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One-Shot Immunomodulatory Nanodiamond Agents for

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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 (siRNAs),[3] hold great promise for treating diseases ranging
wide range of diseases. Nevertheless, it remains highly desir- from inherited disorders to acquired conditions and cancers.
able to develop new delivery systems to improve their trans- Unmethylated cytosine-phosphate-guanine (CpG) ODNs are a
fection efficiency and in vivo stability for sustained biological type of immunostimulatory nucleic acids[4,5] that are commonly
activities. Here, we report on a type of functional nanodiamond found in viral and bacterial DNA.[6] Synthetic CpG ODNs have
(fND)-based agent for facile assembly and efficient delivery of been exploited as immune modulators for many applications
immunostimulatory cytosinephosphateguanine (CpG) oli- including vaccine adjuvants and immunotherapy for cancers,
gonucleotides (ODNs). We found that complexation of CpG infectious, and allergic diseases.[4,5,7]
ODNs with fNDs remarkably increased their cellular uptake Clinical trials of CpG ODNs have been performed from
by approximately three orders of magnitudes. Internalized phase I to phase III.[810] However, the observation of several
CpG-fNDs were localized in lysosomes, which bound to Toll- adverse effects (e.g., sepsis-like events) did not support contin-
like receptor 9 (TLR9) and induced prominent cytokine secre- uation of the study.[10] These side effects are largely associated
tion. More significantly, these immunostimulatory nanoagents with the use of high-dose CpG ODNs and repeated administra-
exhibited long-term immunoregulatory activities lasting at least tion, which are possibly due to low cellular uptake and rapid
3 d at the cellular level and 2 d in mice, which arise from the clearance of naked ODNs.[11] Hence, it is highly demanding to
protection and sustained release properties of fNDs sponge- develop a new delivery system that can increase both the cel-
like porous structure. We further tail-vein injected CpG-fNDs lular internalization and the stability against nuclease degrada-
once in every 3 d in two murine tumor models, i.e., B16 mela- tion for sustained functioning of CpG ODNs.
noma and 4T1 breast carcinoma xenografts. Importantly, mon- Nanomaterials have shown great potential as controllable
otherapy using this immunostimulatory agent significantly carriers for drug delivery.[12] Various types of nanomaterials
suppressed the tumor growth in mice. Given this performance, ranging from inorganic, organic to biomolecular structures,
demonstrable biocompatibility and the low cost of fNDs, the have been employed to carry CpG ODNs to stimulate immu-
fNDs-based nanoagents open new opportunities for immuno- nological reactions in cells.[1316] However, the high cost and
therapy as well as clinical applications of various types of thera- potential toxicological concerns of these nanomaterials largely
peutic nucleic acids. hamper their in vivo applications. Among the wide spectrum
Therapeutic nucleic acids, including functional oligonu- of nanomaterials-based vehicles, nanodiamonds (NDs) have
cleotide (ODNs),[1] aptamers,[2] and small interfering RNAs shown remarkable delivery ability, flexible functionality, and
excellent biocompatibility.[17] In particular, NDs can spontane-
Y. Zhang, Z. Cui, H. Kong, K. Xia, L. Pan, Profs. J. Li, ously form porous nanoclusters under physiological condi-
Y. Sun, L. Wang, Y. Zhu, Prof. C. Fan tions,[18] which show sponge-like effects for the delivery of
Division of Physical Biology and Bioimaging Center small-molecule chemotherapeutic drugs[19] as well as biomac-
Shanghai Synchrotron Radiation Facility romolecules.[20] In this work, we employed poly(D-lysine) (PDL)-
CAS Key Laboratory of Interfacial Physics and Technology
Shanghai Institute of Applied Physics coated functional NDs (fNDs) for intracellular and in vivo
Chinese Academy of Sciences delivery of CpG ODNs and explored their immunostimulatory
Shanghai 201800, China effects for cancer therapy (Figure 1).
E-mail: zhuying@sinap.ac.cn; fchh@sinap.ac.cn To design a therapeutic delivery vector for ODNs, we first
Prof. J. Shi coated NDs with a cationic polymer, poly(D-lysine) (PDL),
Kellogg College
to form PDL-NDs, which was subsequently complexed with
University of Oxford
Banbury Road, Oxford OX2 6PN, UK negatively charged unmethylated CpG ODNs via the simple
Prof. J. Shi electrostatic interaction. NDs exist in the form of clustered
UCB Pharma porous structures.[18] After the PDL coating, the average size
208 Bath Road, Slough SL1 3WE, UK of functionalized NDs (fNDs) was increased from 247 to
Prof. C. Fan 325 nm, and their zeta potential was shifted from 33.0 to
School of Life Science and Technology 67.3 eV (Figure 1). Transmission electron microscopy (TEM)
ShanghaiTech University
Shanghai 201200, China images showed that the sharp surface of NDs was rounded
with the PDL functionalization (Figure S1, Supporting Infor-
DOI: 10.1002/adma.201506232 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] Having substantiated the efficient cellular uptake of CpG-
(Table S1, Supporting Information) for 8 h. Adsorption studies fNDs, we next tracked the internalization of Cy3-labeled CpG-
revealed that fNDs were saturated with CpG ODNs when the fNDs by using real-time live-cell imaging with a total internal
w/w ratio of fNDs/CpG was 10:1 (Figure 1). After the DNA reflection fluorescence (TIRF) microscope. We observed
adsorption, the mean size of ND clusters was further increased that, when a fast-moving red-colored particle approached the
to 338 nm, whereas the zeta potential was decreased to cell membrane, it was abruptly slowed down and stayed in
40.6 eV (Figure 1), suggesting their complexation with CpG the membrane for 300 s and eventually came into the cyto-
ODNs. plasm (Figure 2b and Video S1, Supporting Information). A
Then, we evaluated the cellular uptake of CpG-fNDs using large portion of CpG-fNDs was internalized into cells after
confocal microscopy. When CpG ODNs labeled with a red-color 6 h incubation (Figure 2a). Under TEM imaging, we found
fluorophore Cy3 were incubated with RAW264.7 cells for 6 h, that fNDs were trapped in endosomal and lysosomal vesi-
no apparent fluorescence was observed in cells, which suggest cles (Figure 2c), suggesting that CpG-fNDs were internalized
inefficient cellular uptake of naked ODNs, possibly due to the via endocytosis and then reached lysosomes via intracellular
presence of the electrostatic barrier of the cytoplasmic mem- traffic. This was confirmed by co-localization studies, which
brane (Figure 2a, left). Significantly, bright fluorescent dots showed that approximately 4050% of red dots corresponding
were observed throughout the cytoplasm of cells when incu- to CpG-fNDs were localized to lysosomes labeled in green
bated with CpG-fNDs, implying that fNDs greatly increased color (Figure 2d).
the uptake efficiency of CpG (Figure 2a, left). We also found We further investigate the clearance of CpG-fNDs in cells.
that fluorescent dots of Cy3-CpG were generally co-localized After the initial 6 h incubation with CpG-fNDs, cells were fluo-
with particles of fNDs under bright-field imaging (Figure 2a, rescently imaged at 24, 48, and 72 h, respectively. We found that
left), indicating that CpG remained to be complexed with fNDs intracellular fluorescent signals slowly decreased along with the
during internalization. Quantitative analysis showed that the time, and a substantial amount of fluorescent dots remained
mean fluorescence intensity (MFI) of internalized CpG was in cells even at 72 h (Figure S4, Supporting Information). We
enhanced by nearly three orders of magnitudes as compared also note that the MFI for intracellular CpG-fNDs were 900-,
with that of naked one (Figure 2a, right). Control studies 830-, and 400-fold higher than that for naked CpG at 24, 48, and
showed that the cellular uptake efficiency for fNDs was 2.4- 72 h, respectively (Figure 2e). Interestingly; uncoated NDs also
fold and 15-fold higher than that for uncoated NDs and PDL showed the similar clearance trend, although the corresponding
alone, respectively, suggesting that PDL coating is important fluorescent signals were generally lower than the fNDs
for the delivery (Figure 2a, right and Figure S3, Supporting (Figure 2e and Figure S3, Supporting Information), suggesting
Information). 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 than high-dose CpG ODNs (Figure 3c). In addition, control
clearance abilities of CpG-fNDs, we next assessed the in studies using NDs coated with either PDL alone, uncoated NDs
vitro immunostimulatory activity induced by CpG-fNDs. or NDs coated with two different cationic polymers, polyethy-
Since CpG-fNDs were localized in lysosomes, they might leneimine (PEI), and poly(allylamine hydrochloride) (PAH)
be released and bound to Toll-like receptor 9 (TLR9) that are showed much poorer stimulation than fNDs (Figure S5 and S6,
translocated from endosomes to lysosomes, and then initiate Supporting Information).
immune responses.[22] Thus, we measured the secreted levels Next, we explored whether CpG-fNDs could sustainably
of cytokines in RAW264.7 cells with enzyme-linked immuno- stimulate immunoresponses. After 6 h incubation, we exten-
sorbent assay (ELISA) (Figure 3a), and found that CpG-fNDs sively washed RAW264.7 cells to remove noninternalized NDs
dramatically induced the production of tumor necrosis factor- and then measured the concentrations of secreted cytokines
alpha (TNF-) and interleukin-6 (IL-6) in RAW264.7 cells. Com- at the intervals of 24, 48, and 72 h (Figure 3a). Significantly,
pared with naked CpG ODNs (5 g mL1), CpG-fNDs increased whereas the immunostimulatory effects CpG-fNDs decreased
the secreted levels of TNF- and IL-6 by 59- and 20-fold, respec- along with the time, we still observed substantial amount of
tively (Figure 3b). In fact, even when we increased the dose of secreted TNF-/IL-6 at 72 h, which were 13- and 26-fold higher
naked CpG ODNs by 60-fold, we still found that the treatment than those induced by naked CpG ODNs (Figure 3d,e). Such
of CpG-fNDs resulted in much higher levels of TNF- secretion 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 secreted levels of IL-12 were still 4-fold higher than that with
structure of fNDs. In contrast, although the secreted levels of naked CpG ODNs (Figure 4b,c). It is also worthwhile to note
cytokines induced by CpG-fNDs were comparable with those that although administration of S-CpG could induce high-level
by the positive control, CpG ODNs bearing a nuclease-resistant serum cytokines, its activity was transient and rapidly dropped
phosphorothioate backbone (S-CpG[11]), the latter's effects to the background level after 24 h (Figure 4b,c). In addition,
diminished even at 24 h (Figure 3b,d,e). Hence, the use of both in vitro and in vivo experiments showed that fNDs or
fNDs as the carrier greatly improves the robustness of intracel- fNDs conjugated with a non-CpG sequence had no effect on
lular CpG and endows sustained immunostimulatory activity cytokine secretion (Figure 3b,d,e and Figure 4b,c), suggesting
over a long period. that the observed immunostimulatory activities were indeed
To further assess the therapeutic potential of CpG-fNDs, we caused by CpG delivered by fNDs.
next investigated their in vivo immunostimulatory activity. ICR Next, we performed a series of in vitro and in vivo biocom-
mice administrated with CpG-fNDs via intravenous injection patibility tests to evaluate the therapeutic safety of CpG-fNDs.
were analyzed by measuring their secreted levels of cytokines MTT assays showed that neither fNDs nor CpG-fNDs showed
in serum (Figure 4a). We found that CpG-fNDs dramatically apparent toxicity to cells after 72 h incubation, even at the highest
induced the production of serum IL-12 and IL-6 at 3 h after exposure dose of 100 g mL1 (Figure S7, Supporting Informa-
administration. Compared with the treatment with naked CpG tion). We further monitored in vivo biodistribution and clearance
ODNs of the same amount, the secreted levels of IL-12 and by using NDs labeled with a near-IR dye, XenoLight CF750 (see
IL-6 were enhanced by 19 and 61 times, respectively (Figure 4b, Supporting text, Table S2, Figure S8 and S9, Supporting Infor-
c). We also compared the in vivo immunostimulatory activity mation), which were administrated via intravenous injection in
of CpG-fNDs and high-dose naked CpG ODNs, and found mice. After 3 h of injection, we observed fluorescent signals were
that the CpG-fNDs treatment excelled even the highest dose predominantly in the liver from whole-body imaging, whereas
(40-fold) employed in this work, suggesting the high efficacy the free dye showed wide distribution in mice (Figure 5a). Quan-
of this nanoagent (Figure 4d). Further, we evaluated the sus- titative analysis showed that NDs were also accumulated in
tained immunostimulatory effect of CpG-fNDs. Significantly, spleen, lung and kidney. The peak accumulation after adminis-
we found that the secreted levels of IL-12 remained nearly the tration in these organs was found at 6 h, and the accumulated
same at 24 h after administration of CpG-fNDs, whereas they NDs were thoroughly cleared in all organs after 72 h (Figure 5b).
dramatically dropped in the case of high-dose naked CpG ODNs Since the liver is a primary organ of NDs accumulation before
(Figure 4d). Even at 48 h after administration of CpG-fNDs, the 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 effects in two murine tumor models, i.e., B16-F0 melanoma
(ALT), aspertate aminotransferase (AST), and alkaline phos- and 4T1 breast carcinoma xenograft models, which are known
phate (ALP).[23] We did not observe significant changes in these to express TLRs and widely used in cancer immunotherapy
parameters in mice exposed to fNDs or CpG-fNDs (Figure 5c), studies.[24] Tumor-bearing mice were treated with CpG-fNDs,
suggesting that CpG-fNDs treatment do not adversely affect CpG ODNs, fNDs, or normal saline (NS) once every 3 d
liver function. Histological analysis in multiple tissues also con- (Figure 6a). We found that the CpG-fNDs treatment led to a
firmed the safety of fNDs and CpG-fNDs (Figure 5d). tumor-weight decrease of 73.3% in the B16-F0 tumor model
Having established the potent in vivo immunostimulatory (Figure 6b1,b2; Figure S10a, Supporting Information) and
activity of CpG-fNDs, we further explored their antitumor 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 as revealed in rigorous in vitro and in vivo toxicological evalua-
or naked CpG ODNs did not significantly change the tumor tions. Hence, fNDs compare favorably with other nanoscale vec-
weight (Figure 6b1,b2 and c1,c2 and Figure S10, Supporting tors.[15,16,30,31] Several types of inorganic nanoparticles including
Information). Given the nonspecific nature of CpG-based gold nanoparticles have proven to be excellent vectors for CpG
immunotherapy, its treatment is considered to be comple- in cellular studies in vitro.[13,15,16] However, their prominent
mentary to chemotherapy. Our data demonstrated that mono- effects are often compromised in vivo due to the complexity of
therapy with CpG-fNDs led to significant tumor suppression blood circulation.[32] More recently, DNA nanostructures have
in two murine models, which reveals the great potential for its emerged as another type of promising biomolecular delivery
combination with chemotherapy. carriers with excellent biocompatibility, whose in vivo applica-
We next measured the secreted levels of cytokines in serum tions are nevertheless hampered by their high cost and short
after the treatment with CpG-fNDs. We found no statistical dif- blood circulation time.[16,30]
ference between normal and tumor-bearing mice in the serum In conclusion, we have demonstrated a novel delivery system
levels of IL-12, whereas the serum levels of IL-6 dramatically for therapeutic nucleic acids, CpG ODNs. CpG-fNDs exhibited
increased in both tumor models (Figure 6b3,b4 and 6c3,c4), remarkable cellular uptake efficiency that exceeded naked CpG
which correlated with the progression of tumors.[25] Interest- ODNs by nearly three orders of magnitudes. More importantly,
ingly, treatment with CpG-fNDs significantly increased the both in vitro and in vivo studies showed that CpG-fNDs could
serum levels of IL-12, and downregulated IL-6 in both tumor sustainably stimulate cytokine secretion for 23 d due to their
models (Figure 6b3,b4 and 6c3,c4). In contrast, fNDs or naked sponge-like porous nanostructures. We also confirmed that
CpG ODNs did not affect serum cytokine levels in tumor- this type of nanoagents had excellent biocompatibility as a drug
bearing mice (Figure 6b 3,b4 and 6c3,c4). Previous studies delivery carrier. Based on these new findings, we employed
showed that advanced-stage tumors resulted in elevated levels CpG-fNDs as a monotherapeutic drug for cancer immuno-
of serum IL-6 whereas minimally perturbed IL-12.[25] After therapy, which showed high antitumor efficacy in two murine
CpG-fNDs treatment, pronounced increase in IL-12 and tumor models. Whereas direct translating this one-shot immu-
decrease in IL-6 in both tumor models suggested that the nomodulatory agent to human use remains challenging, the
immunomodulatory effects of CpG-fNDs played an essen- success of CpG-fNDs-based therapy in animal models shed
tial role in the observed antitumor effects. In addition, since new light on cancer immunotherapy, and more broadly, design
nude mice are devoid of T cells, the tumor suppression effect and application of nanomedicine.
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- Experimental Section
uated under phase IIII clinical trials approved by U.S. Food Functionalization of NDs and Complexation with CpG ODNs: NDs with
and Drug Administration (FDA).[810] In these trials, immune individual sizes of 210 nm synthesized with the detonation technique
activation by CpG ODNs has shown therapeutic efficacies in were obtained from Gansu Gold Stone Nano. Material. Co. Ltd. (Gansu,
mice and in patient volunteers.[810,27] However, high-dose China). The unique crystal profile of the NDs was confirmed using an
ODNs ranging from several to tens of milligrams for patients 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
(or tens to hundreds of microgram per kilogram) are required
clusters was about 40200 nm. Characterization of the NDs has been
for treatment due to their low drug bioavailability and stability, described in our previous work.[18]
which brings about not only high treatment cost but also severe NDs were functionalized with poly(D-lysine) (PDL), polyethyleneimine
side effects.[9,10] Although the use of S-CpG by substituting the (PEI), or poly(allylamine hydrochloride) (PAH). See the Supporting
phosphodiester linkage by a phosphorothioate greatly increase Information for detailed preparation process. Prior to usage, CpG ODNs
the resistance to enzymatic degradation and increase the biolog- 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
ical activity of CpG ODNs,[11] these chemically modified ODNs
resuspended in cell culture medium for cell experiment and in NS for
posed significant safety concerns, e.g., renal damage.[28] In our animal experiment as required.
studies, we have demonstrated the advantages of CpG-fNDs Cell Line and Treatment: RAW264.7 macrophage-like cells were grown
for immunotherapy. By exploiting the high cellular uptake effi- in the Dulbeccos modified Eagles medium (DMEM) supplemented
ciency of fNDs, we can reduce the dose down to several micro- with 10% heat-inactivated FBS, 1.5 g L1 NaHCO3, 100 units/mL
grams, which is one-to-two orders magnitude lower than that 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.
for naked CpG ODNs. More importantly, the sustained immu-
Cells were seeded in 24-well plates at a density of 105 cells per well
nostimulatory effects of CpG-fNDs for up to 48 h largely avoid and incubated overnight to allow for cell adherence. fNDs (50 g cmL1),
repeated administration in therapeutic use. CpG ODNs (5, 50, 100, 200, 300 g mL1), non-CpG-fNDs (50 g mL1),
The distinctive porous nanostructure of fNDs plays a key role CpG-fNDs (50 g mL1), CpG-NDs (50 g mL1), CpG-PDL (5 g mL1
in their superior delivery ability. Due to the high surface energy CpG mixed with 2.5 g mL1 PDL), and S-CpG (50 109 M) were added
of NDs, they tend to form clusters with numerous nanoscale into the plate wells, respectively. It should be noted here that high-dose
pores.[18,29] Such sponge-like nanostructures not only pro- 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.
vide high capacity for accommodating CpG ODNs and protec-
Following 6 h incubation, the media were collected and centrifuged at
tion in the blood circulation system, but also endow sustained 13 000 rpm for 15 min at 4 C. Then, all samples were washed twice
release of CpG for long-term immunostimulatory responses. with phosphate buffered saline (PBS) and fresh cell culture medium
We also note that CpG-fNDs exhibit excellent biocompatibility, 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 [5] J. Vollmer, A. M. Krieg, Adv. Drug Delivery Rev. 2009, 61, 195.
the course of 3 d. All of the supernatants were collected and stored at [6] a) H. Hemmi, O. Takeuchi, T. Kawai, T. Kaisho, S. Sato, H. Sanjo,
80 C before assays. M. Matsumoto, K. Hoshino, H. Wagner, K. Takeda, S. Akira, Nature
Animals and Treatment: ICR mice (male, 1822 g) and nude mice 2000, 408, 740; b) A. M. Krieg, Annu. Rev. Immunol. 2002, 20, 709.
(female, 1822 g) were purchased from Shanghai SLAC Laboratory [7] B. Gungor, F. C. Yagci, G. Tincer, B. Bayyurt, E. Alpdundar, S. Yildiz,
Animal Co. Ltd., China. ICR mice were kept in conventional conditions M. Ozcan, I. Gursel, M. Gursel, Sci. Transl. Med. 2014, 6, 235ra61.
and nude mice were kept in pathogen-free conditions. Permission of [8] A. Carpentier, F. Laigle-Donadey, S. Zohar, L. Capelle, A. Behin,
the local ethics committee was obtained, and all animal experiments A. Tibi, N. Martin-Duverneuil, M. Sanson, L. Lacomblez,
were performed according to Chinese law and accepted international S. Taillibert, L. Puybasset, R. Van Effenterre, J. Y. Delattre,
standards in biomedical research.
A. F. Carpentier, Neuro-Oncology 2006, 8, 60.
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