Nanoscale Coordination Polymers for Synergistic NO and

Subscriber access provided by UNIV OF NEWCASTLE

Communication

Nanoscale Coordination Polymers for Synergistic NO and Chemodynamic Therapy of Liver Cancer Yihui Hu, Tian Lv, Yu Ma, Junjie Xu, Yihua Zhang, Yanglong Hou, Zhangjian Huang, and Ya Ding Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.9b01093 • Publication Date (Web): 28 Mar 2019 Downloaded from http://pubs.acs.org on March 28, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

Nanoscale Coordination Polymers for Synergistic NO and Chemodynamic Therapy of Liver Cancer Yihui Hu,†,# Tian Lv,†,# Yu Ma,† Junjie Xu,‡ Yihua Zhang,† Yanglong Hou,‡ Zhangjian Huang,†,* Ya Ding†,*

†

State Key Laboratory of Natural Medicines, Department of Pharmaceutical Analysis

and Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, China

Pharmaceutical University, Nanjing 210009, China

‡

Beijing Key Laboratory for Magnetoeletric Materials and Devices (BKL-MEMD), Beijing

Innovation Center for Engineering Science and Advanced Technology (BIC-ESAT),

Department of Materials Science and Engineering, College of Engineering, Peking

University, Beijing 100871, China

ACS Paragon Plus Environment

1

Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 37

* Corresponding authors: E-mail: [email protected], Tel&Fax: +86-25-

83271072 (Z. J. Huang), E-mail: [email protected], Tel&Fax: +86-25-83271326 (Y.

Ding)


2

Page 3 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

ABSTRACT. Nitric oxide (NO) induces a multitude of antitumor activities, encompassing

the induction of apoptosis, sensitization to chemo-, radio-, or immune-therapy, and

inhibition of metastasis, drug resistance, angiogenesis, and hypoxia, thus attracting

much attention in the area of cancer intervention. To improve the precise targeting and

treatment efficacy of NO, a glutathione (GSH)-sensitive NO donor (1,5-bis[(L-proline-1yl)diazen-1-ium-1,2-diol-O2-yl]-2,4-dinitrobenzene, BPDB) coordinates with iron ions to form the nanoscale coordination polymer (NCP) via a simple precipitation and then

partial ion exchange process. The obtained Fe(II)-BNCP shows desirable solubility,

biocompatibility, and circulation stability. Quick NO release triggered by high concentrations of GSH in tumor cells improves the specificity of NO release in situ, thus

avoiding side effects in other tissues. Meanwhile, under high concentrations of H2O2 in tumors, Fe2+ ions in BPDB-based NCP, named Fe(II)-BNCP, exert Fenton activity to

generate hydroxyl radicals (·OH), which is the main contribution for chemodynamic therapy (CDT). In addition, ·O2- generated by the Haber-Weiss reaction of Fe2+ ions with H2O2 can quickly react with NO to produce peroxynitrite anion (ONOO-) that is more


3

Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 37

cytotoxic than ·O2- or NO only. This synergistic NO-CDT effect has been proved to retard the tumor growth in Heps xenograft ICR mouse models. This work not only

implements a synergistic effect of NO-CDT therapy, but also offers a simple and efficient strategy to construct a coordination polymer nanomedicine via rationally

designed prodrug molecules such as NO donors.

KEYWORDS. nanoscale coordination polymer, NO therapy, chemodynamic therapy,

synergistic therapy, liver cancer

Nitric oxide (NO), a “star” gasotransmitter, plays prominent roles in various

physiological and pathological processes, such as cardiovascular homeostasis, neurotransmission, and immune responses.1-5 In the realm of cancer biology,

accumulating evidence reveals that relatively high levels of NO act as a cytotoxic and

apoptosis-inducing agent. After reacting with oxygen molecules (O2), superoxide ions (·O2-), and transition metals, NO forms various reactive nitrogen oxygen species (RNOS), such as NO2-, N2O3, ONOO-, etc., which oxidizes DNA and induces single-


4

Page 5 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

strand breaks, leading to apoptosis of tumor cells.6-8 Meanwhile, they also regulate the

activity of various metabolic enzymes, oxidoreductases, proteases, protein kinases, and phosphoproteinases in the tumor cells via nitration and nitrosylation reactions.8,9

Particularly, the nitration of the tyrosine residue in the ABC transporter (P-gp) causes

the loss of the transport function of P-gp and reduce the efflux of the drug from tumor cells, thereby inhibiting drug resistance.10,11 Therefore, NO has also been discovered to enhance the efficacy of other treatments such as chemotherapy,12,13 photodynamic therapy (PDT),14-17 radiotherapy,18,19 and ultrasound (US) therapy.20,21

Although the rate of NO generation from its donors is readily tuned by pH,22,23 light,10,24,25 and/or temperature26 via tactful design of the donor structure,27,28 the

application of NO-based cancer therapy remains a challenge in the precise targeting

and location-controlled release of NO in tumor site. As such, polymeric nanocarriers (e.g. films, hydrogels, and nanoparticles),24,29-33 silica particles,34-36 gold nanoparticles,37-39 and CaCO3 nanoparticles40 have been employed to carry NO donors in their interior or on their surface to overcome the drawback of small molecule NO


5

Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 37

donor. These nanocarriers improve the circulation stability of NO donors, avoid their systematic distribution, and increase NO accumulation in tumor via the enhanced

permeability and retention (EPR) effect. However, the shortcomings of the current

nanocarriers for NO donor delivery, including (1) low donor loading efficiency, (2) poor atom economy due to inactive nanocarriers,41 (3) the complexity in the structure that

limits potential clinic utilizations, and (4) the limited degradability and release efficiency,

made the efficacy insufficient in the synergistic therapy.

In this work, to solve the problems mentioned above in a NO-associated chemodynamic therapy (CDT) system,42-44 we designed a nanoscale coordination

polymer (NCP, Figure 1), a soft material constructed from metal ion connectors and polydentate bridging ligands.45-47 Different from encapsulating NO donor in

nanocarriers, the rational designed NO donor here with bidentate organic structure acts as building blocks and conjugates with Fe ions via coordination interaction using an

extremely facile mixture method. It is worth noting that the resultant NCPs are

composed of only NO donor and Fe ions that play their own roles and synergistic effects


6

Page 7 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters


Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 37

BPDB was characterized by 1H NMR and MS spectra (Figures S2 and S3, Supporting

Information). Two carboxyl groups in the chemical structure of BPDB endow active sites

for the coordination with metal ions, for instance, Fe ions used here. To avoid the instability of Fe2+ ions that are easily oxidized and influence the yield in the preparation,

aqueous solutions of BPDB (20 mM, pH~3) were mixed with equal volume of FeCl3·6H2O (150 mM) for 10 min at pH ~5.4. The Fe3+-conjugated BPDB, named Fe(III)BNCP, was precipitated by pouring methanol into the mixed solution under vigorous

stirring. And then, the Fe(III)-BNCP was isolated by centrifugation, washed with

methanol/water, and ultrasonically dispersed for the further use (yield 78.4%). The

framework of BNCP would be degraded by high concentration of GSH in tumor cells (~10 mM).49 4 molecules of NO can be liberated from each BPDB molecule triggered by 2 molecules of GSH (Figure S4, Supporting Information).48 To make a meaningful

comparison, (4,6-dinitro-1,3-phenylene)diproline (DNDP, Figures S5-S7, Supporting

Information) without diazeniumdiolate (“NONO”) NO donor moiety was synthesized as a

control ligand to prepare Fe(III)-DNCP without NO release under the stimuli of GSH.


8

Page 9 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

It has proved that cancer cells produce high amounts of H2O2 due to their rapid growth and abnormal metabolism.50 In the intracellular reduction conditions in tumor cells, Fe3+ ion is expected to be reduced into Fe2+ that exerts Fenton activity to generate hydroxyl radical (·OH) and hydroxide ion (OH-) in the presence of H2O2 (Figure 1 and Figure S8, Supporting Information). The creation of ·OH is the basic mechanism of Fenton reaction for CDT.42,51,52 Moreover, based on the Haber-Weiss cycle, ·OH is further oxidized by H2O2 to produce ·O2- that quickly reacts with NO to form peroxynitrite anion (ONOO-). As a RNOS, ONOO- is more cytotoxic than ·O2- or NO only and further enhances the efficacy of NO and CDT (Figure S8, Supporting Information). To accelerate the rate of Fenton reaction and enhance the generation of ONOO-, part of Fe3+ ions in Fe(III)BNCP and Fe(III)-DNCP were replaced by an aqueous solution of FeCl2·4H2O via the ion exchange reaction.53 Fe(II)-BNCP and Fe(II)-DNCP were rinsed with water,

precipitated upon adding of methanol, and then centrifuged for 3 times and stored at 4 oC

until use (yield 63.8%). Similarly, zinc ion without Fenton activity was employed

instead of the function of iron ions in NCPs. Thus, Zn(II)-DNCP, Fe(II)-DNCP, and


9

Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 37

Zn(II)-BNCP (characterized in Figures S11-S14, Supporting Information) were used as

the controls of Fe(II)-BNCP.

As shown in Figures 2A and S9 (Supporting Information), both Fe(III)-BNCP and

Fe(II)-BNCP displayed many coordinated clusters and they were structurally amorphous

due to the broad X-ray powder diffraction (XRD) patterns (Figure S10, Supporting

Information). The diameters of all prepared NCPs (~ 200 nm) showed no significant

difference both in water and phosphate buffer saline (PBS, 0.01 M, pH 7.4), determined

by dynamic light scattering (DLS) method (Figure S11A, Supporting Information). These

data suggest that the amorphous NCPs were in the nanoscale that may offer EPR effect

in vivo.


10

Page 11 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

Figure 2. (A) TEM images, (B) EDS chemical mapping, showing the distribution for the elements of C, N, O, and Fe (C: red, N: orange, O: yellow and Fe: green), and (C) XPS spectra of (a) Fe(III)BNCP and (b) Fe(II)-BNCP.

The organic skeleton of NCPs was determined by Fourier transform infrared (FT-IR)

spectra (Figure S12, Supporting Information). Characteristic peaks of BPDB were

shown in FT-IR spectra of Zn(II)-BNCP and Fe(II)-BNCP, including the stretching vibration of -NO2 (1350 and 1530 cm-1), -N=N- (1400 cm-1), and benzene rings (1500 and 1600 cm-1) (Figure S12A, Supporting Information). Similarly, the DNDP-based


11

Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 37

NCPs showed the FT-IR peaks of DNDP without the absorption of -N=N- (1400 cm-1)

due to the lack of “NONO” moiety in DNDP compared with BPDB (Figure S12B, Supporting Information). These peaks confirmed that the coordination ligands, e.g.

BPDB and DNDP, are the essential organic ingredient of NCPs and maintains its

intrinsic structure after the NCP preparation. To detect inorganic elements in NCPs,

chemical mapping of energy dispersive spectrometer (EDS) were obtained on a Tecnai-

G2-F30 (FEI) transmission electron microscope at an acceleration voltage of 300 kV

(Figures 2B and S13). Besides C, N, and O elements, either Fe or Zn content is evenly

distributed in the whole structures of NCPs, demonstrating the metallic ions are also the

important constituents in the NCP skeleton. In addition, the positive zeta potentials of

NCPs (Figure S11B, Supporting Information) imply the existence of metallic ions on the

surface of coordination polymers, which has been proved by X-ray photoelectron

spectroscopy (XPS) spectra of Fe 2p regions for Fe(III)-BNCP and Fe(II)-BNCP (Figure

2C, a and b). From surface species concentrations of Fe summarized by XPS (Table

S1, Supporting Information), Fe content in both BNCPs is similar (~12.5%). 13.52% of Fe3+ were reduced into Fe2+ in the preparation of Fe(III)-BNCP, while the ion exchange


12

Page 13 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

process replaces another 29.70% of Fe3+, resulting in 43.22% of Fe2+ in total iron

content of Fe(II)-BNCP. Based on thermal gravimetric analysis (TGA) results (Figure

S14A, Supporting Information), the composition of Fe(III)-BNCP is 66.4 % organic and

24.3 % metallic iron and Fe(II)-BNCP is 64.1 % organic and 26.6% metallic iron. Since the molar mass of BPDB and Fe are 514 g mol-1 and 56 g mol-1, respectively,

Fe4(BPDB)1(H2O)2 is the empirical formula for Fe-terminated BNCPs.

Combining TGA results with XPS data, the formulae for Fe(III)-BNCP and Fe(II)-

BNCP can be approximately deduced to be Fe(III)3.4Fe(II)0.6(BPDB)1(H2O)2 and Fe(III)2.3Fe(II)1.7(BPDB)1(H2O)2, respectively. Since 4 molecules of NO can be liberated from each BPDB molecule, the molar rate of Fe: NO in the NCPs prepared here is 1:1.54 The drug loading efficiency of Fe(II)-BNCP is calculated to be 66.4% BPDB (e.g. 15.5% NO), much higher than other nanocarriers of NO donor delivery systems.24-41

Compared with small molecule BPDB, the corresponding NCPs exhibited the

following advantages. BPDB is insoluble in neutral aqueous solution, whereas Fe- or

Zn-coordinated BPDB or DNDP were well-dispersed in water, which could facilitate


13

Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 37

intravenous injection. In addition, all NCPs except for Fe(II)-DNCP show good and

comparable biocompability in hemolysis assay (2 mM), the increase of GSH concentration caused the

decrease of detectable NO in solution (Figure 3A). One possible reason could be that high concentration of GSH captured NO to produce S-nitrosoglutathione (GSNO)55-58 under the catalysis of metal ions, thus decreasing the conversion of NO to NO2-. In this regard, 1 mM GSH, although much lower than the reported value (ca. 10 mM),49 was used in the following in vitro NO release studies to mimic the GSH level in tumor cells.


15

Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 37

Figure 3. In vitro NO and free radical release from NCPs at a dose of 20 Y Q) < (A) NO release at different levels of GSH, the production of (B) .OH and (C) .O2- from Zn(II)-DNCP, Fe(III)-DNCP, Fe(II)-DNCP, Zn(II)-BNCP, Fe(III)-BNCP, and Fe(II)-BNCP, and (D) the generation of ONOO- from Zn(II)-DNCP, Fe(II)-DNCP, Zn(II)-BNCP, and Fe(II)-BNCP at H2O2 concentration of 400 M. Data presented in (A)-(D) is the mean ± SD (n=3).

At the extracellular GSH concentration (2 Y 4 58 Fe(II)-BNCP and Zn(II)-BNCP

showed very low NO release in 24 h, only 0.07-fold and 0.11-fold of the data triggered

by 1 mM GSH (Figure S17, Supporting Information). It reflected the high stability of

BPDB-based NCPs in circulation and selective NO release inside tumor cells. Different


16

Page 17 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

from the rapid NO release of free BPDB, the coordinated BPDB exhibited a sustained

NO release behavior in 24 h at the GSH concentration of 1 mM. Excitingly, the relatively

slow NO release rate didn’t affect the drug release efficiency of BPDB-based NCPs. A

very high NO release percentage comparable to free BPDB, nearly 100%, was obtained

(Figure S17, blue and red curves, Supporting Information).

In addition, the efficient generation of .OH and .O2- via Fenton reaction and HaberWeiss cycle (Figure S8, Supporting Information) was detected by the fluorescent probe

p-phthalic acid (PTA) and hydroethidine (HE), respectively. In the presence of 400 M

H2O2, almost no free radical was detected in Zn(II)-based NCP groups, F/F0 value close to 1 (Figures 3B and 3C, black and blue histograms). In contrast, the increased

fluorescence intensities at 430 nm and 600 nm in Fe-containing NCP solutions represented the production of .OH and .O2-, respectively. It is noted that the fluorescence intensities of .OH and .O2- generated from Fe-containing NCPs after Fe2+ ion exchange were much higher than those original NCPs prepared by Fe3+ ions (Figures 3B and 3C). It demonstrates a higher proportion of Fe2+ ions in NCPs could


17

Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 37

increase the generation of .OH and .O2- at the same endogenous H2O2 level in tumor, namely the necessity of ion exchange using ferrous ion.

Moreover, ONOO- generated from NCPs was also detected by a specific peroxynitrite probe, PONP, according to a previous report.59 The synthesis procedure, 1H NMR and

MS spectra of PONP were presented in Figures S18-S20 (Supporting Information). It is interesting that, as shown in Figure 3D, only Fe(II)-BNCP produced remarkable ONOOamong all test samples, indicating the simultaneous NO release and .O2- generation from the unique structure of Fe(II)-BNCP. It reveals the mechanism that, under the

simulated tumor microenvironments, the efficacy of NO therapy would be enhanced with the help of Fe2+.

The above results demonstrate the stability of Fe(II)-BNCP under the low GSH and/or H2O2 conditions, as well as the effective generation of NO, .OH, .O2-, and ONOO- from this NCP in simulated tumor environments. To further confirm the intracellular

generation of NO and total amount of ROS, two fluorescent probes, diaminofluorescein-

FM diacetate (DAF-FM DA) and 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA),


18

Page 19 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

were employed, respectively. Cell imaging of liver normal (L02) and liver hepatocellular

carcinoma (HepG2) cell lines with the incubation of NCPs was carried out by confocal

laser scan microscopy (CLSM, Figure S21, Supporting Information, and Figure 4). In

L02 cells, no obvious fluorescence was found for all NCPs (Figure S22, Supporting Information), while consistent results with in vitro detection of NO and free radicals

shown in Figure 3 appeared in HepG2 cells. Red fluorescence of NO and green

fluorescence of ROS were observed in BPDB- and Fe(II)-based NCPs, respectively,

and Fe(II)-BNCP showed the strongest fluorescence of both signals (Figure 4). This

study confirmed again the synergistic NO-CDT effect of Fe(II)-BNCP in the specific

microenvironment of tumor cells.

This NO-CDT effect with high selectivity we proved above led to the lowest IC50 value of Fe(II)-BNCP, 14.3 ± 0.7 g/mL, against the highly sensitive HepG2 cells (vs. IC50, Zn(II)-BNCP=20.7

± 1.4 g/mL) (Figure S22A, Supporting Information). However, there was

almost no cytotoxicity in L02 cells of all these NPCs. It demonstrated that Fe(II)-BNCP

played NO-CDT synergistic effect in tumor cells and owned high biosafety in normal


19

Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 37

cells. Meanwhile, we further exploited the apoptosis and necrosis of HepG2 cells

induced by NCPs. As shown in Figure S22B (Supporting Information), Fe(II)-BNCP

playing NO-CDT synergistic role had highest necrosis rate (21.0%) compared with

Zn(II)-DNCP (5.9%), Fe(II)-DNCP (6.6%), and Zn(II)-BNCP (7.1%). Thus, institute of

cancer research (ICR) mouse model bearing Heps tumors were further established for

in vivo evaluation of antitumor efficacy of Fe(II)-BNCP.


20

Page 21 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

Figure 4. CLSM images of NO release and ROS generation of Zn(II)-DNCP, Fe(II)-DNCP, Zn(II)-BNCP, and Fe(II)-BNCP in HepG2 cells. The scale bar is 20 m.

Antitumor efficacy was evaluated in ICR mice bearing Heps tumors at a dose of 20

mg BPDB or DNDP/kg. The changes of tumor volume, body weight, survival rate, and

final tumor weight of mice treated with saline, Zn(II)-DNCP, Fe(II)-DNCP, Zn(II)-BNCP,

and Fe(II)-BNCP were presented in Figure B \3< Tumor growth was significantly


21

Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 37

suppressed in the group treated with Fe(II)-BNCP compared to other NCPs (Figure 5A,

only 0.33- and 0.40-folds of saline and Zn(II)-DNCP, respectively) after12-day treatment. On the 12th day, the tumor weight of the Fe(II)-BNCP group was not only

significantly smaller than those of other NCP groups (Figure 5B), but the tumor has

disappeared in one Fe(II)-BNCP-treated mice (Figure 5C). The body weight of mice

treated with Fe(II)-BNCP showed no significant difference with other groups (Figure

5D). It indicated that the mice lived a high-quality life and did not have much discomfort

after the treatment of Fe(II)-BNCP. Tumor-bearing mice in the survival rate experiments

continued to receive intravenous injections in the indicated formulation at 2-day intervals

(Figure 5E). After the 12-day treatment, the survival rates of mice in the saline (37.5%),

DNDP-based NCPs (50%), and Zn(II)-BNCP (62.5%) groups were lower than the

survival rate of Fe(II)-BNCP (87.5%), demonstrating a lower toxicity of the Fe(II)-BNCP

formulations. Therefore, the changes of tumor size, body weight, and survival rate in

Heps heterotopic xenograft mice clearly demonstrate higher tumor selectivity and

biosafety of Fe(II)-BNCP than other test groups. To demonstrate this hypothesis,

histological sections were stained by hematoxylin and eosin (H&E) and TdT-mediated


22

Page 23 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

dUTP nick end labeling (Figure 5F) to detect the cell-killing and apoptosis degree of

tumor and other normal tissues in Heps heterotopic liver tumor mice. Fe(II)-BNCP

resulted in the largest region of necrosis and the most significant apoptosis in the tumor

tissue, while no apparent damage and apoptosis were found in the normal tissues

(Figures S23 and S24, Supporting Information). These results demonstrated that the

treatment with Fe(II)-BNCP not only effectively inhibit the growth of tumor, but also reduced systemic toxicity of NO donor in vivo.


23

Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 37

Figure 5. Antitumor efficacy of (a) saline, (b) Zn(II)-DNCP, (c) Fe(II)-DNCP, (d) Zn(II)-BNCP, and (e) Fe(II)-BNCP in Heps tumor xenograft ICR mouse models. The changes of (A) tumor volume, (B) tumor tissue weight, (C) tumor photos after 12 days’ treatment, (D) body weight, and (E) survival rates of the five groups of tumor-bearing mice during the 12-day treatment. Each point in panels A, B, D, and E represents the mean ± SD (n = 8). (F) Histological observation of tumor tissue sections (stained with H&E) and detection of apoptosis in tumor tissue sections (stained by TdT-mediated dUTP nick end labeling using a TUNEL kit, Beyotime, Shanghai, China) from control and test groups 12 days post-injection. The scale bar is 50 Y)

Nanoscale Coordination Polymers for Synergistic NO and

Recommend Documents