Intracellular NO-Generator Based on Enzyme Trigger for Localized

Subscriber access provided by YORK UNIV

Biological and Medical Applications of Materials and Interfaces

Intracellular NO-Generator Based on Enzyme Trigger for Localized Tumor-Cytoplasm Rapid Drug Release and Synergetic Cancer Therapy Lin Hou, Yinling Zhang, Xuemei Yang, Chunyu Tian, Yingshan Yan, Hongling Zhang, Jinjin Shi, Huijuan Zhang, and Zhenzhong Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b17750 • Publication Date (Web): 18 Dec 2018 Downloaded from http://pubs.acs.org on December 23, 2018

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 34 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

ACS Applied Materials & Interfaces

Intracellular

NO-Generator

Based

on

Enzyme

Trigger

for

Localized

Tumor-Cytoplasm Rapid Drug Release and Synergetic Cancer Therapy

Lin Hou

a, b, c,

Yinling Zhang

a, d #,

Xuemei Yang

a, d #,

Chunyu Tian

a, d,

Yingshan Yan

a, d,

Hongling Zhang a, b, c, Jinjin Shi a, b, c, Huijuan Zhang a, b, c,*, Zhenzhong Zhang a, b, c,*

a

School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, China

b

Key Laboratory of Targeting Therapy and Diagnosis for Critical Diseases, Henan Province

c

Collaborative Innovation Center of New Drug Research and Safety Evaluation, Henan Province,

Zhengzhou, China d

Modern Analysis and Computer Center of Zhengzhou University

*Corresponding

Author:

Email

address:

[email protected]

(Zhenzhong

Zhang),

[email protected] (Huijuan Zhang); #These authors contributed equally.

Keywords: Nitric oxide, enzyme-responsive,

micelle,

synergistic therapy

ACS Paragon Plus Environment

glutathione S-transferase π,

ACS Applied Materials & Interfaces 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

Abstract Nitric oxide (NO) is an important biological messenger implicated in tumor therapy. However, current NO release systems suffer from some disadvantages, such as hydrolysis during blood circulation, poor specificity and robust irradiation for stimuli. Accordingly, we constructed an intracellular enzyme-triggered NO-generator to achieve tumor cytoplasm-specific disruption and localized rapid drug release. Diethylamine NONOate (DEA/NO) was used as a NO donor and conjugated with hyaluronic acid (HA) to form self-assembly micelle (HA-DNB-DEA/NO), and encapsulate chemotherapeutic agent (doxorubicin (DOX)) into its hydrophobic core (DOX@HA-DNB-DEA/NO). After HA receptor mediated internalization into tumor cells, HA shell would undergo digestion into small conjugated pieces by hyaluronidase. Meanwhile, DOX@HA-DNB-DEA/NO also responded to the intratumoral over-expressed glutathion and glutathione S-transferase π, leading to the intracellular NO production and controlled DOX rapid release. In vitro and in vivo results proved the enzyme-dependent and enhanced targeting delivery profile, and demonstrated that NO and DOX could co-locate in specific tumor site, which provided a precondition for exerting their synergistic efficacy. Moreover, expression of p53 protein was upregulated in tumor tissue after treatment, indicating that NO induced cell apoptosis mediated by tumor suppressor gene p53. Overall, this intelligent drug loaded NO-generator might perform as an enhancer to realize better clinical outcomes.


Page 2 of 34

Page 3 of 34 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


1. Introduction Nitric oxide (NO) is a gaseous signal molecule involved in many physiological and pathological processes, including vasodilatation, wound healing, antimalarial activity, diabetes and immune response1-3. In recent years, increasing evidences showed that NO possessed a great potential in cancer therapy4-8. It has been reported that NO played dual roles in tumor progression in a concentration-dependent manner9. Low concentration of NO (i.e., picomolar) can stimulate tumor growth by promoting angiogenesis, whereas high concentration (i.e., micromolar) will cause tumor regression and metastasis inhibition. The main mechanism of NO against tumor cells is generating reactive nitrogen species (RNS) which can damage DNA as well as mitochondria, induce lipid peroxidation and deplete stored antioxidants10-12. NO also increases the expression of tumor suppressor gene p53 and eventually results in tumor cell apoptosis13-14. In addition, co-delivery of NO with chemotherapeutic agents can significantly enhance the antitumor effects due to the chemosensitization of NO15-18. However, because of the gaseous state and short half-life in the body19, it is difficult to use NO as a therapeutic agent directly. To solve this problem, a series of NO donors have been designed, such as N-diazeniumdiolates (NONOates), sodium nitroprusside (SNP), S-nitrosothiols (RSNOs) and nitric oxide-donor doxorubicins (NO–DOXOs)1,

20-23.

Unfortunately, these low molecular weight compounds lack

specificity and are easily cleared during blood circulation. In order to realize tumor targeting NO delivery and prolong its life in vivo, NO-generating nanoparticles that can release NO by some exogenous stimulating factors such as X-ray24, ultraviolet25 and near-infrared (NIR) light16 have been constructed. Nevertheless, the shallow penetration depth of ultraviolet and NIR light as well as the harmful ionizing radiation of X-ray to adjacent normal tissue limited their application in cancer treatment26. It is pressing to develop a nano-platform that can response to endogenous stimuli at tumor sites to achieve NO pinpointed release. PABA/NO

[O2-(2,4-dinitro-5-(4-(N-methylamino)benzoyloxy)phe-

nyl)

1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate] and JS-K [O2-(2, 4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2- diolate] are two typical



Page 4 of 34

NO-release prodrugs that can be metabolized by intracellular enzymes27-30. For example, PABA/NO is able to release NO catalyzed by glutathione S-transferase π (GSTπ) which is over-expressed in many human tumor cells and closely related to poor prognosis together with drug resistance31-34. However, the poor solubility and relatively instability restricted the further research and application of these prodrugs35. Inspired by PABA/NO and JS-K, we conceived a NO-generator with hydrophobic NO donors in its core, which could be able to release NO upon the nucleophilic attack of the sulfur atom of GSH catalyzed by GSTπ. Hyaluronic acid (HA) was introduced as a hydrophilic shell and conjugated with diethylamine NONOate (DEA/NO) via 1,5-Difluoro-2,4-dinitrobenzene (FFDNB) to form a novel amphiphilic polymer (HA-DNB-DEA/NO) which could be self-assembled into micelles. Herein, the biodegradable HA can recognize and bind to CD44 receptor overexpressing on numerous kinds of tumor cells36-39. Furthermore, it has been reported that NO holds potential in enhancing tumor EPR effects, and thus can improve targeting delivery40. After specific internalization into tumor cells, HA shell could be digested by hyaluronidase (HAase) and facilitate the NO-generator exposing to the GSH and GSTπ. Based on above consideration, we hypothesized that HA-DNB-DEA/NO could keep stable during blood circulation due to lack of HAase and GSTπ. Since NO can enhance the antitumor activity of chemotherapeutic drugs with direct DNA interference41, doxorubicin (DOX) is chosen as a model drug and encapsulated into the hydrophobic core. Once DOX loaded HA-DNB-DEA/NO (DOX@HA-DNB-DEA/NO) is internalized into cancer cells, this system can be responsively triggered by serial enzymes to generate NO, instantaneously burst, accordingly release DOX, and eventually lead to the synergistic antitumor effects (As shown in Figure1). In

this

investigation,

the

self-assembled

DOX

loaded

NO-generator

(DOX@HA-DNB-DEA/NO) was prepared and characterized by transmission electron microscope (TEM) and dynamic light scattering (DLS). The amount of NO triggered by GSTπ and the enzyme-dependent profile were detected in vitro using Griess reagent. The receptor-mediated cellular uptake, cytotoxicity and apoptosis for


Page 5 of 34 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


DOX@HA-DNB-DEA/NO were also evaluated on human hepatoma cell line SMMC-7721. Afterwards, the tumor targeting ability and antitumor efficacy of DOX@HA-DNB-DEA/NO were examined on human hepatocellular carcinoma (HCC) tumor xenograft models. In addition, the generation of NO in tumor site after intravenous injection was assessed through frozen section staining. Western blot, immunohistochemistry and hematoxylin-eosin (H&E) staining methods were further employed

to

estimate

tumor

progression

after

treatment

with

the

DOX@HA-DNB-DEA/NO.

Figure 1. Scheme of enzyme-triggered NO-generator enhanced tumor-targeting and synergetic cancer

therapy.



Page 6 of 34

2. Results and discussion 2.1.

Synthesis

and

characterization

of

HA-DNB-DEA/NO

and

DOX@HA-DNB-DEA/NO HA-DNB-DEA/NO was synthesized as shown in Figure 2. The structure of HA-DNB-DEA/NO was characterized by 1H NMR (Figure 3A) and FT-IR spectra (Figure 3B). In 1H NMR spectrum of HA-FDNB, the two doublet sharp peaks at δ= 6.40 and 8.83 ppm were assigned to ArH, suggesting the coupling of HA and FFDNB. By contrast, two singlet peaks belonging to ArH (δ= 7.22 and 8.67 ppm) and the emerging peaks of -CH3- (δ= 0.91 ppm) and -CH2- (δ= 2.83 ppm) in HA-DNB-DEA/NO spectrum indicated DEA/NO was successfully conjugated with HA-FDNB. For FT-IR analysis, HA-FDNB exhibited the specific absorptions at 1621, 1568, and 1537 cm-1 arising from aromatic ring skeleton vibration of FFDNB, and the emerging peak at 1773 cm-1 might be attributed to ester bond, which further verified the formation of HA-FDNB. Moreover, nucleophilic substitution reaction between F in HA-FDNB and O grafted on DEA/NO was confirmed by characteristic peak at 2979, 2877 cm-1 (-CH3-) and 2934 cm-1 (-CH2-). In addition, the successful conjugation between HA and FFDNB could be demonstrated with the appearance of the absorption at 267 nm owing to aromatic ring from FFDNB in UV-vis spectrum (Figure S1A). By comparison, the absorption peak of HA-DNB-DEA/NO red shifted to 276 nm. The critical micelle concentration (CMC) of amphiphilic HA-DNB-DEA/NO was determined by fluorescence spectroscopy using pyrene as a probe, and CMC value of HA-DNB-DEA/NO was 15.52 μg/mL (calculated from the curve in Figure S1B). Such low value indicated of the high thermodynamic stability of this system42-43 and guaranteed the self-assembled micelles to retain their original morphology even under the highly diluted conditions during blood circulation. As shown in Figure 3C and D, TEM image of HA-DNB-DEA/NO displayed spherical shape with a distinct core-shell structure, and the particle size was around 150 nm, which was similar to that determined by using DLS.


Page 7 of 34 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


After formation of micelles, HA-DNB-DEA/NO had a great potential to load hydrophobic drugs into its inner core. DOX, a broad-spectrum chemotherapeutic drug44-45, was selected as a model agent in this subject. According to TEM (Figure 3C, b) and DLS (Figure 3D, b) analysis, DOX@HA-DNB-DEA/NO showed larger size about 190 nm compared with HA-DNB-DEA/NO, which might be attributed to the entrapment of DOX into HA-DNB-DEA/NO. It is well documented that suitable size (20-200 nm) of nanoparticles are readily accumulated in tumor regions due to enhanced permeability and retention (EPR) effect46, so DOX@HA-DNB-DEA/NO would possess a favorable tumor targeting efficiency. The surface zeta potentials of HA-DNB-DEA/NO and DOX@HA-DNB-DEA/NO were -38.5 and -23.3 mV (Figure 3E, b) respectively, indicating this system could avoid aggregation and rapid clearance by mononuclear phagocyte system (MPS) during blood circulation47. The amount of DOX loaded into HA-DNB-DEA/NO was calculated by measuring the UV-vis absorption at 481 nm. The drug-loading (DL) of DOX was 20.12% on the basis of above-mentioned feed ratio, which implied that HA-DNB-DEA/NO could act as an outstanding vehicle for drug delivery. F

O2N

O

O

O OH

OH O

O OH

O

+

O

OH O

F

F

NO2

NH

O2N

Na2CO3

O

O O

NO2

OH O

O

OH

O

OH

n

O

OH

NH

O

n

FFDNB

HA

HA-FDNB

N N

O

N

+ O

N

N

O

O2N

t-BuOH N2

N O

Na

O2N

O

O O

OH

OH O

O OH

O

O

OH O

NH

n

DEA/NO

HA-DNB-DEA/NO polymer

Figure 2. The synthesis of HA-DNB-DEA/NO.



Page 8 of 34

Figure 3. Characterization of polymer conjugates and micelles. (A) 1H NMR spectra of HA, HA-FDNB, and HA-DNB-DEA/NO; (B) FT-IR spectra of HA (a), HA-FDNB (b), and HA-DNB-DEA/NO

(c);

(C)

TEM

images

of

HA-DNB-DEA/NO

(a)

and

DOX@HA-DNB-DEA/NO (b); (D, E) Size and zeta potential distribution of HA-DNB-DEA/NO (a)

and

DOX@HA-DNB-DEA/NO


(b)

by

DLS.

Page 9 of 34 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


2.2. In vitro NO release with response to serial enzymes As a structural analogue of NO-prodrug, we presumed that HA-DNB-DEA/NO was able to release NO under high level of GSH catalyzed by GSTπ which was abundantly expressed in many tumor cells. Moreover, HAase highly expressed in tumor cells would also affect the outer shell of HA-DNB-DEA/NO to facilitate the exposure to GSTπ. In order to verify this hypothesis, the release behavior of NO from HA-DNB-DEA/NO was monitored by using Griess reagent in different PBS medium (Figure 4A). After 40 h incubation, 6.74 μM of NO was released from 100 μg/mL of HA-DNB-DEA/NO in PBS containing 10 mM GSH and 5 μg/mL GSTπ, while only 0.338 and 0.547 μM of NO were detected in PBS or PBS with 10 mM GSH even after 96 h, respectively.

More importantly, the release of NO was effectively accelerated

with the increasing concentration of GSTπ from 50 ng/mL to 5 μg/mL, suggesting the enzyme-induced and transferase-dependent properties of this NO-generator. However, when GSH was removed from the release medium, NO was scarely generated which was similar with the behavior in PBS, even in the presence of GSTπ. This was probably because NO was generated by nucleophilic substitution reaction of the thiol functionality from GSH to the electrophilic center of HA-DNB-DEA/NO under the catalysis of GSTπ. In other words, it further confirmed that NO release should be under simultaneous stimulus of GSH and GSTπ, which are always interdependent and coexistence in the tumor cells. Subsequently, HAase was introduced into buffer solutions with GSH and GSTπ, and we found that the amount of NO released was significantly increased compared with that in corresponding medium without HAase. For instance, 8.82 μM of NO was detected after incubation with HAase for 96 h , which was significantly higher than that without influence of HAase. The change in the particle size of HA-DNB-DEA/NO was also observed by DLS and TEM. As shown in Figure S2 and Figure 4B, NO-generator retained their structural integrity in the absence of enzymes. Conversely, the particle size sharply increased (Figure 4B) and the nano-structure was destabilized after touch with HAase/GSH/GSTπ for 12 h (Figure S2), indicating that



the release of NO might cause rupture of this system, which provided a fundamental premise for internal drug release. It should be mentioned that NO was released a little slowly in the first few hours, which might be because the core-shell structure of micelles prevented the progress of catalytic reaction, thus delaying the release of NO.

500

**

8 7 6

*** *** ***

Nitrite (μM)

9

5 4 3 2

400

*** *** ***

10

GSH+GSTπ（ 5μg/mL) GSH PBS HAase+GSH+GSTπ（ 5μg/mL) GSTπ（ 5μg/mL) GSH+GSTπ（ 50ng/mL) GSH+GSTπ（ 1μg/mL)

600

Particle size (nm)

11

700

B


12

**

13

A

300 200 100

1 0

0 0

20

40

60

80

100

0

20

Time (h)

40

60

80

100

Time (h)

100

C


90 80

*

70 60 50

*** *** ***

Cumulative DOX release (%)

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 34

40 30 20 10 0 0

20

40

60

80

100

Time (h)

Figure 4. In vitro responsive release under the catalysis of HAase and GSTπ. (A) Release profile of NO from HA-DNB-DEA/NO in the presence or absence of GSH,

HAase and GSTπ in PBS at

37 ℃ (n=3); (B) The variation of particle size for HA-DNB-DEA/NO micelles in different release media (n=3); (C) Release curve of DOX from DOX@HA-DNB-DEA/NO with or without the presence of GSH,

HAase and GSTπ. The error bars in the graph represent standard deviations

(n=3). ** P < 0.01, *** P < 0.001.

From the results as above, it could be inferred that HA-DNB-DEA/NO might maintain stable and would not cause NO release in the normal physiological condition. Once they arrived at tumor cells, the HA compact outer shell could be degraded by HAase highly active in tumor cells, resulting in reduced spatial barrier on the micelles,


Page 11 of 34 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


and then HA-DNB-DEA/NO could respond to the overexpressed GSH/GSTπ and trigger NO tumor cytoplasm-specific release. 2.3. In vitro drug release Having confirmed the desired enzyme responsiveness of NO-generator, we further investigated the release profile of DOX in different medium. As shown in Figure 4C, DOX released slowly from DOX@HA-DNB-DEA/NO in PBS (pH 7.4) during the whole process. For example, only 2.17% and 3.16% of DOX released within 24 h and 96 h, respectively. Similar behavior of DOX was observed in the medium only containing 10 mM GSH, which implied that there would be almost no drug leakage during the systemic circulation. On the contrary, a rapid release occurred in the presence of 10 mM GSH and 5 μg/mL of GSTπ, and accumulative percentage reached 42.36% and 65.29% after 24 and 96 h. This might be due to the fact that formation of NO from the generator could lead to the partial dissociation of micelles, which eventually caused a continuous and considerable leakage of DOX. Similar with the experiments in section 2.2, HAase was further introduced into release medium containing GSH/GSTπ, and DOX release rate was accordingly promoted compared with that in the homologue medium withou HAase. It can be seen that DOX relase percentage was increased from 36.54% to 60.35% at 20 h. Cotriggered release by HAase and GSTπ was validated to be faster than single triggered release with only GSTπ. After HAase-mediated degradation, the remaining conjugated small pieces of HA could be easily catalyzed by GSTπ through GSH nucleophilic attack, subsequently further promoting DOX release. These results confirmed that DOX@HA-DNB-DEA/NO could realize cytoplasm-selective NO and DOX release after uptake by cancer cells where HAase, GSH and GSTπ was overexpressed, and then achieve a synergistic cancer therapy. 2.4. Cellular experiments 2.4.1. In vitro cellular uptake study Cellular uptake of DOX@HA-DNB-DEA/NO was investigated on SMMC-7721 cells overexpressing GSTπ to evaluate the specific tumor cell targeting ability and intracellular DOX release34. As can be seen in Figure 5A, after 2 and 6 h incubation,



free DOX (red fluorescence) was completely internalized into cell nucleus which was the target of DOX. Nevertheless, most of DOX@HA-DNB-DEA/NO was localized in the cytoplasm at 2 h, suggesting that DOX was still wrapped in the NO-generator at this time point. With incubation time extending to 6 h, DOX was released and subsequently entered into cell nucleus. This could be explained by HA shell degradation and destruction properties of micelles after NO release under the catalysis of GSTπ, then leading to the leakage of DOX. To further prove the function of HA in facilitating the cellular internalization via interaction with CD44 receptors, SMMC-7721 cells were pretreated with free HA for 2 h prior to the incubation with DOX@HA-DNB-DEA/NO. As SMMC-7721 cells abundantly expressed CD44 receptor48-49 that could bind with HA, the cellular uptake of DOX@HA-DNB-DEA/NO might be based on HA-receptor mediated endocytosis. Accordingly, it was found that the fluorescent intensity of DOX was significantly inferior to that of untreated one, suggesting that free-HA could competitively bind to CD44 receptor against the HA-DNB-DEA/NO resulting in the decreased fluorescence signals. Collectively, DOX@HA-DNB-DEA/NO allows cytoplasm-selective delivery and enzymed-triggered drug release favoring nuclei-specific accumulation. 2.4.2. Intracellular NO detection Considering the novel NO donor system in this work could inherently generate NO in the presence of GSH and GSTπ, which could be further enhanced by HAase, the NO release properties in SMMC-7721 cells was examined by DAF-FM DA probe that could react with NO to display a obviously green fluorescence50. As shown in Figure 5B, faint fluorescent signals appeared in cells after treatment with HA-DNB-DEA/NO for 2 h. With incubation time prolonged to 6 h, noticeable and strong green fluorescence was observed, suggesting that HA-DNB-DEA/NO entered into SMMC-7721 cells and then released NO. Together with the results that SMMC-7721 showed the time-dependent DOX fluorescence increase after incubation with DOX@HA-DNB-DEA/NO, we supposed that efficient cellular uptake of NO-generator and serial enzymes catalysis resulted in specifically intracellular co-delivery of NO and drug.


Page 12 of 34

Page 13 of 34 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


Figure 5. Fluorescence microscope images. (A) Cellular uptake study after treatment with free DOX for 2 h (a) and 6 h (d) or pretreated cells with free HA for 2 h then incubated with DOX@HA-DNB-DEA/NO for 2 h (b) and 6 h (e) or incubated with DOX@HA-DNB-DEA/NO for 2 h (c) and 6 h (f ) (n=3); (B) Intracellular NO detection in SMMC-7721 cells (a) and HL-7702 cells (b) after incubating with HA-DNB-DEA/NO for 2 h and 6 h by using DAF-FM DA probe (n=3). * P < 0.05, ** P < 0.01, *** P < 0.001.

As HAase, GSH and GSTπ are overexpressed in cancer cells, it can be assumed that NO would be generated selectively in tumor cells rather than normal cells after



treatment

with

HA-DNB-DEA/NO.

Therefore,

NO

Page 14 of 34

delivery

behavior

of

HA-DNB-DEA/NO was also evaluated on HL-7702 cells as the normal control. As expected, HL-7702 cells remained invisible of green fluorescence even at a micelles concentration increased up to 100 μg/mL. It meant that HA-DNB-DEA/NO indeed generated much higher level of NO in tumor cells than normal cells, indicating of specific anticancer activity and low toxicity on normal site. 2.4.3. In vitro cytotoxicity evaluation To investigate the selectivity in antitumor efficacy of DOX@HA-DNB-DEA/NO to tumor cells and normal cells, the cytotoxicity on SMMC-7721 cells were tested by SRB

assay

after

treatment

with

free

DOX,

HA-DNB-DEA/NO,

or

DOX@HA-DNB-DEA/NO. According to Figure 6A, cell viability displayed drug concentration- and time-dependent patterns. Interestingly, we found that drug free HA-DNB-DEA/NO demonstrated an antitumor efficacy at high concentration with IC50 value of 58.128, 33.888, 22.226 μg/mL at 24, 48 and 72 h, respectively (Table S1). It has been well documented that high concentration of NO can kill cancer cells directly while low concentration of NO is more likely to promote tumor growth1, 9, and thus a slight proliferation of SMMC-7721 cells was observed at extremely low HA-DNB-DEA/NO concentrations (equivalently 0.039~0.156

μg/mL of DOX in

Figure 6A) after 24 h incubation. By contrast, free DOX was able to transport into cells by passive diffusion, leading to a great cytotoxicity. As shown in Figure 5A, there was no significant difference in the uptake of DOX@HA-DNB-DEA/NO and free DOX by SMMC-7721 cells. Nevertheless, the cell viability for DOX@HA-DNB-DEA/NO group remarkably decreased in comparison with free DOX group, and the IC50 value were 2.59-, 2.42-, and 3.45-folds lower than free DOX group at 24, 48 and 72 h. This might

be

attributed

to

simultaneous

release

of

NO

and

DOX

from

DOX@HA-DNB-DEA/NO, in which NO could react with intracellular molecules such as superoxide anion and oxygen to produce reactive nitrogen species (RNS)10, 12. On one hand, RNS could cause DNA base deamination, inhibit mitochondrial respiration, nitrify many functional proteins and induce lipid peroxidation, thus


Page 15 of 34 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


resulting in a powerful tumor killing ability1. On the other hand, RNS could also inhibit the activity of DNA repair enzymes to augment the cytotoxicity of DOX41. These results suggested that antitumor activity would be significantly enhanced by co-delivery of NO and DOX.

Figure 6. In vitro antitumor activity of different formulations. (A) In vitro cytotoxicity of HA-DNB-DEA/NO, free DOX, and DOX@HA-DNB-DEA/NO after incubating with SMMC-7721 cells for 24, 48, and 72 h. The concentration of blank HA-DNB-DEA/NO was consistent with the carriers in DOX@HA-DNB-DEA/NO; (B) Cell apoptosis of SMMC-7721 cells after treatment with blank control (a), HA-DNB-DEA/NO (b), free DOX (c), and DOX@HA-DNB-DEA/NO (d). * P < 0.05, ** P < 0.01, *** P < 0.001.



2.4.4. Cell apoptosis assay Flow cytometry was employed to investigate cell apoptosis after different treatments. Compared with the control group, a total apoptosis rate of SMMC-7721 cells incubated with HA-DNB-DEA/NO for 24 h was 10.6% (4.2% and 6.4% for early and late apoptosis, respectively), while free DOX group showed an apoptosis rate of 22 % (Figure 6B). NO was reported to exert antitumor activity by increasing the expression of tumor suppressor gene p53 to induce cell apoptosis13-14. Hence, cell apoptosis caused by HA-DNB-DEA/NO might be owing to the release of NO under the catalysis of GSTπ in cells. However, there were 74.7% of early and late apoptosis after treatment with DOX@HA-DNB-DEA/NO, which was significantly higher than free DOX and HA-DNB-DEA/NO alone. It could be explained by the fact that NO not only could induce tumor cell apoptosis, but also enhance the pro-apoptotic capacity of DOX via inhibiting the activity of DNA repairing enzymes, and eventually leading to a noticeable cell apoptosis. 2.5. In vivo experiments 2.5.1 In vivo imaging analysis In order to estimate the biodistribution of HA-DNB-DEA/NO after intravenous injection, a noninvasive near-infrared (NIR) optical imaging technique was applied. DiR, a hydrophobic NIR dye, was encapsulated into HA-DNB-DEA/NO (DiR@HA-DNB-DEA/NO) for tracking the carrier in vivo. The tumor-bearing nude mice were divided into three groups: saline, free DiR and DiR@HA-DNB-DEA/NO, and the real-time images at different time points were recorded in Figure 7A. For free DiR group, DiR was lack of tumor targeting ability and mainly accumulated in liver. In addition, the fluorescence intensity weakened gradually with time, suggesting that free DiR could be cleared easily in vivo. On the contrary, DiR labeled HA-DNB-DEA/NO could accumulate in tumor region at 1 h post-injection, and signals were still strong up to 24 h. This notable tumor targeting capacity could be elucidated by NO-mediated EPR effect as well as receptor-mediated endocytosis. Additionally, it was worth mentioning that the vasodilatory effect of NO released in


Page 16 of 34

Page 17 of 34 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


tumor site would dilate blood vessels and boost EPR to enhance the passive tumor target ability40. The fluorescent signals of DiR@HA-DNB-DEA/NO lasted for a long time even after 24 h post-injection, indicating its prolonged circulation feature. This characteristics was due to the hydrophilic surface and negative charge of this system which could avoid rapid clearance by MPS during blood circulation47. A

B

4265.

5733.5

7201.9

8670.4

1013

Figure 07. In vivo tumor-targeting evaluation. (A) Fluorescence imaging of tumor-bearing nude 8.9 mice treated with saline (a), free DiR (b), and DiR@HA-DNB-DEA/NO micelles (c) at different time points. (B) Ex vivo fluorescence imaging of the major organs and tumors harvested from the mice after treatment with saline (a), free DiR (b), and DiR@HA-DNB-DEA/NO (c) for 24 h.

After 24 h, mice were sacrificed and major organs and tumors were harvested for ex vivo imaging. As shown in Figure 7B, DiR labeled HA-DNB-DEA/NO displayed an intense fluorescent intensity in tumor site, while free DiR preferred to accumulate in liver and spleen. Taken together, it proved that HA-DNB-DEA/NO possessed a strong tumor targeting ability and enhanced tumor retention time.



2.5.2 NO detection in tumors Having confirmed the intracellular NO release properties of HA-DNB-DEA/NO, we further verified the generation of NO in solid tumors by frozen section images (Figure 8A). After intravenous administration with HA-DNB-DEA/NO and DOX@HA-DNB-DEA/NO in tumor-bearing mice, distinctly green fluorescence signals belonging to NO were noticed under CLSM, while saline control and free DOX groups didn’t present any fluorescence. These results proved that HA-DNB-DEA/NO possessed the capability to release NO at tumor site even after systemic injection. For DOX@HA-DNB-DEA/NO group, NO and DOX signals both appeared in the same location, indicating that DOX@HA-DNB-DEA/NO could co-deliver NO and DOX to the tumor. Moreover, DOX@HA-DNB-DEA/NO group exhibited much stronger DOX fluorescence than free DOX group, which further certified the enhanced tumor targeting ability of DOX@HA-DNB-DEA/NO. The major mechanism of NO for killing cancer cells was involved in generating RNS which can nitrate tyrosine residues of many proteins to produce 3-NT (an indicator for NO-medicated cell damage) 6. Therefore, 3-NT was adopted as an indirect marker to examine the presence of NO. Immunohistochemistry analysis for 3-NT in tumor tissues were shown in Figure 8B. Tumor tissues treated with HA-DNB-DEA/NO and DOX@HA-DNB-DEA/NO both revealed 3-NT signals obviously. This result testified the production of NO in tumor site after treatment with HA-DNB-DEA/NO or DOX@HA-DNB-DEA/NO, which was consistent with the results of immunofluorescence analysis.


Page 18 of 34

Page 19 of 34 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


Figure 8. NO detection in tumors.

(A) Frozen section images of NO release in tumor tissues

after intravenous injection with saline (a), HA-DNB-DEA/NO (b), free DOX (c), and DOX@HA-DNB-DEA/NO (d); (B) Tumor slices stained by 3-NT immunohistochemistry after treatment with saline, HA-DNB-DEA/NO, free DOX, and DOX@HA-DNB-DEA/NO. * P < 0.05, ** P < 0.01, *** P < 0.001.



Page 20 of 34

2.5.3 In vivo anti-tumor efficacy evaluation To investigate in vivo antitumor effect of different drug formulations, we established SMMC-7721 tumor xenograft models on nude mice. During 12 days of treatment, changes of relative tumor volume (V/V0) for each group were summarized in Figure 9A, and HA-DNB-DEA/NO demonstrated a modesty tumor growth inhibition, owing to the antitumor activity of NO released in tumor site. Free DOX group demonstrated similar trend of tumor growth with HA-DNB-DEA/NO group, with

a

relative

tumor

volume

of

2.44

±

0.29.

Nevertheless,

DOX@HA-DNB-DEA/NO displayed the best therapeutic efficacy with a reduced relative tumor volume of 0.69 ± 0.17 after day 12, which was much lower than free DOX or HA-DNB-DEA/NO group alone. At the endpoint, tumor tissues were isolated and then weighed. The tumor inhibition ratio of HA-DNB-DEA/NO, free DOX and DOX@HA-DNB-DEA/NO was 29.69% ± 2.2%, 43.75% ± 3.4%, and 84.38% ± 4.1%, respectively (Figure 9B). This excellent antitumor activity of DOX@HA-DNB-DEA/NO, on one hand, could be attributed to the enhanced EPR effects caused by NO and active tumor targeting caused by HA, which increased the accumulation of nanoparticles in tumor regions36. On the other hand, the released NO and DOX from DOX@HA-DNB-DEA/NO in tumor site could synergistically enhance the therapeutic efficacy16-17, 41. What’s more, the histological changes of tumor tissues were tested through H&E staining (Figure 9C). It could be observed a compact cell arrangement in saline group, whereas

cell

shrinkage

HA-DNB-DEA/NO

and

and free

expanding DOX

intercellular

groups.

By

space

contrast,

the

emerged

in

images

of

DOX@HA-DNB-DEA/NO group displayed apparent cell lysis and necrosis, implying a remarkable antitumor activity. Furthermore, the status of cell survival and proliferation

in

tumor

tissues

were

evaluated

by

TUNEL

and

PCNA

immunohistochemistry. Figure 9C displayed large amounts of apoptotic cells in TUNEL images and barely proliferating cells in PCNA images after treatment with DOX@HA-DNB-DEA/NO. It has been reported that NO could induce tumor cell apoptosis mainly by increasing the expression of p53 which belonged to a tumor


Page 21 of 34 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


suppressor gene13-14. Therefore, western blot was employed to analyze the expression level of p53 protein in tumor tissues (Figure 9D). Compared with saline control group, p53 protein was up-regulated in HA-DNB-DEA/NO and DOX@HA-DNB-DEA/NO groups, indicating that NO could activate and induce the expression of p53. Moreover, the expression of Bax and cleaved caspase-3 proteins were up-regulated significantly, and the expression of Bcl-2 protein was decreased. It could be inferred that p53 might up-regulate Bax gene and down-regulate Bcl-2 gene and eventually lead to the activation of Caspase-3, which were consistent with previous reports51-52. This might be a signaling pathway for NO-inducing apoptosis. The toxicity of different formulations was further examined. Mice treated with free

DOX

exhibited

continuous

weight

loss

(Figure

S3A),

but

DOX@HA-DNB-DEA/NO induced slight increase in body weight. This negligible systemic toxicity in vivo might be owing to tumor targeting ability of carriers. The toxicity of main organs was also evaluated by H&E staining (Figure S3B). It was noteworthy that free DOX caused myocardial fibers swell, distortion and deformation due

to

its

severe

cardiotoxicity,

while

no

breakage

was

observed

in

DOX@HA-DNB-DEA/NO group. These results demonstrated that DOX loaded in NO-generator could not only improve the therapeutic efficacy, but also reduce the non-targeting distribution in normal organs.



Figure 9. In vivo antitumor efficacy evaluation. (A) The variation of relative tumor volume during treatment in different groups; (B) Tumor inhibition ratio after treatment with HA-DNB-DEA/NO, free DOX, and DOX@HA-DNB-DEA/NO; (C) Tumor slices stained by H&E, TUNEL as well as PCNA immunohistochemistry after treatment with saline, HA-DNB-DEA/NO, free DOX, and DOX@HA-DNB-DEA/NO; (D) Western blotting

for the expression of proteins in tumor tissues


Page 22 of 34

Page 23 of 34 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


after treatment with saline, HA-DNB-DEA/NO, free DOX, and DOX@HA-DNB-DEA/NO. * P < 0.05, ** P < 0.01, *** P < 0.001.

3. Conclusion In this subject, HA-DNB-DEA/NO was successfully synthesized as a novel NO-generator to achieve intracellular enzyme-triggered NO and DOX tumor cytoplasm-selective release. In vivo imaging analysis proved that HA-DNB-DEA/NO preferentially targeted to the tumor region without accumulation in normal organs, via NO-mediated EPR effect as well as receptor-mediated endocytosis. With a high expression of HAase, GSH and GSTπ in tumor sites, HA outer shell of HA-DNB-DEA/NO was degraded by HAase, and NO donor core was catalyzed by GSH/GSTπ to release abundant NO to kill tumor cells. More importantly, DOX@HA-DNB-DEA/NO displayed a remarkable tumor growth inhibition in vitro and in vivo comparing with free DOX and substantially mitigated side effects on normal tissues, revealing that the combination of DOX and NO could cause a synergistic antitumor effect. Therefore, HA-DNB-DEA/NO could be used as a promising NO donor delivery system and further enhanced anticancer activity of chemotherapeutic drugs.

4. Experimental Section 4.1. Materials Sodium hyaluronic acid (HA, molecular weight 48 kDa) was purchased from Bloomage

Freda

Biopharm

Co.,

Ltd.

(Shandong,

China).

1,5-Difluoro-2,4-dinitrobenzene (FFDNB) was obtained from Aladdin Industrial Corporation (Shanghai, China). Diethylamine NONOate sodium salt hydrate (DEA/NO) was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Doxorubicin hydrochloride (DOX·HCl) was purchased from Dalian Meilun Biological Technology Co., Ltd. (Liaoning, China). Griess reagent was purchased from Promega Corporation (Madison, WI USA). Glutathione S-transferase π (GSTπ) was obtained from ProSpec-Tany TechnoGene Ltd. DAF-FM DA was obtained from



Beyotome Institute of Biotechnology (China). Annexin V-FITC Apoptosis Detection Kit was purchased from KeyGen Biotech. Inc. (Nanjing, China). Rabbit Anti-3-Nitrotyrosine antibody and Rabbit Anti-PCNA antibody were purchased from Beijing Biosynthesis Biotechnology Co., Ltd. (Beijing, China). p53 antibody was obtained from Proteintech Group, Inc. (Wuhan, China). Bax, Bcl-2, and active caspase-3 were purchased from Abcam Inc. (Cambridge, UK). All other reagents were of analytical grade or higher. 4.2. Synthesis of HA-DNB-DEA/NO 4.2.1. Synthesis of HA-FDNB. HA-FDNB was synthesized by a nucleophilic substitution reaction. Briefly, HA (100 mg) and FFDNB (150.27 mg) were placed in a mortar, ground with anhydrous sodium carbonate for 5 min, and then stirred at 78 ℃ for 12 h. After the reaction completed, the resultant products (HA-FDNB) were washed with dichloromethane to remove the excess FFDNB and the precipitate was dried in the air to give a 53% yield of the products . 4.2.2. Synthesis of HA-DNB-DEA/NO. 21.5 mg of HA-FDNB were dissolved in 1 mL of N,N-dimethylformamide (DMF) and 1 mL of tert-butyl alcohol, following cooled to 0 ℃ under nitrogen. 20 mg/mL of DEA/NO in methanol was added dropwise to the above solution with a syringe. After that, the mixture was raised to room temperature gradually and stirred for 48 h. The reaction solution was treated with excess pre-cooled acetone, and precipitate was obtained by centrifuging at 3000 rpm for 15 min. Finally, the product was reconstituted, dialyzed against distilled water for 8 h, and freeze-dried in vacuum for 48 h to give a 42.1% yield of the products . 4.3. Preparation of DOX loaded HA-DNB-DEA/NO 10 mg of HA-DNB-DEA/NO were dissolved in 4 mL of distilled water and stirred for 30 min at room temperature to obtain empty micelles. Next, 5 mg of DOX·HCl was dispersed in DMF and deprotonated by triethylamine. The DOX solution was added to above-mentioned micelles drop by drop under stirring, sonicated for 30 min and posteriorly dialyzed against water for 12 h. Resulting solution was centrifuged at 3000 rpm for 15 min. Supernatant was filtered with 0.45 μm micro porous membranes and DOX@HA-DNB-DEA/NO were acquired. The


Page 24 of 34

Page 25 of 34 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


amount of DOX loaded in micelles was measured using UV-Vis spectrometer after destroying the structure of micelles by DMSO. Drug-loading (DL) was calculated by the following formulas:

4.4. Characterization The chemical structure of HA-DNB-DEA/NO was analyzed by 1H nuclear magnetic resonance spectra (1H NMR, Bruker BioSpin GmbH 400, Germany) and fourier transform infrared spectroscopy (FT-IR, Thermo, USA). Surface morphology was characterized by transmission electron microscopy (TEM, FEI Tecnai G20). The particle size and zeta potential distribution were measured using dynamic light scattering (DLS, Zetasizer Nano ZS-90, Malvern, UK). 4.5. In vitro NO release The amount of NO released from HA-DNB-DEA/NO with or without the presence of GSH, GSTπ and HAase was detected by Griess reagent. Briefly, PBS (pH 7.4, 0.01M) buffer containing 100 μg/mL of HA-DNB-DEA/NO, 10 mM of GSH, 5 μg/mL of GSTπ and 0.5 mg/mL of HAase was incubated in a water bath shaker (37 ℃ and 100 rpm). At predetermined time intervals, 50 μL of release medium was collected and placed in 96-well plates. Then experiment was proceeded in accordance with the instructions of griess assay kit and the absorbance value was recorded at 540 nm using a microplate reader. Finally, the concentration of nitrite could be measured based on the standard curve which was established previously, and thus the amount of NO released from HA-DNB-DEA/NO could be calculated out. 4.6. In vitro drug release In vitro release profile of DOX from DOX@HA-DNB-DEA/NO was investigated

in

the

presence

or

absence

of

GSH,

GSTπ

and

HAase.

DOX@HA-DNB-DEA/NO (50 μg of DOX) was dissolved in PBS buffer containing 10 mM GSH, 5 μg/mL GSTπ, and 0.5 mg/mL of HAase and shaken gently at 37 ℃. Subsequently, the released DOX was taken at predetermined time intervals and quantified by fluorescence spectrophotometer (RF-5301PC, Shimadzu).



Page 26 of 34

4.7. Cellular experiments 4.7.1. Cell culture. Human hepatoma SMMC-7721 cells and HL-7702 cells were cultured in RPMI-1640 culture medium containing 10% fetal bovine serum (FBS) and 1% antibiotics (100 μg/mL streptomycin and 100 U/mL penicillin) at 37 ℃ in a humidified incubator under an atmosphere of 5% CO2. 4.7.2. Cellular uptake. SMMC-7721 cells were seeded at a density of 1 × 105 cells per well on glass cover slips in 6-well plates and cultured for 24 h. Then fresh medium containing DOX or DOX@HA-DNB-DEA/NO (5 μg/mL of DOX) was added into the wells and incubated for 2 and 6 h, respectively. At the predetermined time points, cells were washed with PBS and fixed with 1 mL of 4% paraformaldehyde for 20 min. Subsequently, cells were stained with DAPI (1 μg/mL) for 6 min at 37 ℃, and observed under fluorescence microscope. To confirm DOX@HA-DNB-DEA/NO was specifically taken up by SMMC-7721 cells through HA-receptor mediated endocytosis, cells were incubated with free HA (5 mg/mL) for 2 h before adding DOX@HA-DNB-DEA/NO. 4.7.3. Intracellular NO release and detection. The generation of NO in SMMC-7721 cells

was

detected

by

3-amino-4-aminomethyl-2’,7’-difluorescein,

diacetate

(DAF-FM DA) probe. SMMC-7721 cells were seeded on glass cover slips in 6-well plates at a density of 1 × 105 cells per well. After incubation for 24 h, the cells were treated with 1 mL of fresh medium containing 5 μM DAF-FM DA for 30 min at 37 ℃. Medium was discarded and cells were washed with PBS for three times to remove the excess probe. Subsequently, cells were incubated with HA-DNB-DEA/NO (100 μg/mL) for 2 h and 6 h, respectively. After that, cells were rinsed with PBS for three times and the fluorescence signal of NO was observed under fluorescence microscope (Eclipse 80i, Nikon Corporation). In addition, studies on HL-7702 cells were used as control. 4.7.4. In vitro cytotoxicity assay. The antitumor activity in vitro was evaluated using sulforhodamine B (SRB) assay. Briefly, SMMC-7721 cells were seeded in 96 well-plates at a density of 5 × 103 cells per well and cultured for 24 h. Then the medium

was

replaced

by

fresh

medium


containing

free

DOX,

Page 27 of 34 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


DOX@HA-DNB-DEA/NO or HA-DNB-DEA/NO at serial DOX concentrations ranging from 0 to 5 μg/mL. After incubation for 24, 48, and 72 h, the viability of SMMC-7721 cells was measured. 4.7.5. Cell apoptosis assay. SMMC-7721 cells (3 × 105 cells/well) were seeded into 6 well-plates and allowed to grow for 24 h. After that, cells were treated with free DOX, DOX@HA-DNB-DEA/NO or HA-DNB-DEA/NO (5 μg/mL of DOX, 19.85 μg/mL of HA-DNB-DEA/NO) for another 24 h at 37 ℃. The treated cells were collected, suspended with binding buffer, and stained with Annexin V-FITC and propidium iodide (PI). Quantitative measurement of apoptosis and necrosis was conducted on the flow cytometry (FCM, Epics XL, COULTER, USA). 4.8. In vivo experiments 4.8.1. In vivo imaging study All animal experiments were carried out in strict accordance with the protocols approved by Henan Laboratory Animal Center. Male BALB/c nude mice at the age of 4-6 weeks were purchased from Hunan SJA Laboratory Animal Co., Ltd (Hunan, China). Animal models were established by inoculating subcutaneously SMMC-7721 cells (1 × 107 cells per mouse) in the armpit region of the mice. In order to observe the biodistribution in vivo, a near-infrared (NIR) dye DiR was loaded into HA-DNB-DEA/NO (DiR@HA-DNB-DEA/NO) in the same method of loading DOX. When the tumor size reached to ~500 mm3, mice were administrated with saline, free DiR, and DiR@HA-DNB-DEA/NO (0.2 mg/kg of DiR) via tail vein, respectively. Images were captured after 0.5, 1, 3, 6, 8, 12, and 24 h post-injection using a Kodak in vivo imaging system FX PRO (Kodak, USA, excitation: 748 nm, emission: 780 nm). At 24 h post-injection, the mice were sacrificed. Then the main organs (heart, liver, spleen, lung, and kidney) and tumors were excised for ex vivo fluorescence imaging. 4.8.2. NO release in tumor Tumor-bearing mice were administrated with saline, HA-DNB-DEA/NO (1.6 mg/mL), DOX (400 μg/mL), and DOX@HA-DNB-DEA/NO (400 μg/mL of DOX) via tail vein. After 12 h, 100 μL of DAF-FM DA (50 μΜ) was intratumorally injected. 1 h later, mice were euthanized and tumors were isolated. The fluorescence signal of



Page 28 of 34

NO and DOX at tumor site were observed by frozen section under confocal laser scanning microscopy (CLSM, Olympus FV1000). 4.8.3. In vivo antitumor evaluation When the tumor volume reached about 200 mm3, mice were separated into the following

groups

(n

=

6):

saline,

HA-DNB-DEA/NO,

DOX,

and

DOX@HA-DNB-DEA/NO at DOX dose of 4 mg/kg every other day for six times. In addition, the body weights and tumor volumes were monitored before injection every time. The tumor volume was calculated as (a × b2)/2, where ‘a’ represents the larger diameter and ‘b’ represents the smaller one. At the endpoint of the inhibition study, tumors were harvested and weighed, and the tumor inhibition ratio was obtained. The inhibition ratio (IR) was defined as IR (%) = [(Wc-Wt)/Wc] × 100%, where Wc and Wt represented for the average tumor weight of control groups and treatment groups, respectively. Furthermore, the tumors were excised, fixed with 4% paraformaldehyde and embedded in paraffin. The tumor tissues were carefully sectioned and stained with hematoxylin and eosin (H&E) to evaluate the histopathological changes. In order to detect the cell apoptosis in tumor tissue, the tumor sections were stained by TUNEL Cell Apoptosis Detection Kit (KeyGen Biotech, Nanjing) according to the specification. Proliferating cell nuclear antigen (PCNA) and 3-nitrotyrosine (3-NT) were analyzed in tumor tissue by immunohistochemistry. All the stained slices were observed and taken photographs under a microscope. 4.8.4. Western blot analysis The expression levels of p53, Bax, Bcl-2, and Cleaved caspase-3 were investigated by western blot, which was performed on proteins extracted from the tumor tissues after treatment in the lysis buffer. The extracted proteins were separated by 4-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membrane. The membrane was blocked in PBS buffer with 0.05% tween-20 and 5% non-fat milk, and incubated with primary antibody against p53 (10442-1-AP; Proteintech Group, Inc.), Bax (ab32503; Abcam), Bcl-2 (ab32124; Abcam) and Active caspase-3 (ab32024; Abcam) overnight at

4

℃.

Subsequently,

membranes

were

incubated


with

horseradish

Page 29 of 34 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


peroxidase-coupled secondary antibody, and signals were observed using the Pierce ECL Western Blotting Substrate (Thermo, Rockford, IL). The relative expression level of protein was quantified with ImageJ software. 4.8.5. In vivo safety evaluation The systemic toxicity for different formulations was assessed by monitoring the weight changes of animals during treatment. At the end of the experiment, animals were euthanized and the major organs (heart, liver, spleen, lung, and kidney) were excised for H&E straining. The histopathological changes of tissues were observed under optical microscope. 4.9. Statistical analysis All the data were expressed as mean ± standard deviation (SD) from three to ten independent measurements in separate independent experiments and were analyzed using descriptive statistic and single-factor analysis variance. Supporting Information The experimental section for CMC, Figures of UV-vis spectra and the Curve of I338/I333 vs LogC for the polymers, TEM images of HA-DNB-DEA/NO with different enzymes, Body weight changes and H&E images in different groups, IC50 value of different formulations against SMMC-7721 cells at 24, 48, and 72 h. Acknowledgements This research was supported by National Natural Science Foundation of China (U1704172), Key Technologies R & D of Henan province (162102310510), Outstanding Young Talent Research Fund of Zhengzhou University (51099255), Key Program for Basic Research of Universities in Henan province (19zx005).

Competing Interests The authors have declared that no competing interest exists.



References (1) Carpenter, A. W.; Schoenfisch, M. H. Nitric Oxide Release: Part II. Therapeutic Applications. Chem Soc Rev 2012, 41 (10), 3742-3752. (2) Kumar, S.; Singh, R. K.; Bhardwaj, T. R. Therapeutic Role of Nitric Oxide as Emerging Molecule. Biomedicine & Pharmacotherapy 2017, 85, 182-201. (3) Bogdan, C. Nitric Oxide and the Immune Response. Nature Immunology 2001, 2, 907-916. (4) Zhang, K.; Xu, H.; Jia, X.; Chen, Y.; Ma, M.; Sun, L.; Chen, H. Ultrasound-Triggered Nitric Oxide Release Platform Based on Energy Transformation for Targeted Inhibition of Pancreatic Tumor. ACS Nano 2016, 10 (12), 10816-10828. (5) Riganti, C.; Miraglia, E.; Viarisio, D.; Costamagna, C.; Pescarmona, G.; Ghigo, D.; Bosia, A. Nitric Oxide Reverts the Resistance to Doxorubicin in Human Colon Cancer Cells by Inhibiting the Drug Efflux. Cancer Research 2005, 65 (2), 516-525. (6) Fu, J.; Zou, Y.; Huang, Z.; Yan, C.; Zhou, Q.; Zhang, H.; Lai, Y.; Peng, S.; Zhang, Y. Identification of Nitric Oxide-Releasing Derivatives of Oleanolic Acid as Potential Anti-Colon Cancer Agents. RSC Advances 2015, 5 (25), 19445-19454. (7) Duong, H. T. T.; Kamarudin, Z. M.; Erlich, R. B.; Li, Y.; Jones, M. W.; Kavallaris, M.; Boyer, C.; Davis, T. P. Intracellular Nitric Oxide Delivery from Stable NO-Polymeric Nanoparticle Carriers. Chemical Communications 2013, 49 (39), 4190-4192. (8) Chung, M. F.; Liu, H. Y.; Lin, K. J.; Chia, W. T.; Sung, H. W. A pH-Responsive Carrier System that Generates NO Bubbles to Trigger Drug Release and Reverse P-Glycoprotein-Mediated Multidrug Resistance. Angewandte Chemie 2015, 127 (34), 10028-10031. (9) Fukumura, D.; Kashiwagi, S.; Jain, R. K. The Role of Nitric Oxide in Tumour Progression. Nature Reviews Cancer 2006, 6, 521-534. (10) Sharma, K.; Chakrapani, H. Site-Directed Delivery of Nitric Oxide to Cancers. Nitric Oxide 2014, 43, 8-16. (11) Korde Choudhari, S.; Chaudhary, M.; Bagde, S.; Gadbail, A. R.; Joshi, V. Nitric Oxide and Cancer: A Review. World Journal of Surgical Oncology 2013, 11 (1), 118-129. (12) Leon, L.; Jeannin, J.-F.; Bettaieb, A. Post-Translational Modifications Induced by Nitric Oxide (NO): Implication in Cancer Cells Apoptosis. Nitric Oxide 2008, 19 (2), 77-83. (13) Tarr, J. M.; Eggleton, P.; Winyard, P. G. Nitric Oxide and the Regulation of Apoptosis in Tumour Cells. Current Pharmaceutical Design 2006, 12 (34), 4445-4468. (14) Mijatovic, S.; Maksimovic-Ivanic, D.; Mojic, M.; Malaponte, G.; Libra, M.; Cardile, V.; Miljkovic, D.; Harhaji, L.; Dabideen, D.; Cheng, K. F.; Bevelacqua, Y.; Donia, M.; Garotta, G.; Al-Abed, Y.; Stosic-Grujicic, S.; Nicoletti, F. Novel Nitric Oxide-Donating Compound (S,R)-3-phenyl-4,5-dihydro-5-isoxazole Acetic Acid–Nitric Oxide (GIT-27NO) Induces p53 Mediated Apoptosis in Human A375 Melanoma Cells. Nitric Oxide 2008, 19 (2), 177-183. (15) Azizzadeh, B.; Yip, H. T.; Blackwell, K. E.; Horvath, S.; Calcaterra, T. C.; Buga, G. M.; Ignarro, L. J.; Wang, M. B. Nitric Oxide Improves Cisplatin Cytotoxicity in Head and Neck Squamous Cell Carcinoma. Laryngoscope 2001, 111 (11 Pt 1), 1896-1900. (16) Tan, L.; Huang, R.; Li, X.; Liu, S.; Shen, Y.-M. Controllable Release of Nitric Oxide and Doxorubicin from Engineered Nanospheres for Synergistic Tumor Therapy. Acta Biomaterialia 2017, 57, 498-510. (17) Song, Q.; Tan, S.; Zhuang, X.; Guo, Y.; Zhao, Y.; Wu, T.; Ye, Q.; Si, L.; Zhang, Z. Nitric Oxide Releasing d-α-Tocopheryl Polyethylene Glycol Succinate for Enhancing Antitumor Activity of Doxorubicin. Molecular Pharmaceutics 2014, 11 (11), 4118-4129.


Page 30 of 34

Page 31 of 34 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


(18) Frederiksen, L. J.; Sullivan, R.; Maxwell, L. R.; Macdonald-Goodfellow, S. K.; Adams, M. A.; Bennett, B. M.; Siemens, D. R.; Graham, C. H. Chemosensitization of Cancer in vitro and in vivo by Nitric Oxide Signaling. Clinical Cancer Research 2007, 13 (7), 2199-2206. (19) Miller, M. R.; Megson, I. L. Recent Developments in Nitric Oxide Donor Drugs. Br J Pharmacol 2007, 151 (3), 305-321. (20) Hrabie, J. A.; Keefer, L. K. Chemistry of the Nitric Oxide-Releasing Diazeniumdiolate ("Nitrosohydroxylamine") Functional Group and Its Oxygen-Substituted Derivatives. Chemical Reviews 2002, 102 (4), 1135-1154. (21) Mayevska, O.; Chen, O.; Karatsai, O.; Bobak, Y.; Barska, M.; Lyniv, L.; Pavlyk, I.; Rzhepetskyy, Y.; Igumentseva, N.; Redowicz, M. J.; Stasyk, O. Nitric Oxide Donor Augments Antineoplastic Effects of Arginine Deprivation in Human Melanoma Cells. Experimental Cell Research 2017, 355 (2), 162-171. (22) Quan, Y.-Y.; Liu, Y.-H.; Lin, C.-M.; Wang, X.-P.; Chen, T.-S. Peroxynitrite Dominates Sodium Nitroprusside-Induced Apoptosis in Human Hepatocellular Carcinoma Cells. Oncotarget 2017, 8 (18), 29833-29845. (23) Gazzano, E.; Chegaev, K.; Rolando, B.; Blangetti, M.; Annaratone, L.; Ghigo, D.; Fruttero, R.; Riganti, C. Overcoming Multidrug Resistance by Targeting Mitochondria with NO-Donating Doxorubicins. Bioorganic & Medicinal Chemistry 2016, 24 (5), 967-975. (24) Fan, W.; Bu, W.; Zhang, Z.; Shen, B.; Zhang, H.; He, Q.; Ni, D.; Cui, Z.; Zhao, K.; Bu, J.; Du, J.; Liu, J.; Shi, J. X-ray Radiation-Controlled NO-Release for On-Demand Depth-Independent Hypoxic Radiosensitization. Angewandte Chemie International Edition 2015, 54 (47), 14026-14030. (25) Fan, J.; He, Q.; Liu, Y.; Zhang, F.; Yang, X.; Wang, Z.; Lu, N.; Fan, W.; Lin, L.; Niu, G.; He, N.; Song, J.; Chen, X. Light-Responsive Biodegradable Nanomedicine Overcomes Multidrug Resistance via NO-Enhanced Chemosensitization. ACS Applied Materials & Interfaces 2016, 8 (22), 13804-13811. (26) Kuo, W.-J.; Wang, Y.-C.; Chen, M.-H.; Tung, F.-I.; Liu, T.-Y. Study of a Novel Vehicle Developed for Enhancing the Efficacy of Radiation Therapy. Ceramics International 2017, 43, S789-S796. (27) Findlay, V. J.; Townsend, D. M.; Saavedra, J. E.; Buzard, G. S.; Citro, M. L.; Keefer, L. K.; Ji, X.; Tew, K. D. Tumor Cell Responses to a Novel Glutathione S-Transferase-Activated Nitric Oxide-Releasing Prodrug. Mol Pharmacol 2004, 65 (5), 1070-1079. (28) Kogias, E.; Osterberg, N.; Baumer, B.; Psarras, N.; Koentges, C.; Papazoglou, A.; Saavedra, J. E.; Keefer, L. K.; Weyerbrock, A. Growth-Inhibitory and Chemosensitizing Effects of the Glutathione-S-Transferase-pi-Activated Nitric Oxide Donor PABA/NO in Malignant Gliomas. Int J Cancer 2012, 130 (5), 1184-1194. (29) Ji, X.; Pal, A.; Kalathur, R.; Hu, X.; Gu, Y.; Saavedra, J. E.; Buzard, G. S.; Srinivasan, A.; Keefer, L. K.; Singh, S. V. Structure-Based Design of Anticancer Prodrug PABA/NO. Drug Des Devel Ther 2008, 2, 123-130. (30) Zhao, G.; Wang, X. Advance in Antitumor Agents Targeting Glutathione-S-Transferase. Curr Med Chem 2006, 13 (12), 1461-1471. (31) Cullen, K. J.; Newkirk, K. A.; Schumaker, L. M.; Aldosari, N.; Rone, J. D.; Haddad, B. R. Glutathione S-Transferase pi Amplification is Associated with Cisplatin Resistance in Head and Neck Squamous Cell Carcinoma Cell Lines and Primary Tumors. Cancer Res 2003, 63 (23), 8097-8102. (32) Aliya, S.; Reddanna, P.; Thyagaraju, K. Does Glutathione S-Transferase Pi (GST-Pi) a Marker Protein for Cancer? Molecular and cellular biochemistry 2003, 253 (1-2), 319-327. (33) Huang, J.; Tan, P. H.; Thiyagarajan, J.; Bay, B. H. Prognostic Significance of Glutathione S-Transferase-pi in Invasive Breast Cancer. Mod Pathol 2003, 16 (6), 558-565.



(34) Fu, J.; Liu, L.; Huang, Z.; Lai, Y.; Ji, H.; Peng, S.; Tian, J.; Zhang, Y. Hybrid Molecule from O2-(2,4-dinitrophenyl)diazeniumdiolate and Oleanolic Acid: A Glutathione S-Transferase pi-Activated Nitric Oxide Prodrug with Selective Anti-Human Hepatocellular Carcinoma Activity and Improved Stability. J Med Chem 2013, 56 (11), 4641-4655. (35) Saavedra, J. E.; Srinivasan, A.; Buzard, G. S.; Davies, K. M.; Waterhouse, D. J.; Inami, K.; Wilde, T. C.; Citro, M. L.; Cuellar, M.; Deschamps, J. R.; Parrish, D.; Shami, P. J.; Findlay, V. J.; Townsend, D. M.; Tew, K. D.; Singh, S.; Jia, L.; Ji, X.; Keefer, L. K. PABA/NO as an Anticancer Lead: Analogue Synthesis, Structure Revision, Solution Chemistry, Reactivity toward Glutathione, and in vitro Activity. J Med Chem 2006, 49 (3), 1157-1164. (36) Choi, K. Y.; Chung, H.; Min, K. H.; Yoon, H. Y.; Kim, K.; Park, J. H.; Kwon, I. C.; Jeong, S. Y. Self-Assembled Hyaluronic Acid Nanoparticles for Active Tumor Targeting. Biomaterials 2010, 31 (1), 106-114. (37) Toole, B. P. Hyaluronan: from Extracellular Glue to Pericellular Cue. Nat Rev Cancer 2004, 4 (7), 528-539. (38) Wang, T.; Hou, J.; Su, C.; Zhao, L.; Shi, Y. Hyaluronic Acid-Coated Chitosan Nanoparticles Induce ROS-Mediated Tumor Cell Apoptosis and Enhance Antitumor Efficiency by Targeted Drug Delivery via CD44. J Nanobiotechnology 2017, 15 (1), 7-19. (39) Platt, V. M.; Szoka, F. C., Jr. Anticancer Therapeutics: Targeting Macromolecules and Nanocarriers to Hyaluronan or CD44, a Hyaluronan Receptor. Mol Pharm 2008, 5 (4), 474-486. (40) Deepagan, V. G.; Ko, H.; Kwon, S.; Rao, N. V.; Kim, S. K.; Um, W.; Lee, S.; Min, J.; Lee, J.; Choi, K. Y.; Shin, S.; Suh, M.; Park, J. H. Intracellularly Activatable Nanovasodilators To Enhance Passive Cancer Targeting Regime. Nano Lett 2018, 18 (4), 2637-2644. (41) Evig, C. B.; Kelley, E. E.; Weydert, C. J.; Chu, Y.; Buettner, G. R.; Burns, C. P. Endogenous Production and Exogenous Exposure to Nitric Oxide Augment Doxorubicin Cytotoxicity for Breast Cancer Cells but not Cardiac Myoblasts. Nitric oxide : biology and chemistry 2004, 10 (3), 119-129. (42) Huo, M.; Zhang, Y.; Zhou, J.; Zou, A.; Yu, D.; Wu, Y.; Li, J.; Li, H. Synthesis and Characterization of Low-Toxic Amphiphilic Chitosan Derivatives and Their Application as Micelle Carrier for Antitumor Drug. Int J Pharm 2010, 394 (1-2), 162-173. (43) Zhang, N.; Wardwell, P. R.; Bader, R. A. Polysaccharide-Based Micelles for Drug Delivery. Pharmaceutics 2013, 5 (2), 329-352. (44) Yan, J.; Su, T.; Cheng, F.; Cao, J.; Zhang, H.; He, B. Multifunctional Nanoparticles Self-Assembled from Polyethylenimine-Based Graft Polymers as Efficient Anticancer Drug Delivery. Colloids Surf B Biointerfaces 2017, 155, 118-127. (45) Zeybek, A.; Sanli-Mohamed, G.; Ak, G.; Yilmaz, H.; Sanlier, S. H. In vitro Evaluation of Doxorubicin-Incorporated Magnetic Albumin Nanospheres. Chem Biol Drug Des 2014, 84 (1), 108-115. (46) Shenoi, M. M.; Shah, N. B.; Griffin, R. J.; Vercellotti, G. M.; Bischof, J. C. Nanoparticle Preconditioning for Enhanced Thermal Therapies in Cancer. Nanomedicine (London, England) 2011, 6 (3), 545-563. (47) Svenson, S.; Case, R. I.; Cole, R. O.; Hwang, J.; Kabir, S. R.; Lazarus, D.; Lim Soo, P.; Ng, P. S.; Peters, C.; Shum, P.; Sweryda-Krawiec, B.; Tripathi, S.; van der Poll, D.; Eliasof, S. Tumor Selective Silencing Using an RNAi-Conjugated Polymeric Nanopharmaceutical. Mol Pharm 2016, 13 (3), 737-747. (48) Gao, Y.; Ruan, B.; Liu, W.; Wang, J.; Yang, X.; Zhang, Z.; Li, X.; Duan, J.; Zhang, F.; Ding, R.; Tao, K.; Dou, K. Knockdown of CD44 Inhibits the Invasion and Metastasis of Hepatocellular Carcinoma both in vitro and in vivo by Reversing Epithelial-Mesenchymal Transition. Oncotarget 2015, 6 (10), 7828-7837.


Page 32 of 34

Page 33 of 34 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


(49) Hou, Y.; Zou, Q.; Ge, R.; Shen, F.; Wang, Y. The Critical Role of CD133(+)CD44(+/high) Tumor Cells in Hematogenous Metastasis of Liver Cancers. Cell research 2012, 22 (1), 259-272. (50) Zhang, X.; Tian, G.; Yin, W.; Wang, L.; Zheng, X.; Yan, L.; Li, J.; Su, H.; Chen, C.; Gu, Z. Controllable Generation of Nitric Oxide by Near-Infrared-Sensitized Upconversion Nanoparticles for Tumor Therapy. Advanced Functional Materials 2015, 25 (20), 3049-3056. (51) Follis, A. V.; Llambi, F.; Merritt, P.; Chipuk, J. E.; Green, D. R.; Kriwacki, R. W. Pin1-Induced Proline Isomerization in Cytosolic p53 Mediates BAX Activation and Apoptosis. Molecular cell 2015, 59 (4), 677-684. (52) Moldoveanu, T.; Follis, A. V.; Kriwacki, R. W.; Green, D. R. Many Players in BCL-2 Family Affairs. Trends in biochemical sciences 2014, 39 (3), 101-111.



Abstract Graphic

The intracellular enzyme-triggered NO-generator preferentially targeted to the tumor region without accumulation in normal organs, via NO-mediated EPR effect as well as receptor-mediated endocytosis. With a high expression of HAase, GSH and GSTπ in tumor sites, HA outer shell of HA-DNB-DEA/NO was degraded by HAase, and NO donor core was catalyzed by GSH/GSTπ to release abundant NO, co-locating with DOX to exert synergistic efficacy.


Page 34 of 34

Intracellular NO-Generator Based on Enzyme Trigger for Localized

Recommend Documents