNs with fNDs remarkably increased their cellular uptake
by approximately three orders of magnitudes. Internalized
CpG-fNDs were localized in lysosomes, which bound to Toll-
like receptor 9 (TLR9) and induced prominent cytokine secre-
tion. More significantly, these immunostimulatory nanoagents
exhibited long-term immunoregulatory activities lasting at least
3 d at the cellular level and 2 d in mice, which arise from the
protection and sustained release properties of fNDs sponge-
like porous structure. We further tail-vein injected CpG-fNDs
once in every 3 d in two murine tumor models, i.e., B16 mela-
noma and 4T1 breast carcinoma xenografts. Importantly, mon-
otherapy using this immunostimulatory agent significantly
suppressed the tumor growth in mice. Given this performance,
demonstrable biocompatibility and the low cost of fNDs, the
fNDs-based nanoagents open new opportunities for immuno-
therapy as well as clinical applications of various types of thera-
peutic nucleic acids.
Therapeutic nucleic acids, including functional oligonu-
cleotide (ODNs),[1] aptamers,[2] and small interfering RNAs
Y. Zhang, Z. Cui, H. Kong, K. Xia, L. Pan, Profs. J. Li,
Y. Sun, L. Wang, Y. Zhu, Prof. C. Fan
Division of Physical Biology and Bioimaging Center
Shanghai Synchrotron Radiation Facility
CAS Key Laboratory of Interfacial Physics and Technology
Shanghai Institute of Applied Physics
Chinese Academy of Sciences
Shanghai 201800, China
E-mail: zhuying@sinap.ac.cn; fchh@sinap.ac.cn
Prof. J. Shi
Kellogg College
University of Oxford
Banbury Road, Oxford OX2 6PN, UK
Prof. J. Shi
UCB Pharma
208 Bath Road, Slough SL1 3WE, UK
Prof. C. Fan
School of Life Science and Technology
ShanghaiTech University
Shanghai 201200, China
DOI: 10.1002/adma.201506232
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(siRNAs),[3] hold great promise for treating diseases ranging
from inherited disorders to acquired conditions and cancers.
Unmethylated cytosine-phosphate-guanine (CpG) ODNs are a
type of immunostimulatory nucleic acids[4,5] that are commonly
found in viral and bacterial DNA.[6] Synthetic CpG ODNs have
been exploited as immune modulators for many applications
including vaccine adjuvants and immunotherapy for cancers,
infectious, and allergic diseases.[4,5,7]
Clinical trials of CpG ODNs have been performed from
phase I to phase III.[810] However, the observation of several
adverse effects (e.g., sepsis-like events) did not support contin-
uation of the study.[10] These side effects are largely associated
with the use of high-dose CpG ODNs and repeated administra-
tion, which are possibly due to low cellular uptake and rapid
clearance of naked ODNs.[11] Hence, it is highly demanding to
develop a new delivery system that can increase both the cel-
lular internalization and the stability against nuclease degrada-
tion for sustained functioning of CpG ODNs.
Nanomaterials have shown great potential as controllable
carriers for drug delivery.[12] Various types of nanomaterials
ranging from inorganic, organic to biomolecular structures,
have been employed to carry CpG ODNs to stimulate immu-
nological reactions in cells.[1316] However, the high cost and
potential toxicological concerns of these nanomaterials largely
hamper their in vivo applications. Among the wide spectrum
of nanomaterials-based vehicles, nanodiamonds (NDs) have
shown remarkable delivery ability, flexible functionality, and
excellent biocompatibility.[17] In particular, NDs can spontane-
ously form porous nanoclusters under physiological condi-
tions,[18] which show sponge-like effects for the delivery of
small-molecule chemotherapeutic drugs[19] as well as biomac-
romolecules.[20] In this work, we employed poly(D-lysine) (PDL)-
coated functional NDs (fNDs) for intracellular and in vivo
delivery of CpG ODNs and explored their immunostimulatory
effects for cancer therapy (Figure 1).
To design a therapeutic delivery vector for ODNs, we first
coated NDs with a cationic polymer, poly(D-lysine) (PDL),
to form PDL-NDs, which was subsequently complexed with
negatively charged unmethylated CpG ODNs via the simple
electrostatic interaction. NDs exist in the form of clustered
porous structures.[18] After the PDL coating, the average size
of functionalized NDs (fNDs) was increased from 247 to
325 nm, and their zeta potential was shifted from 33.0 to
67.3 eV (Figure 1). Transmission electron microscopy (TEM)
images showed that the sharp surface of NDs was rounded
with the PDL functionalization (Figure S1, Supporting Infor-
mation). To prepare the immunostimulatory agent CpG-fNDs,
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Figure 1. Preparation of fNDs and their immunomodulatory activity for cancer therapy. Upper left: average particle size and potential of NDs, fNDs,
and CpG-fNDs suspended in water (concentration: 50 g mL1). These ND clusters showed reasonably narrow size distributions with polydispersity
indices ranging from 0.1 to 0.3. Upper right: plot of the w/w ratio of CpG-to-fNDs to the complexation ratios (CpG ODN loaded on fNDs). Data rep-
resented as mean SD (n = 3). Middle: schematic showing of the preparation of fNDs and CpG-fNDs. Lower panels: schematic showings of in vitro
and in vivo immunostimulatory effects, and one-short immunostimulatory agents for cancer therapy.

we incubated fNDs with a mouse-specific CpG ODN-1826[4,21]
(Table S1, Supporting Information) for 8 h. Adsorption studies
revealed that fNDs were saturated with CpG ODNs when the
w/w ratio of fNDs/CpG was 10:1 (Figure 1). After the DNA
adsorption, the mean size of ND clusters was further increased
to 338 nm, whereas the zeta potential was decreased to
40.6 eV (Figure 1), suggesting their complexation with CpG
ODNs.
Then, we evaluated the cellular uptake of CpG-fNDs using
confocal microscopy. When CpG ODNs labeled with a red-color
fluorophore Cy3 were incubated with RAW264.7 cells for 6 h,
no apparent fluorescence was observed in cells, which suggest
inefficient cellular uptake of naked ODNs, possibly due to the
presence of the electrostatic barrier of the cytoplasmic mem-
brane (Figure 2a, left). Significantly, bright fluorescent dots
were observed throughout the cytoplasm of cells when incu-
bated with CpG-fNDs, implying that fNDs greatly increased
the uptake efficiency of CpG (Figure 2a, left). We also found
that fluorescent dots of Cy3-CpG were generally co-localized
with particles of fNDs under bright-field imaging (Figure 2a,
left), indicating that CpG remained to be complexed with fNDs
during internalization. Quantitative analysis showed that the
mean fluorescence intensity (MFI) of internalized CpG was
enhanced by nearly three orders of magnitudes as compared
with that of naked one (Figure 2a, right). Control studies
showed that the cellular uptake efficiency for fNDs was 2.4-
fold and 15-fold higher than that for uncoated NDs and PDL
alone, respectively, suggesting that PDL coating is important
for the delivery (Figure 2a, right and Figure S3, Supporting
Information).
Having substantiated the efficient cellular uptake of CpG-
fNDs, we next tracked the internalization of Cy3-labeled CpG-
fNDs by using real-time live-cell imaging with a total internal
reflection fluorescence (TIRF) microscope. We observed
that, when a fast-moving red-colored particle approached the
cell membrane, it was abruptly slowed down and stayed in
the membrane for 300 s and eventually came into the cyto-
plasm (Figure 2b and Video S1, Supporting Information). A
large portion of CpG-fNDs was internalized into cells after
6 h incubation (Figure 2a). Under TEM imaging, we found
that fNDs were trapped in endosomal and lysosomal vesi-
cles (Figure 2c), suggesting that CpG-fNDs were internalized
via endocytosis and then reached lysosomes via intracellular
traffic. This was confirmed by co-localization studies, which
showed that approximately 4050% of red dots corresponding
to CpG-fNDs were localized to lysosomes labeled in green
color (Figure 2d).
We further investigate the clearance of CpG-fNDs in cells.
After the initial 6 h incubation with CpG-fNDs, cells were fluo-
rescently imaged at 24, 48, and 72 h, respectively. We found that
intracellular fluorescent signals slowly decreased along with the
time, and a substantial amount of fluorescent dots remained
in cells even at 72 h (Figure S4, Supporting Information). We
also note that the MFI for intracellular CpG-fNDs were 900-,
830-, and 400-fold higher than that for naked CpG at 24, 48, and
72 h, respectively (Figure 2e). Interestingly; uncoated NDs also
showed the similar clearance trend, although the corresponding
fluorescent signals were generally lower than the fNDs
(Figure 2e and Figure S3, Supporting Information), suggesting
that the use of NDs slowed down the clearance of CpG ODNs.

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Figure 2. fNDs greatly increased CpG ODN accumulation and slowed down the clearance of CpG ODNs in cells. a) Cellular uptake of Cy3-CpG-fNDs.
Left: confocal images of RAW264.7 cells after 6 h incubation with 5 g mL1 Cy3-CpG and 50 g mL1 Cy3-CpG-fNDs. Scale bar: 5 m. Right: mean fluo-
rescence intensity (MFI) inside the cells. Data are represented as means SD. b) Cellular uptake of Cy3-CpG-fNDs as visualized in real time with a TIRF
microscope. Upper: three stages (I: approaching the membrane; II: staying in the membrane; III: inside the cytoplasm) and a representative trajectory
of internalization of the CpG-fNDs particle (see also Movie S1, Supporting Information). Scale bar: 5 m. Lower: speed analysis of the representative
trajectory. c) TEM images of internalized CpG-fNDs. Black arrow indicates the location of the internalized CpG-fNDs. Scale bar: 200 nm. d) Confocal
images corresponding to co-localization of CpG-fNDs (red) and lysosomes (green) in RAW264.7 cells incubated with 50 g mL1 Cy3-CpG-fNDs for
6 h. Scale bar: 5 m. e) MFI inside the cells at 24, 48, and 72 h after the initial 6 h incubation. Data are represented as means SD.
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Figure 3. Sustained in vitro immunoregulatory activities of CpG-fNDs. a) General experimental design. b,d,e) RAW264.7 cells were treated with indi-
cated materials with concentrations of 50 g mL1 [DNA equivalent (5 g mL1)] for 6 h. S-CpG of 50 109 M was used as positive control. c) RAW264.7
cells were treated with CpG ODNs with varied concentrations for 6 h. The concentrations of b,c,d) TNF-a and b,e) IL-6 in culture media were measured
at b,c) 6 h or at d,e) the intervals of 24, 48, and 72 h after the initial 6 h incubation with ELISA. Data are represented as means SD. **p < 0.01;
***p < 0.001, significantly different from CpG ODNs; ##p < 0.01, ###p < 0.001, significantly different from CpG-fNDs.

Having established the efficient cellular uptake and slow
clearance abilities of CpG-fNDs, we next assessed the in
vitro immunostimulatory activity induced by CpG-fNDs.
Since CpG-fNDs were localized in lysosomes, they might
be released and bound to Toll-like receptor 9 (TLR9) that are
translocated from endosomes to lysosomes, and then initiate
immune responses.[22] Thus, we measured the secreted levels
of cytokines in RAW264.7 cells with enzyme-linked immuno-
sorbent assay (ELISA) (Figure 3a), and found that CpG-fNDs
dramatically induced the production of tumor necrosis factor-
alpha (TNF-) and interleukin-6 (IL-6) in RAW264.7 cells. Com-
pared with naked CpG ODNs (5 g mL1), CpG-fNDs increased
the secreted levels of TNF- and IL-6 by 59- and 20-fold, respec-
tively (Figure 3b). In fact, even when we increased the dose of
naked CpG ODNs by 60-fold, we still found that the treatment
of CpG-fNDs resulted in much higher levels of TNF- secretion
than high-dose CpG ODNs (Figure 3c). In addition, control
studies using NDs coated with either PDL alone, uncoated NDs
or NDs coated with two different cationic polymers, polyethy-
leneimine (PEI), and poly(allylamine hydrochloride) (PAH)
showed much poorer stimulation than fNDs (Figure S5 and S6,
Supporting Information).
Next, we explored whether CpG-fNDs could sustainably
stimulate immunoresponses. After 6 h incubation, we exten-
sively washed RAW264.7 cells to remove noninternalized NDs
and then measured the concentrations of secreted cytokines
at the intervals of 24, 48, and 72 h (Figure 3a). Significantly,
whereas the immunostimulatory effects CpG-fNDs decreased
along with the time, we still observed substantial amount of
secreted TNF-/IL-6 at 72 h, which were 13- and 26-fold higher
than those induced by naked CpG ODNs (Figure 3d,e). Such
sustained immunostimulatory effects should arise from the

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Figure 4. Sustained in vivo immunoregulatory activities of CpG-fNDs. a) General experimental design. b,c) ICR mice were administered intravenously
(i.v.) with indicated materials with a dose of 80 g/mice (20 g) [DNA equivalent (8 g/mice)] (n = 8). Equal amount of S-CpG was used as positive
control. d) ICR mice were administered intravenously (i.v.) with CpG ODNs with varied doses (n = 9). Blood samples were obtained at bd) 3, 24, and
48 h after injection, and b,d) serum IL-12 and c) Serum IL-6 levels were analyzed with ELISA. Data are represented as means SD. *p < 0.05; **p < 0.01,
***p < 0.001, significantly different from NS; #p < 0.05,##p < 0.01, ###p < 0.001, significantly different from CpG ODNs.

sustained release of CpG ODNs from the sponge-like porous
structure of fNDs. In contrast, although the secreted levels of
cytokines induced by CpG-fNDs were comparable with those
by the positive control, CpG ODNs bearing a nuclease-resistant
phosphorothioate backbone (S-CpG[11]), the latter's effects
diminished even at 24 h (Figure 3b,d,e). Hence, the use of
fNDs as the carrier greatly improves the robustness of intracel-
lular CpG and endows sustained immunostimulatory activity
over a long period.
To further assess the therapeutic potential of CpG-fNDs, we
next investigated their in vivo immunostimulatory activity. ICR
mice administrated with CpG-fNDs via intravenous injection
were analyzed by measuring their secreted levels of cytokines
in serum (Figure 4a). We found that CpG-fNDs dramatically
induced the production of serum IL-12 and IL-6 at 3 h after
administration. Compared with the treatment with naked CpG
ODNs of the same amount, the secreted levels of IL-12 and
IL-6 were enhanced by 19 and 61 times, respectively (Figure 4b,
c). We also compared the in vivo immunostimulatory activity
of CpG-fNDs and high-dose naked CpG ODNs, and found
that the CpG-fNDs treatment excelled even the highest dose
(40-fold) employed in this work, suggesting the high efficacy
of this nanoagent (Figure 4d). Further, we evaluated the sus-
tained immunostimulatory effect of CpG-fNDs. Significantly,
we found that the secreted levels of IL-12 remained nearly the
same at 24 h after administration of CpG-fNDs, whereas they
dramatically dropped in the case of high-dose naked CpG ODNs
(Figure 4d). Even at 48 h after administration of CpG-fNDs, the
secreted levels of IL-12 were still 4-fold higher than that with
naked CpG ODNs (Figure 4b,c). It is also worthwhile to note
that although administration of S-CpG could induce high-level
serum cytokines, its activity was transient and rapidly dropped
to the background level after 24 h (Figure 4b,c). In addition,
both in vitro and in vivo experiments showed that fNDs or
fNDs conjugated with a non-CpG sequence had no effect on
cytokine secretion (Figure 3b,d,e and Figure 4b,c), suggesting
that the observed immunostimulatory activities were indeed
caused by CpG delivered by fNDs.
Next, we performed a series of in vitro and in vivo biocom-
patibility tests to evaluate the therapeutic safety of CpG-fNDs.
MTT assays showed that neither fNDs nor CpG-fNDs showed
apparent toxicity to cells after 72 h incubation, even at the highest
exposure dose of 100 g mL1 (Figure S7, Supporting Informa-
tion). We further monitored in vivo biodistribution and clearance
by using NDs labeled with a near-IR dye, XenoLight CF750 (see
Supporting text, Table S2, Figure S8 and S9, Supporting Infor-
mation), which were administrated via intravenous injection in
mice. After 3 h of injection, we observed fluorescent signals were
predominantly in the liver from whole-body imaging, whereas
the free dye showed wide distribution in mice (Figure 5a). Quan-
titative analysis showed that NDs were also accumulated in
spleen, lung and kidney. The peak accumulation after adminis-
tration in these organs was found at 6 h, and the accumulated
NDs were thoroughly cleared in all organs after 72 h (Figure 5b).
Since the liver is a primary organ of NDs accumulation before
clearance, we then measured several liver damage-associated

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Figure 5. In vivo biocompatibility of NDs and CpG-fNDs. a) Representative whole-body images of ICR mice after tail vein administration with 1.2 mg
of XenoLight CF750-labeled NDs or 30 fmol of XenoLight 750. b) Quantification of NDs accumulated in mouse tissues at different time points after
tail vein administration with XenoLight 750-labeled NDs with a dose of 80 g per mouse (20 g) (n = 5 at each time point). Data are represented as
means SD. The insets show organ-specific imaging of ICR mice at 6 h after injection (Li, liver; S, spleen; Lu, lung; K, kidney). c,d) ICR mice were tail
vein administered with fNDs or CpG-fNDs with a dose of 80 g/mice (20 g). c) Analysis of biochemical parameters in serum (n = 5); d) hematoxylin
and eosin (H&E) histopathological sections of liver, spleen, lung, and kidney tissue (n = 3) were analyzed at 48 h after injection. Scale bar: 100 m.

serum biochemical parameters including alanine transferase
(ALT), aspertate aminotransferase (AST), and alkaline phos-
phate (ALP).[23] We did not observe significant changes in these
parameters in mice exposed to fNDs or CpG-fNDs (Figure 5c),
suggesting that CpG-fNDs treatment do not adversely affect
liver function. Histological analysis in multiple tissues also con-
firmed the safety of fNDs and CpG-fNDs (Figure 5d).
Having established the potent in vivo immunostimulatory
activity of CpG-fNDs, we further explored their antitumor
effects in two murine tumor models, i.e., B16-F0 melanoma
and 4T1 breast carcinoma xenograft models, which are known
to express TLRs and widely used in cancer immunotherapy
studies.[24] Tumor-bearing mice were treated with CpG-fNDs,
CpG ODNs, fNDs, or normal saline (NS) once every 3 d
(Figure 6a). We found that the CpG-fNDs treatment led to a
tumor-weight decrease of 73.3% in the B16-F0 tumor model
(Figure 6b1,b2; Figure S10a, Supporting Information) and
28.4% in the 4T1 tumor models (Figure 6c1,c2; Figure S10b,

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Figure 6. Potent antitumor effects of CpG-fNDs in murine tumor models. a) General experimental design. b) B16-F0 and c) 4T1 tumor-bearing nude
mice were administered intravenously (i.v.) with NS (200 L), fNDs (80 g), CpG ODNs (8 g), or CpG-fNDs (80 g) once every 3 d for continuous
17 d (n = 8). b1,c1) Photo and b2,c2) weight ratio of tumor tissues from nude mice after treatment. b3,b4,c3,c4) Blood samples were obtained
on the second day after the final injection, and b3,c3) serum IL-12 and b4,c4) serum IL-6 levels were analyzed with ELISA. Data are represented
as means SD. *p < 0.05; **p < 0.01; ***p < 0.001, significantly different from NS; #p < 0.05,##p < 0.01, significantly different from CpG ODNs;
**p < 0.01; ***p < 0.001, significantly different from normal.

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Supporting Information). In contrast, the treatment with fNDs
or naked CpG ODNs did not significantly change the tumor
weight (Figure 6b1,b2 and c1,c2 and Figure S10, Supporting
Information). Given the nonspecific nature of CpG-based
immunotherapy, its treatment is considered to be comple-
mentary to chemotherapy. Our data demonstrated that mono-
therapy with CpG-fNDs led to significant tumor suppression
in two murine models, which reveals the great potential for its
combination with chemotherapy.
We next measured the secreted levels of cytokines in serum
after the treatment with CpG-fNDs. We found no statistical dif-
ference between normal and tumor-bearing mice in the serum
levels of IL-12, whereas the serum levels of IL-6 dramatically
increased in both tumor models (Figure 6b3,b4 and 6c3,c4),
which correlated with the progression of tumors.[25] Interest-
ingly, treatment with CpG-fNDs significantly increased the
serum levels of IL-12, and downregulated IL-6 in both tumor
models (Figure 6b3,b4 and 6c3,c4). In contrast, fNDs or naked
CpG ODNs did not affect serum cytokine levels in tumor-
bearing mice (Figure 6b 3,b4 and 6c3,c4). Previous studies
showed that advanced-stage tumors resulted in elevated levels
of serum IL-6 whereas minimally perturbed IL-12.[25] After
CpG-fNDs treatment, pronounced increase in IL-12 and
decrease in IL-6 in both tumor models suggested that the
immunomodulatory effects of CpG-fNDs played an essen-
tial role in the observed antitumor effects. In addition, since
nude mice are devoid of T cells, the tumor suppression effect
of CpG was T-cell-independent and relied on innate immune
responses.[26]
CpG ODNs have been employed as immunostimulatory
agents for infectious diseases and cancers, and rigorously eval-
uated under phase IIII clinical trials approved by U.S. Food
and Drug Administration (FDA).[810] In these trials, immune
activation by CpG ODNs has shown therapeutic efficacies in
mice and in patient volunteers.[810,27] However, high-dose
ODNs ranging from several to tens of milligrams for patients
(or tens to hundreds of microgram per kilogram) are required
for treatment due to their low drug bioavailability and stability,
which brings about not only high treatment cost but also severe
side effects.[9,10] Although the use of S-CpG by substituting the
phosphodiester linkage by a phosphorothioate greatly increase
the resistance to enzymatic degradation and increase the biolog-
ical activity of CpG ODNs,[11] these chemically modified ODNs
posed significant safety concerns, e.g., renal damage.[28] In our
studies, we have demonstrated the advantages of CpG-fNDs
for immunotherapy. By exploiting the high cellular uptake effi-
ciency of fNDs, we can reduce the dose down to several micro-
grams, which is one-to-two orders magnitude lower than that
for naked CpG ODNs. More importantly, the sustained immu-
nostimulatory effects of CpG-fNDs for up to 48 h largely avoid
repeated administration in therapeutic use.
The distinctive porous nanostructure of fNDs plays a key role
in their superior delivery ability. Due to the high surface energy
of NDs, they tend to form clusters with numerous nanoscale
pores.[18,29] Such sponge-like nanostructures not only pro-
vide high capacity for accommodating CpG ODNs and protec-
tion in the blood circulation system, but also endow sustained
release of CpG for long-term immunostimulatory responses.
We also note that CpG-fNDs exhibit excellent biocompatibility,
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as revealed in rigorous in vitro and in vivo toxicological evalua-
tions. Hence, fNDs compare favorably with other nanoscale vec-
tors.[15,16,30,31] Several types of inorganic nanoparticles including
gold nanoparticles have proven to be excellent vectors for CpG
in cellular studies in vitro.[13,15,16] However, their prominent
effects are often compromised in vivo due to the complexity of
blood circulation.[32] More recently, DNA nanostructures have
emerged as another type of promising biomolecular delivery
carriers with excellent biocompatibility, whose in vivo applica-
tions are nevertheless hampered by their high cost and short
blood circulation time.[16,30]
In conclusion, we have demonstrated a novel delivery system
for therapeutic nucleic acids, CpG ODNs. CpG-fNDs exhibited
remarkable cellular uptake efficiency that exceeded naked CpG
ODNs by nearly three orders of magnitudes. More importantly,
both in vitro and in vivo studies showed that CpG-fNDs could
sustainably stimulate cytokine secretion for 23 d due to their
sponge-like porous nanostructures. We also confirmed that
this type of nanoagents had excellent biocompatibility as a drug
delivery carrier. Based on these new findings, we employed
CpG-fNDs as a monotherapeutic drug for cancer immuno-
therapy, which showed high antitumor efficacy in two murine
tumor models. Whereas direct translating this one-shot immu-
nomodulatory agent to human use remains challenging, the
success of CpG-fNDs-based therapy in animal models shed
new light on cancer immunotherapy, and more broadly, design
and application of nanomedicine.
Experimental Section
Functionalization of NDs and Complexation with CpG ODNs: NDs with
individual sizes of 210 nm synthesized with the detonation technique
were obtained from Gansu Gold Stone Nano. Material. Co. Ltd. (Gansu,
China). The unique crystal profile of the NDs was confirmed using an
automatic X-ray diffractometer equipped with Cu K (1.541 ) radiation
(40 kV, 40 mA). TEM images showed that the size of the majority of ND
clusters was about 40200 nm. Characterization of the NDs has been
described in our previous work.[18]
NDs were functionalized with poly(D-lysine) (PDL), polyethyleneimine
(PEI), or poly(allylamine hydrochloride) (PAH). See the Supporting
Information for detailed preparation process. Prior to usage, CpG ODNs
were incubated with fNDs at a w/w ratio of 1:10 in Millipore water for
8 h, and then centrifuged at 13 000 rpm for 10 min. Precipitation was
resuspended in cell culture medium for cell experiment and in NS for
animal experiment as required.
Cell Line and Treatment: RAW264.7 macrophage-like cells were grown
in the Dulbeccos modified Eagles medium (DMEM) supplemented
with 10% heat-inactivated FBS, 1.5 g L1 NaHCO3, 100 units/mL
penicillin, 100 g mL1 streptomycin, 4.5 g L1 glucose, and 4 103 M
L-glutamine at 37 C in a humidified 5% CO2-containing atmosphere.
Cells were seeded in 24-well plates at a density of 105 cells per well
and incubated overnight to allow for cell adherence. fNDs (50 g cmL1),
CpG ODNs (5, 50, 100, 200, 300 g mL1), non-CpG-fNDs (50 g mL1),
CpG-fNDs (50 g mL1), CpG-NDs (50 g mL1), CpG-PDL (5 g mL1
CpG mixed with 2.5 g mL1 PDL), and S-CpG (50 109 M) were added
into the plate wells, respectively. It should be noted here that high-dose
S-CpG induces excessive immune activation, leading to cell damage and
even death.[28] Hence, we used 50 109 M S-CpG for cell experiments.
Following 6 h incubation, the media were collected and centrifuged at
13 000 rpm for 15 min at 4 C. Then, all samples were washed twice
with phosphate buffered saline (PBS) and fresh cell culture medium
was added into the plate wells. After 24 h incubation, the media were
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collected and centrifuged. This process was repeated every 24 h over
the course of 3 d. All of the supernatants were collected and stored at
80 C before assays.
Animals and Treatment: ICR mice (male, 1822 g) and nude mice
(female, 1822 g) were purchased from Shanghai SLAC Laboratory
Animal Co. Ltd., China. ICR mice were kept in conventional conditions
and nude mice were kept in pathogen-free conditions. Permission of
the local ethics committee was obtained, and all animal experiments
were performed according to Chinese law and accepted international
standards in biomedical research.
In the in vivo immunostimulatory experiments, ICR mice were tail-vein-
injected with 80 g of fNDs, 8, 80, 160, 320 g of CpG ODNs, 80 g of
non-CpG-fNDs, 80 g of CpG-fNDs, 8 g of S-CpG (positive control), or
200 L of NS (negative control), respectively. The blood samples were taken
from retro-orbital plexus of mice at 3, 24, and 48 h after injection (eight per
group at each time point), and their sera stored at 80 C until assays.
In antitumor experiments, B16-F0 and 4T1 tumors were generated by
subcutaneous injection of 6 105 B16-F0 cells and 1 106 4T1 cells in
100 L of NS in the right forelimbs of nude mice (day 0), respectively. Two
days after tumor inoculation, mice were tail vein injected with 80 g of
fNDs, 8 g of CpG ODNs, 80 g of CpG-fNDs, or 200 L of NS (eight per
group) once every 3 d, from day 2 to day 17. After the final injection, all of
the mice were sacrificed on the second day and the stripped subcutaneous
sarcoma tumors were weighed to evaluate the antitumor effect. The blood
samples were taken from retro-orbital plexus of mice and the IL-12 and
IL-6 concentration in serum was determined using ELISA.
Supporting Information
Supporting Information is available from the Wiley Online Library or
from the author.
Acknowledgments
Y.Z. and Z.C. contributed equally to this work. The authors thank Yan
Peng from Gansu Gold Stone Nano. Material Co. Ltd for providing
nanodiamonds. The authors thank Associate Prof. Yongjun Li at the
Shanghai Institute of Organic Chemistry, CAS for his assistance in
NDs-CF750 preparation and Congxi Song at the Soochow University for
whole-body imaging. This work was supported by the Ministry of Science
and Technology of China (Grant Nos. 2012CB825805, 2013CB933800,
and 2012CB932602), the National Natural Science Foundation of China
(Grant Nos. 11179004, 11275251, U1232113, 21390414, 31371015
and 11575278), the special project of Shanghai Nano-technology
(Grant No. 13NM1402300), Shanghai Rising-Star Program (Grant No.
14QA1404400), and Youth Innovation Promotion Association of CAS.
Received: December 15, 2015
Revised: January 10, 2016
Published online: February 2, 2016
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