Light-Activated ROS-Responsive Nanoplatform Co-delivering Apatinib

Subscriber access provided by University of Winnipeg Library

Biological and Medical Applications of Materials and Interfaces

Light-Activated ROS-Responsive Nanoplatform Codelivering Apatinib and Doxorubicin for Enhanced ChemoPhotodynamic Therapy of Multidrug-Resistant Tumors Xiao Wei, Lingqiao Liu, Xing Guo, Yi Wang, Jingya Zhao, and Shaobing Zhou ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b04163 • Publication Date (Web): 08 May 2018 Downloaded from http://pubs.acs.org on May 8, 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 42 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

Light-Activated ROS-Responsive Nanoplatform Codelivering Apatinib and Doxorubicin for Enhanced Chemo-Photodynamic Therapy of Multidrug-Resistant Tumors Xiao Wei,† Lingqiao Liu,‡ Xing Guo,† Yi Wang,† Jingya Zhao,† and Shaobing Zhou†,* † Key Laboratory of Advanced Technologies

of Material, Minister of Education, School

of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, Sichuan, P.R. China ‡

School of Life Science and Engineering, Southwest Jiaotong University, Chengdu

610031, P.R. China KEYWORDS: ROS-response  nanoplatform  chemo-photodynamic therapy  multidrug resistance  co-delivery

ABSTRACT

Clinical chemotherapy confronts a challenge resulting from cancer-related multidrug resistance (MDR), which can directly lead to treatment failure. To address it, an innovative approach is proposed to construct a light-activated reactive oxygen species (ROS)-responsive nanoplatform based on a protoporphyrin (PpIX)-conjugated and dual chemotherapeutics-loaded polymer micelle. This system combines chemotherapy and photodynamic therapy (PDT) to defeat the MDR of tumors. Such an intelligent nanocarrier can prolong the circulation time in blood because of the negative

ACS Paragon Plus Environment

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

polysaccharide component of chondroitin sulfate (CS), and subsequently being selectively internalized by MCF-7/ADR cells (DOX-resistant). When exposed to 635nm red light, this nanoplatform generates sufficient ROS through the photoconversion of PpIX, further triggering the disassociation of the micelles to release the dual cargoes. Afterwards, the released apatinib, serving as a reversal inhibitor of MDR, can recover the chemosensitivity of DOX by competitively inhibiting the P-glycoprotein drug pump in drug-resistant tumor cells, and the excessive ROS has a strong capacity to exert its PDT effect to act on the mitochondria or the nuclei, ultimately causing cell apoptosis. As expected, this intelligent nanosystem successfully reverses tumor MDR via the synergism between apatinib-enhanced DOX sensitivity and ROS-mediated PDT performance.

INTRODUCTION

Chemotherapy, as an orthodox antitumor protocol in the clinic, has been confronted by the enormous dilemma of intricate multidrug resistance (MDR),1, 2 which largely plays a limited and lethal role in cancer treatment and originates from the resistance to drugs that are not related with structure and mechanism.3, 4 The latent mechanism of MDR is known to be P-glycoprotein (P-gp) overexpressed in drug-resistant tumor cell membrane, which directly excludes diverse small molecular drugs, leading to ineffectiveness in killing tumor cells.1, 5-7 To address this puzzle, diverse classes of drug delivery systems have been designed to escape from the recognization of the P-gp .2, 811

Nonetheless, the cargoes of these nanosystems have been generally delivered to the


Page 2 of 42

Page 3 of 42 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


cytoplasm, in which the cargoes can be still facilely identified and eliminated by P-gp pump, eventually leading to the low content of drugs acting on the target site.12-14 In light of this issue, incorporating a reversal inhibitor of the MDR into the system enables the chemotherapeutics to successfully bypass the MDR mechanism. For example, apatinib, a tyrosine kinase inhibitor that commonly serves as an antineoplastic drug, can also notably weaken or reverse P-gp-induced MDR and potentiate the chemosensitivity in MDR tumor cells.15 Photodynamic therapy (PDT) can also be integrated into all-in-one system due to its insusceptibility to drug resistance. PDT is usually considered the accessional power of typical chemotherapy to further realize the synergism in treating cancer.16-18 Generally, PDT has mainly been employed as the photosensitizer to produce reactive oxygen species (ROS) via the energy conversion, directly inducing the eradication of tumor cells through irreversible destruction to bio-macromolecules, such as lipids, proteins or DNA.19-26 Excessive ROS can directly destroy the mitochondria by lowering the mitochondrial membrane potential (MMP) or influencing the expression of membrane related proteins,18, 27, 28 and acts in the nuclei to break the DNA helix and evoke the inactivation of enzymes involved in DNA repair,29, 30 which ultimately causes cell apoptosis. Accordingly, an ideal scenario that integrates of a PDT system with a competitive inhibitor of P-gp transporter can substantially overcome the MDR of tumors for enhanced antitumor therapy. Herein, we design a new light-activated ROS-responsive nanoplatform based on a protoporphyrin-conjugated polymer micelle co-delivering



dual chemotherapeutics, aiming to reverse MDR by combining chemotherapy and PDT. As illustrated in Scheme 1A, this formulation, AC-CS-PpIX (ACP), comprises acetylated-chondroitin sulfate (AC-CS) as the hydrophilic block, and protoporphyrin IX (PpIX) grafted on AC-CS via an ester bond as the hydrophobic block.20 Doxorubicin (DOX) and apatinib (Apa) can be simultaneously encapsulated into amphipathic ACP micelles through hydrophobic interaction and π-π stacking force, designated ACPDox+Apa. A red light-induced ROS generation from the ACP micelles is expected to disassociate micelles,21 leading to the robust and fast release of the dual drugs. The negatively charged polysaccharide component of CS can help the drug-loaded micelles extend their blood circulation,20, 31 further improving their effective concentrations at tumor regions.14, 32 Thereafter, as presented in Scheme 1B, the ACP-Dox+Apa micelles are selectively uptaken by MDR cells via the affinity between the CS and CD44. When exposed to light irradiation, the micelles can generate ROS for photochemical rupture of the endo-/lysosomal membrane.19, 33 Simultaneously, the rich ROS can also cause the dissociation of ACP-Dox+Apa micelles due to the oxidation destruction of the CS polysaccharide backbone, which then releases the dual drugs. The released Apatinib can competitively combine with the P-gp transporter to reduce its enzyme catalysis activity,15 thus retaining DOX abundantly inside the MDR tumor cells. Later, the retained DOX or generated ROS locates in the nuclei to induce DNA damage-mediated cell death along with the enhanced expression of phosphorylated H2A.X (p-H2A.X, a marker of double-strand breaks).29, 34 Meanwhile, the excessive ROS also exerts its PDT effect on the mitochondrion via decreasing its mitochondrial membrane potential


Page 4 of 42

Page 5 of 42 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


(MMP; ΔΨm) or upregulating the expression of its outer membrane protein (VADC1),27,

28, 35

further leading to mitochondria-dependent apoptosis followed by the

decreased or increased expression of apoptosis-associated protein. Ultimately, the combined chemo-PDT is expected to successfully combat tumor MDR to promote synergistic antitumor potency.

RESULTS AND DISCUSSION

Characterization of ACP polymer. The formation of ROS-sensitive ACP polymer was performed as shown in Figure S1 in Supporting Information (SI). First, to improve the hydrophobicity of chondroitin sulfate sodium salt (CS) and its solubility in dimethyl sulfoxide (DMSO), the hydroxyl groups of CS were acetylated. To substantiate the successful decoration of acetyl groups, fourier transform infrared spectroscopy (FT-IR) was performed as presented in Figure S2 in SI. Subsequently, the PpIX, as a photosensitizer with good PDT potency, was conjugated to the acetylated CS (AC-CS) via a carbodiimide reaction. Then, 1H nuclear magnetic resonance (1H NMR) spectroscopy and FT-IR analyses were utilized to demonstrate the molecular structure of AC-CS-PpIX (ACP) polymer (Figure S3A, B). The loading content of PpIX in copolymer was determined by UV-vis method and presented as 6.1% (Table S1 in SI). The resultant ACP was an amphipathic polymer, which could self-assemble into micelles. Therefore, 1H NMR detection of ACP micelles in D2O was further employed to verify the conventional core-shell components of the micelles (Figure S4 in SI), and it was found that all the peaks of the hydrophilic AC-CS were presented. Moreover, as



displayed in Figure S5, the critical micelle concentration (CMC) was measured for the formation of the micelles, and the CMC value of 0.001472 mg/mL suggested the micelles should be stable during the circulation in blood. It was clearly observed that ACP micelles owned its uniform distribution and spherical structure (Figure 1A). As shown in Table S1, the size of 104 ±2 nm with the potential of -40.4 ±0.9 mV enabled the micelles to effectively stack in tumor regions.14, 36, 37 Next, both doxorubicin (DOX) and apatinib (Apa), as two anticancer reagents, were encapsulated to the inner core of ACP micelles via a hydrophobic interaction and/or π– π stacking. As shown in Figure S6A-D and Table S2 in SI, it certified the drug-loaded micelles still maintained their spherical shape and monodispersion. Moreover, the DOX-loadedcontent (LC) and efficiency (EE) of the ACP-Dox mcielles presented separately 5.8% and 63.8%, and the LC of 5.4% and EE of 66.8% likewise presented in the ACP-Dox+Apa group along with the LC of 0.7% and EE of 68.3 % of Apatinib. The mean size of approximately 130 nm with the PDI of about 0.2 facilitated tumortargeted delivery,14, 38-40 and the obvious negative potential of the micelles elongates the circulation time in blood, presumably due to avoiding the scavenging system in vivo.41-44 Light-activated ROS-responsive ACP Micelles. The self-assemble formation of ACP micelles notably decreased the aggregation of PpIX induced by π–π stacking, which was confirmed by UV-vis spectra (Figure 1B). Compared to free PpIX, it was cleared found that there was a broadened Soret band at approximately 400 nm in ACP micelles, probably owning to weak π-π conjugation between the PpIX molecules. Like


Page 6 of 42

Page 7 of 42 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


the UV-vis absorbance of PpIX in DMSO (Figure S7), the characteristic peak at approximately 635 nm of PpIX was also dramatically found in ACP micelles (Figure 1B), implying that the visible light at 635 nm could be used to induce the generation of ROS. Furthermore, the generation of ROS induced by ACP micelles upon light irradiation was determined and exhibited in Figure 1C,20 the relative ROS level of free PpIX under light illumination retained a low content along with time increasing and similar data were also presented in the blank control of PBS. Conversely, the ACP micelles upon light irradiation significantly increased the content of ROS, uncovering that the self-assembly behavior of ACP micelles effectively evaded self-quenching of PpIX due to its hydrophobicity and enhanced ROS production, contributing to subsequent PDT potency. On account of the ROS-sensitive property of the ACP polymer, the production of ROS from ACP micelles under illumination could break the CS polysaccharide backbone and degrade them into oligosaccharide or monosaccharide as illustrated in Figure 1D.21 To further verify this design, the 1H NMR assessment of ACP was performed as displayed in Figure 1E, providing evidence that the cleaved proton peaks were observed under light irradiation for preset times, implying ROS induced the degradation of the ACP polymer. In addition, the evolution of the particle size was detected for the ROS-mediated disassembly of the drug-loaded micelles (Figure 1F, G). The size of ACP-Dox and ACP-Dox+Apa micelles obviously increased with irradiation time, suggesting that ROS generating from the drug-loaded micelles directly disrupted the ACP polymer, followed by drug release triggered by the disassociation of micelles. To prove it, the ACP-Dox+Apa micelles were illuminated



Page 8 of 42

with a 635-nm laser before monitoring DOX release. As exhibited in Figure 1H, I, the released DOX or Apa at pH 7.4 or 5.0 without irradiation was less than 20% within 72 h of incubation, which revealed the stability of the micelles. Conversely, the ACPDox+Apa micelles displayed a fast release at pH 7.4 or 5.0 with irradiation, which strongly supported abovementioned results of 1H NMR and particle size distribution. Therefore, it can be concluded that light-activated ROS generation significantly induced the disassociation of drug-loaded micelles to

release anticancer

chemotherapeutics. In addition, it was reported that the released Apa dramatically overcame multidrug resistance.15 To probe its mechanism, the inhibition action of Apa was evaluated according to Lineweaver-Burk plots.45 As presented in Figure 1J, the y-intercept of the presence of Apa increased and its x-intercept unaffected, indicating that Apa was a competitive inhibitor of DOX efflux. Intracellular ROS Generation Mediated Anti-MDR in Vitro. As shown in Figure S8A-D in SI, the cytotoxic data of blank micelles certified that there was no cytotoxicity toward EC, MCF-7 and MCF-7/ADR cells. It is known that chondroitin sulfate (CS) can

target

CD44-overexpressed

tumor

cells.46-48

Fluorescence

microscopic

observations were used to testify the targeting ability of micelles (Figure S9 and S10A). It was transparently found that the PpIX content of the ACP group was obviously more than that of the DEP group within the same incubation time, suggesting that the abundant micelles accumulated in tumor cell via its targeting function. Additionally, the content of intracellular PpIX was quantified by flow cytometry (Figure S10B, C).


Page 9 of 42 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


In contrast to the DEP group, the fluorescence intensity of PpIX in the ACP group significantly enhanced, which was consistent with the outcome of CLSM. The ROS production property of ACP in aqueous solution was confirmed. Hence, the generation of intracellular ROS from ACP micelles upon light irradiation was further investigated as exhibited in Figure S11A, B in SI and Figure 2A, B. It was observed that the relative ROS level in ACP micelles with prolonged irradiation time was significantly increased in comparison with other groups. Furthermore, the intracellular ROS generation in MCF-7/ADR cells was observed by FM (Figure S12) and CLSM (Figure 2D). The qualitative outcome showed that the weak green fluorescence was presented in control and AC-CS groups, and the ROS fluorescence of PpIX group also remained at a reduced level, which implied the drug resistance. Conversely, the remarkable fluorescence of ROS was observed in ACP group (Figure 2D, Figure S13A), testifying to the production of intracellular ROS upon illumination. Importantly, it was found that the green fluorescence of ROS abundantly distributed in the cytoplasm and nucleus, implying that the generated ROS could act in both the cytoplasm and nucleus. In addition, the flow cytometric evaluation was also employed to quantify the intracellular ROS generation (Figure 2C, Figure S13B). The quantitative data of the mean fluorescence intensity of ROS in ACP group was remarkably enhanced in contrast with that in other group, which was in accordance with the data from CLSM (Figure S13A). The ROS production from ACP micelles under illumination could directly influence the mitochondrial integrity by oxidizing phospholipids and protein in the membrane.



Moreover, mitochondrial disruption is always accompanied by the decline of mitochondrial membrane potential (ΔΨm), which was assessed via JC-1 staining. Therefore, the ΔΨm analysis was performed (Figure 2E). Clearly, with the increased irradiation time, the green fluorescence of the J-monomer enhanced gradually while the J-aggregates declined evidently, which confirmed that the illuminated time-dependent ROS generation induced the loss of ΔΨm. Subsequently, to evaluate PDT of ACP micelles under illumination induced anti-MDR potency, the ROS-mediated cell death was further investigated by the AlamarBlue (AB) assay and live/dead staining. As a control, 47.3% of cytotoxicity appeared at 400 μg mL−1 in MCF-7 cells (Figure S14A), suggesting that the ROS generation could induce cell death due to the PDT of PpIX. Moreover, PDT-mediated toxicity of MCF-7 cells was evidently observed by fluorescence microscopy (FM) (Figure S14B). Similarly, as presented in Figure 2F and 2G, 39.3% of cytotoxicity and the increased dead cells (Figure 2G) demonstrated that the ROS overcame MDR by directly leading to the death of MCF-7/ADR cells. Intracellular DOX Release Triggered by Illumination-activated ROS. Next, the ROS-responsive DOX release and nuclear localization in tumor cells were evaluated under prolonging irradiation time. It was distinctly found that the red fluorescent PpIX nearly merged with the green fluorescence of DOX after 1 min of illumination, which was in line with the fluorescence intensity profiles (Figure 3B1, B2). These results testified that the intracellular ACP-Dox+Apa micelles remained intact and detained in the cytoplasm. Moreover, little DOX fluorescence distributed in the nuclei and maintained delocalized (Figure 3C1, C2). To quantitatively assess the colocalization


Page 10 of 42

Page 11 of 42 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


between the two types of fluorescence, the Pearson’s correlation coefficients (Rr) were further utilized to analyze intracellular dug release and the nuclear localization, which is a measurement of the strength of the association between the two variables.49 The mean Rr values of red-green are 0.69 and 0.57 (Figure 3D1, D2), respectively, indicating that DOX was still localized in the ACP micelles. Moreover, the 0.16 and 0.18 of Rr values between blue-green certified that there was no drug entering the nuclei. After 3 min of irradiation, the majority of green fluorescence of DOX separated from the PpIX fluorescence of ACP micelles (Figure 3A3), and this phenomenon was similarly observed as analyzed in Figure 3B3, implying that the disassembly of ACPDox+Apa micelles upon light irradiation contributed to the controlled release of DOX. Furthermore, from Figure 3C3, it was found that the released DOX appeared in the nuclei. In addition, as shown in Figure 3D3, the 0.32 of Rr values between red-green gave strong evidence for the drug isolated from the micelles. The colocalization coefficient of blue-green is 0.38 (Figure 3D3), suggesting that there was no remarkable nuclear localization. The best ROS-responsive efficacy occurred after 5 min of illumination as presented in Figure 3A4. The merged fluorescent image proved that a majority of the DOX located outside the micelles, moreover, it could diffuse into the nuclei. This outcome was consistent with the corresponding fluorescence intensity analysis (Figure 3B4, C4). Additionally, the colocalization coefficient of red-green is 0.16 and the Rr value of the blue-green is 0.58 (Figure 3D4), further suggesting that the light-activated intracellular ROS promotes the DOX release and effectively enters the nuclei.



DOX Accumulation and Efflux Analyses in vitro. To further confirm the mechanism of intracellular DOX release under illumination, we next studied whether the Apa could combat the efflux of DOX to increase the content of DOX inside the MCF-7/ADR cells. CLSM was first employed to qualitatively analyze the intracellular DOX distribution and nuclear localization. As shown in Figure 4A1, it presented the weak fluorescence intensity in free DOX-treated group, which is mainly due to pumping out the DOX. Conversely, when DOX combined with Apa (Figure 4A2), the red fluorescence intensity of DOX was significantly enhanced and a considerable quantity of DOX located in nuclei, proving that the Apa reversing MDR enabled the rich DOX detained inside the MDR cells. From the corresponding fluorescence intensity profiles of DOX and nuclei, it was observed that little DOX in free DOX group entered the nuclei (Figure 4B1); however, the majority of DOX in DOX+Apa group distributed in nuclei (Figure 4B2). The ACP-Dox+Apa micelles without irradiation revealed the a majority of DOX presented in cytoplasm and scarcely localized in nuclei (Figure 4A3), demonstrating that the micelles were internalized abundantly by CD44 receptor-mediated endocytosis, and there was hardly any disassociation of the micelles so that the DOX was retained in cytoplasm and delocalized in the cell nucleus. Likewise, the fluorescence intensity peaks of DOX barely overlapped with those of DAPI (Figure 4B3), indicating that there was no colocalization between DOX and nuclei. Specifically, in Figure 4A4, the light-irradiated ACP-Dox+Apa micelles presented the best potency for the DOX accumulation and nuclear localization (Figure 4A4). Similarly, the fluorescence intensity profile displayed that the retained DOX could enter the nuclei


Page 12 of 42

Page 13 of 42 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 4B4). Additionally, the Pearson’s correlation coefficients (Rr) were used to quantify the nuclear localization of DOX. As exhibited in Figure 4C, the mean Rr values of the free DOX- and the ACP-Dox+Apa (-L)-treated groups are separately 0.19 and 0.28, indicating that there was no colocalization between DOX and nuclei. In contrast, the colocalization coefficients of other groups are more than 0.5, implying that the DOX significantly located in nuclei, which was consistent with the observation of CLSM and the fluorescence intensity analyses. Figure 4D shows the semi-quantitative data from the outcome of CLSM with the same as the flow cytometric detection (Figure 4E, F), testifying that Apa overcome the MDR in order to make the DOX be retained effectively in MDR cells, and ACP-Dox+Apa micelles were notably internalized via targeting the cells. To measure the DOX efflux, the cells were first treated with different formulations, and then the micelles-treated groups received light irradiation. Thereafter, the contents of the DOX efflux and DOX retained inside the MDR cells were measured by fluorescence spectrophotometry and flow cytometry, respectively. As displayed in Figure 4G, the mass of DOX efflux was large in the free DOX-treated group and constantly enhanced along with extending incubation time, reflecting the P-gpmediated drug exclude. Likewise, there was an increased pumping content of DOX in ACP-Dox-treated group, because the drug released in the cytoplasm was also excluded outside of MDR cells. However, in DOX+Apa-treated group, the extracellular DOX content remained lower due to the Apa competitively inhibiting the P-gp to recover the sensitivity of DOX. Especially, the lowest quantity of DOX was pumped out in the



ACP-Dox+Apa micelles, indicating they could overcome the MDR via the micelles evading the recognition of drug pump and the introduced Apa inhibiting the drug transporter. These means ensured the increased retention of DOX inside the MDR cells. Additionally, flow cytometric analysis further confirmed the accumulation content of DOX inside MDR cells (Figure 4H), which dramatically reflected the DOX efflux in an indirect way. Cytotoxicity and Cell Apoptosis Assays. The toxicity of MCF-7/ADR cells incubated with free DOX or Apa was first determined. Free DOX showed remarkable drug resistance for the MDR cells because its IC50 value is 119.44 μg mL−1 (Figure S15A). Otherwise, free Apatinib revealed 21.19 μg mL−1 (Figure S15B). Based on the cytotoxicity curves, 3.0 μg mL−1 of Apa was chosen as a maximum concentration, which displayed that more than 90% of the cells were viable and used for the following MDR reversal evaluation. As shown in Figure S15C, Apa with increased concentrations enhanced the cytotoxicity of DOX, indicating that Apa notably recovered the DOX sensitivity. Accordingly, when Apa maintains 3 μg mL−1, DOX revealed a 24.14 μg mL−1; a DOX/Apa ratio of 8:1 was selected for the following studies. Then, the toxicities DOX-sensitive or resistant MCF-7 cells treated with other samples were tested. As shown in Figure S16, the toxicities of MCF-7 cells treated with DOX+Apa, ACP-Dox (light irradiation) and ACP-Dox+Apa (light irradiation) were nearly the same as free DOX-treated cells, attributed to drug sensitivity and PDT effect from PpIX via photodynamic conversion (Figure S8B and Figure S14A). In the meantime, the ACPDox (no irradiation) and ACP-Dox+Apa (no irradiation) treated groups showed lower


Page 14 of 42

Page 15 of 42 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


cytotoxicity, resulting from the micelles without disassembly. As revealed in Figure 5A, B, the DOX treated MCF-7/ADR cells remained much less toxic without IC50 value within limits, proving that robust resistance of the cells to free DOX. When DOX mixed with Apa, this group (IC50, 39.31 μg mL−1) achieved significantly higher cytotoxicity, implying that Apatinib reversed the MDR and sensitized the cells to DOX. In addition, both the ACP-Dox- and ACP-Dox+Apa-treated groups with illumination displayed enhanced cytotoxicity, especially in the illuminated ACP-Dox+Apa-treated group, which had a minimal IC50 value of 17.34 μg mL−1, suggesting that the ROS generation caused the drug release and released Apa simultaneously promoted the DOX retention inside the MDR cells, meanwhile, the excessive ROS also brought about PDT toxicity that was strongly demonstrated in Figure S8C and Figure 2F. However, the ACP-Doxand ACP-Dox+Apa-treated groups without illumination had lower cytotoxicity, possibly owing to little drug release from the undecomposed micelles. Collectively, these results demonstrated that the ACP-Dox+Apa micelles under light irradiation could obviously combat the MDR using a combination of PDT and ROS-triggered chemotherapy. Next, the apoptotic detection was conducted to confirm the apoptosis-inducing abilities of ACP-Dox+Apa micelles with or without light irradiation. After illumination, flow cytometry was utilized to analyzed different treatments-treated cells (Figure 5C, D). There was no obvious apoptosis in MCF-7/ADR cells incubated with DOX (a total apoptotic ratio of 13.64%), probably due to pumping out of the drug mediated by the P-gp. Similar case occured in ACP-Dox+Apa group (no irradiation) (apoptotic ratio of



19.74%), which was possibly ascribed to the undecomposed micelles. Conversely, DOX combining with Apa presented an increased apoptotic ratio of 29.14%, implying that Apa improved the apoptosis-inducing concentration of DOX inside the cells by overcoming the MDR mechanism. Particularly, the illuminated ACP-Dox+Apa-treated group displayed the best apoptotic potency (apoptotic ratio of 45.34%), which was approximately 3.3-fold of free DOX, verifying the light-triggered drug release, Apaenhanced DOX accumulation and PDT-synergistic apoptotic efficacy. In Vivo Anti-MDR Efficacy and Apoptotic Mechanism Study. To evaluate the therapeutic potency in vivo of ACP-Dox+Apa micelles for combating the multidrug resistance, the tumor-bearing nude mice were intravenously administered saline, ACP micelles (with light), free DOX, DOX+Apa, ACP-Dox micelles (with light), ACPDox+Apa micelles and ACP-Dox+Apa micelles (with light), respectively. As shown in Figure 6A, B, the growth of the tumor was significantly suppressed at 15 d in the ACPDox+Apa micelles (with light) group, indicating that a combination of PDT and ROStriggered chemotherapy could effectively overcome the MDR. The tumor treated with ACP micelles upon irradiation also exhibited a moderated inhibition efficacy compared to saline group due to the ROS production by ACP micelles with illumination. Likewise, the ACP-Dox micelles with light irradiation brought about the stronger anticancer effect, resulting from ROS-responsive DOX release activated by light. By comparison, it was obvious that the illuminated ACP-Dox+Apa micelles best suppressed the tumor growth, implying that ROS-induced Apa release promoted the DOX retention in MDR tumors. In addition, both the free DOX and DOX+Apa groups displayed no notable inhibition


Page 16 of 42

Page 17 of 42 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


efficacy due to the short circulation time in blood. Correspondingly, the tumor weights of various groups were presented in Figure 6C, which provided strong evidence for the above-mentioned outcomes in Figure 6A, B. Additionally, the body weight of all mouse groups was evaluated (Figure 6D). There was obvious weight decrease in nude mice administered with free DOX or free DOX+Apa, possibly resulting from the remarkable side effect of free DOX. No apparent weight change of drug-loaded micelle groups with or without light irradiation was observed, indicating there was negligible systemic toxicity produced by nanomedicine or PDT. The systemic toxicity of free drug was further analyzed by histopathological analyses of normal tissues, performed with H&E staining (Figure S17). Correspondingly, in free DOX or DOX+Apa group, it revealed evident neutrophil accumulation and myocardial fiber damage in those hearts, implying DOX probably brought about acute cardiotoxicity.50,

51

In contrast, undetectable

physiological morphology changes were visualized in normal organs of ACP-Dox+Apa treatment group (with light), implying that this nanomedicine reduced cardiotoxicity by decreasing the accumulation of DOX in heart, and the phototoxicity produced by the PDT was limited to the irradiated area, which notably decreased the systemic toxicity. Furthermore, the antitumor efficacy was further confirmed by executing the staining of tumor tissues. Figure 6E shows that both the saline and unilluminated ACPDox+Apa groups presented compact tumor cells. Similarly, free DOX-treated group also maintained a normal state of tumor growth, which was probably attributed to its short blood circulation time and drug resistance. In contrast, the ACP, ACP-Dox and ACP-Dox+Apa groups under light irradiation showed massive tumor necrosis and



extensive nuclear shrinkage and fragmentation.52 Especially, the illuminated ACPDox+Apa group revealed the largest necrotic region of MDR tumor cells, certifying that it exerted its best anti-MDR efficacy by chemo-photodynamic therapy. Likewise, the results of TUNEL staining also reflected the most obvious apoptotic efficacy induced by ACP-Dox+Apa micelles receiving light. In addition, the IHC analysis related with Ki67 (a marker of cell proliferation) was performed to further assess tumor cell proliferation. Compared to other groups, the expression level of Ki67 in the illuminated ACP-Dox+Apa group presented the lowest, implying that the synergistic chemo-PDT prominently inhibited tumor cell proliferation by overcoming the MDR of tumor. Overall, these histological analyses gave evidence that the ACP-Dox+Apa micelles upon light irradiation had high anti-MDR activity. To confirm the enhanced anti-MDR potency of ACP-Dox+Apa in depth, the associated mechanism of apoptosis was studied in vivo. As shown in Figure 6F, G, the illuminated ACP-Dox+Apa treatment group markedly downregulated or upregulated the level of apoptosis related protein, suggesting that it evoked obvious mitochondriadependent apoptosis. At the meantime, high levels of ROS could cause DNA damage in nuclei, which is recognized as an apoptotic signal.53 The expression of phosphorylated H2AX (p-H2A.X, a marker of DNA double-strand breaks) was significantly elevated when the MDR tumor was exposed to the ACP-Dox+Apa micelles under light irradiation (Figure 6H,I).29 Moreover, the excessive ROS may act on mitochondria by inducing the oligomerization of VDAC1 (mitochondrial outer membrane protein), leading to formation of pore structures in the mitochondrial


Page 18 of 42

Page 19 of 42 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


membrane, which can mediate cytochrome C release to further activate caspasedependent apoptosis.28, 35 Obviously, the greatly increased expression of VDAC1 in the ACP-Dox+Apa group upon illumination was observed compared to the other groups (Figure 6H, I). These outcomes certified that the synergism of chemo-PDT triggered by the light-irradiated ACP-Dox+Apa micelles caused notable MDR cell apoptosis.

CONCLUSION

We designed a new light-triggered ROS-responsive nanoplatform based on a protoporphyrin-conjugated polymer micelle co-delivering chemotherapeutics, which effectively combines both chemotherapy and PDT for overcoming the MDR of tumor. Such an intelligent nanocarrier prolongs the blood circulation time via the negative polysaccharide component of CS and enhances the specific internalization by MDR tumor cells via CD44 receptor. When exposed to red light, this nanomedicine abundantly produces ROS and induces the disassembly of the micelles to release the dual drugs. After that, the released Apatinib recovers the chemosensitivity of DOX via competitively suppressing the drug transporter on the tumor cell membrane, and the sufficient ROS had great capacity to exert its PDT effect to act on the mitochondria or the nuclei, eventually resulting in cell death. This nanoplatform successfully combats the tumor MDR via the combination of chemotherapy and PDT. Therefore, this research supplies a novel strategy in the development of a smart light-activated nanocarrier for fighting tumor multidrug resistance. EXPERIMENTAL SECTION



Materials. Protoporphyrin IX (PpIX) was purchased from Sigma-Aldrich (Merck, Germany) and Chondroitin sulfate sodium salt (CS, Mn=1526.03Da) was bought from TCI Development Co. Ltd. (Shanghai). N-Hydroxy succinimide (NHS), 4(dimethylamino)

pyridine

(DMAP)

and1-Ethyl-3-(3-dimethylaminopropyl)

carbodiimide hydrochloride (EDC•HCl), were obtained from Biokem Chemical Reagent Co. (Chengdu, China). Doxorubicin hydrochloride (DOX•HCl) was obtained from Zhejiang Hisun Pharmaceutical Co., Ltd. (Taizhou, China). Apatinib (YN-968D1) (Mn=493.58) was supplied by Hanxiang Biotechnology Co., Ltd. (Shanghai, China). Annexin V-FITC Apoptosis Detection Kits was purchased from KeyGEN Biotech Co. Ltd (Nanjing, China). All other chemicals were purchased from KeLong Chemical Reagent Co. (Chengdu, China) and used without further purification. Synthesis of PpIX Conjugated Acetylated CS (ACP). First, CS was acetylated according to a previous report.21 In brief, CS (50 mg) was dispersed in 10 mL formamide by vigorously stirring. Additionally, pyridine (25 μL) and acetic anhydride (20 μL) were supplemented. The above mixture solution was continued to react until the fluid became transparent. The reaction solution was then transferred into a dialysis bag (MWCO 1000) to dialyze for 3 d. The acetylated CS (AC-CS) was gained by lyophilization. Following, to execute the graft of AC-CS with PpIX, AC-CS (50 mg, 0.03 mmol) and DMAP (40 mg, 0.33 mmol) were dissolved in 60 mL dimethyl sulfoxide (DMSO). Moreover, NHS (80 mg, 0.70 mmol), EDC (80 mg, 0.42 mmol) and PpIX (20 mg, 0.04 mmol), were separately added to the above mixture. The reaction solution was stirred at room temperature in the dark until the PpIX completely reacted,


Page 20 of 42

Page 21 of 42 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


which was assessed according to the result of thin layer chromatography (TLC). Next, the reactant solution was placed in a dialysis bag (MWCO 1000) and dialyzed for 3 d to purification. The AC-CS-PpIX (ACP) was collected by lyophilization and stored in a desiccator for subsequent use. Formation of Blank micelles and Drug-loaded Micelles. Namely, the lyophilized powder of ACP (5 mg) was dispersed in the tetrahydrofuran (THF) (5 mL). Afterwards, the mixture was added dropwise into 5 mL of deionized water under high-speed stirring. The blank ACP micelles gradually assembled by evaporating the THF. For the preparation of the DOX- or DOX+Apatinib-loaded micelles (ACP-Dox or ACPDox+Apa), the operation procedure was nearly same as the above mentioned method. Additionally, the DMSO solution of DOX•HCl added a drop of trimethylamine and Apa was added to the THF solution together with ACP polymer. Finally, the ACP-Dox or ACP-Dox+Apa micelles were obtained by dialyzing (MWCO 1000) for 3 d and subsequently lyophilizing. Characterization of ACP Polymers and Micelles. To characterize the polymers, Fourier transform infrared (FT-IR) spectra were recorded by a Nicolet 5700 spectrometer. 1H Nuclear magnetic resonance (1H NMR) spectra were detected by a Bruker AM 300 apparatus. DMSO-d6 and D2O were used as solvents, and tetramethylsilane (TMS) was utilized as the internal reference. Dynamic light scattering (DLS) was employed to measure the size and potential of micelles at 25 °C. A transmission electron microscopy (TEM) observation of micelles was performed with a JEOL 2010F instrument (JEOL Ltd., Japan) operated at 200 kV.



The critical micelle concentration (CMC) was determined by a fluorescence spectrophotometer (F-7000, Hitachi, Japan) using pyrene as a probe. The fluorescence emission intensity was examined at 333 and 339 nm via an excitation wavelength at 390 nm. UV-vis spectrophotometer (UV-2550, Shimadzu, Japan) was utilized to analyze the UV-vis spectra of ACP micelles. To investigate the production of reactive oxygen (ROS) from the illuminated ACP micelles, fluorescence spectrophotometer was used to detect the ROS intensity using DCFH-DA probe.17 A UV-vis spectrophotometer was employed to examine drug-loading content (LC) and encapsulation efficiency (EE) of drug-loaded micelles. According to a calibration curve, the concentration of DOX or Apa was calculated by the absorbance of DOX at 481 nm or Apa at 271 nm. Investigation of ROS Sensitivity of Micelles under Light Irradiation. To determine the ROS sensitivity, the 1H NMR was first executed to confirm whether the composition variation of the polymer occurred upon light irradiation. Namely, ACP micelles were respectively irradiated for predetermined time by a 635-nm laser. Then, the reactant solutions were freeze-dried, which was analyzed by 1H NMR. Additionally, DLS was identically used to analyze the ROS response of micelles. Likewise, the drugloaded micelles were irradiated with light for appointed time periods. Afterwards, DLS detection was performed to detect the mean diameter of the micelles. In Vitro Light-Activated ROS-triggered Drug Release under Light Irradiation. For evaluating the ROS-responsive drug release under light irradiation, the lyophilized powders of micelles were resuspended in buffer solution with different pH values. All of the above media contained Tween-80 (0.8% w/w). Thereafter, the solution was


Page 22 of 42

Page 23 of 42 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


dialyzed in the relevant dispersion medium, irradiated with a 635-nm laser, and gently shaken at 100 cycles per minute in a temperature-controlled incubator. One milliliter of solution outside the dialysis bag was withdrawn at appointed time points and supplemented with fresh buffer. The cumulative drug release curves were assessed based on the UV-vis concentration of DOX and Apatinib. The Inhibitory Mechanism of DOX Efflux by Apatinib. In detail, 1 ×105 of MCF7/ADR cells were added to a 6-well plate, then, treated with various doses of free DOX with or without Apatinib for 10 min at 37 °C. Subsequently, the treated cells were washed, centrifuged and collected to measure the intracellular concentration of DOX by a FACSCalibur flow cytometer (BD Biosciences, U.S.A.). The apical uptake of DOX was maintained at 0 °C, used as a control. The inhibitory mechanism of DOX efflux by Apatinib was assessed via Lineweaver-Burk plots.45 Cytocompatibility Assay. The alamar blue (AB) assay and live/dead staining were performed to estimate the cytotoxicity of blank micelles. For the analysis of cell viability by AB, EC, MCF-7 and MCF-7/ADR cells (2 ×104) were seeded into 48-well plates, respectively. Then, the cells were incubated with the different concentrations of ACP micelles for additional 48 h. After that, each well was rinsed three times by PBS and then replaced with 300 μL AB solution for further 4 h reaction. Subsequently, the AB solution of each well was measured by an automated microplate spectrophotometer. For live/dead staining, the above three kinds of cells were inoculated into 24-well plates, incubated with blank micelles, then stained with 2 mM calcein acetoxymethylester (Calcein-AM) for 10 min. Lastly, fluorescence microscopy was used to observe the live



cells stained green fluorescence. Determination of Targeting Ability of ACP Micelles. To investigate the ACP micelles targeting the MCF-7/ADR cells, qualitative fluorescent observations (FM and CLSM) and quantitative flow cytometric analyses were used to evaluate the cellular uptake of micelles, respectively. For time-dependent cellular uptake, MCF-7/ADR cells treated with the micelles were utilized as FM observation (Nikon, Japan). For CLSM, 1 ×105 cells per dish of MCF-7/ADR cells were planted into petri dishes, administrated with free PpIX (10 μg mL−1), Dex-PpIX micelles (non-targeted) and ACP micelles (targeted). Dex-PpIX polymer was synthesized as our previous report.20 The cells were then fixed and stained with DAPI. Images were obtained by a Leica Microsystems CMS Gmbh (TCS SP5, Germany). The excitation and emission wavelengths of DAPI were 405 nm and 488 nm, and the excitation and emission wavelengths of PpIX were 633 nm and 650 nm. To quantify the intracellular uptake, the fluorescence intensity of PpIX inside the cells was measured by flow cytometry. Various formulations treated MCF7/ADR cells were collected to measure the fluorescence intensity of PpIX by flow cytometry. Intracellular ROS Detection. DCFH-DA as ROS sensor was used to confirm the intracellular ROS generation mediated by irradiated micelles. MCF-7 or MCF-7ADR cells (5000 cells) were planted in a 96-well plate, and then treated with different formulations after 24 h culture. After 4 h treatment, DCFH-DA (1 ×10-5 M) was added to stain the ROS. Thereafter, all the groups were performed to illuminate for appointed time. A microplate spectrophotometer was employed to determine the fluorescence of


Page 24 of 42

Page 25 of 42 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


ROS. The excitation and emission wavelengths of DCF were 488 nm and 530~550 nm. Besides, FM and CLSM was separately utilized to test the generation of intracellular ROS. Various formulations treated cells were stained with DCFH-DA for 20 min, and received 5 min light irradiation (635 nm laser, 10 mW/cm2), and were observed by FM and CLSM, respectively. Image-Pro Plus 6.0 software was used to quantify the DCF fluorescence of ROS. Furthermore, flow cytometry was also utilized to quantitatively detect the intracellular ROS. Different samples incubated MCF-7/ADR cells were stained with DCFH-DA, illuminated under a 635 nm laser, and performed flow cytometric assay using flow cytometer. Investigation of Mitochondrial Membrane Potential and In Vitro Photodynamic Therapy (PDT). The mitochondrial membrane potential was confirmed by CLSM. Firstly, MCF-7/ADR cells (1 ×105) were planted in petri dishes. Then, the cells were treated with ACP micelles for 4 h, rinsed and illuminated under a 635 nm laser (10 mW/cm2). Thereafter, the cells were stained with JC-1 (2.5 µg mL–1) after additional 1h incubation and analyzed by CLSM. The excitation wavelength of JC-1 was 488 nm. The green fluorescence of J-aggregate was excitated at 510-540 nm (green) and the red fluorescence of J-monomer was excitated at 570-600 nm. The AB assay and live/dead staining was carried out to evaluate the toxicity toward MCF-7/ADR cells by PDT. For the assesment of cell viability by AB, different concentrations of micelles treated cells were received light irradiation for 5 min and incubated for another 24 h. The 300 μL of AB solution was added to incubate for 3 h, and measured by an automated microplate spectrophotometer. The data were analyzed using Gen5software (Biotek). The relative



cell viability was confirmed by comparing with untreated control cells. In addition, the cytotoxicity brought from PDT was evaluated by live/dead staining. ACP micelles treated cells were stained by Calcein-AM and PI for 15 min, and then visualized by fluorescence microscopy. ROS-responsive DOX Release and Nuclear Localization. To determine light irradiation mediated ROS-triggered DOX release and further nuclear localization, MCF-7/ADR cells (1× 105) seeded in petri dishes were treated with ACP-Dox+Apa micelles for 4 h, received light irradiation for preset time, and further incubated for 1 h. Then, the cells were fixed and stained with DAPI. Images were obtained using a CLSM. DAPI, PpIX and DOX were excitated at 405 nm, 633 nm and 485 nm, respectively. The fluorescence emissions of DAPI, PpIX and DOX were 488 nm, 650 nm and 595 nm. The fluorescence intensity of intracellular DOX and the nuclear localization analysis were performed via the Image-Pro Plus 6.0 software. For nuclear localization, 20 cells from five randomly selected areas of each image were chosen and analyzed, and the corresponding Pearson's correlation coefficients (Rr) were statistically analyzed as a colocalization reference. DOX Accumulation and Efflux Studies. MCF-7/ADR cells (1×105) seeded in petri dishes were administrated with free DOX (10 μg mL−1), DOX+Apatinib (10 μg DOX/mL and 1.25 μg Apatinib/mL), ACP-Dox+Apa (- laser) and ACP-Dox+Apa (+ laser) (10 μg DOX/mL). After 4 h incubation, the group of ACP-Dox+Apa with irradiation received 5-min illumination and cultured for another 1 h. Afterwards, the cells were fixed and stained with DAPI for 15 min. Then, CLSM was used to observe


Page 26 of 42

Page 27 of 42 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


the intracellular fluorescence and nuclear localization of DOX. The excitation and emission wavelengths of DAPI were 405 nm and 488 nm, respectively. The excitation and emission wavelengths of DOX were 485 nm and 595 nm. Image-Pro Plus 6.0 software was employed to analyze the intracellular fluorescence of DOX and the nuclear localization carried out by 20 cells from five randomly selected areas of each image were chosen and analyzed, and the corresponding Pearson's correlation coefficients (Rr) were statistically analyzed as a nuclear localization reference. Besides, cellular uptake of DOX was quantitatively analysed by flow cytometry, MCF-7/ADR cells treated with different formulations were collected to measure the fluorescence intensity of intracellular DOX by flow cytometry. Untreated group was considered as a blank control. For DOX efflux study, MCF-7/ADR cells were incubated with various formulations for 4 h. Among of them, ACP-Dox and ACP-Dox+Apa treated groups were received 5-min light irradiation and further cultured for preset time. Subsequently, the medium from each well was collected and detected by a fluorescence spectrophotometer, then calculated as the mass of DOX exclusion. Meanwhile, the cells were collected and measured via flow cytometry. In Vitro Cytotoxicity and Apoptosis Assays. Cytotoxicity was evaluated by the means of AB assay. The tumor cells (2 × 104) grown in 48-well plates were incubated with free DOX, DOX+Apatinib, ACP-DOX (-laser), ACP-DOX (+laser), ACPDox+Apa (-laser) and ACP-Dox+Apa (+laser) at different drug concentrations containing 0.625, 1.25, 2.5, 5, 10, 25, 50, 100 μg mL−1. Afterwards, ACP-DOX (+laser) and ACP-Dox+Apa (+laser) treated cells were received 5-min light irradiation (635 nm



laser) and cultured for further 48 h. Whereafter, 300 μL of AB solution was added to each well for additional 3 h incubation, then measured by a microplate spectrophotometer. The inhibitory concentration (IC50) values were calculated using Origin 8.0 (OriginLab, Northampton, MA) according to the fitted data. For cell apoptotic analysis, 1 × 105 cells of MCF-7/ADR cells incubated with different formulations were harvested, then stained with apoptosis detection reagents and detected by flow cytometry. In Vivo Antitumor Efficacy. MCF-7/ADR cells (1 × 107) in 200 μL serum-free medium were subcutaneously inoculated to the right-lower leg of mice to develop tumor models. When the tumor grown about 150 mm3, all the nude mice were randomly divided into seven groups (n=5). Then, tumor-bearing nude mice were intravenously treated with saline, ACP (+ laser), free DOX, DOX+Apatinib, ACP-Dox (+ laser), ACP-Dox+Apa (- laser), ACP-Dox+Apa (+ laser). The dosage of DOX was 5 mg/kg. After 6 h-injection, the illuminated groups were performed to illuminate by 635 nm laser for 10 min, respectively. All of treatments were administrated in vein on days 0, 3 and 6 at 2 d intervals. The measurements of tumor size and mice body weight were performed every three days, and the tumor volume could be calculated as following: equation: V = 0.5 × a × b2 (a indicates the length and b indicates the width). At 15th day of the treatment, the tumors of each group were isolated, weighted and photographed. Histological evaluation. The tumor-bearing nude mice were sacrificed at 15th day of antitumor, tumor tissues and their normal organs were then isolated, fixed and


Page 28 of 42

Page 29 of 42 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


embedded. After deparaffinization using xylene, a 5 μm thickness of tissue section was stained with hematoxylin and eosin (H&E) and terminal deoxynucleotidyltransferase mediated UTP end labeling (TUNEL), or immunostained with a rabbit polyclonal antibody for Ki-67 (Abcam, Cambridge) and immunohistochemical SP Kit (Zhongshan Goldbridge Biotechnology, Beijing, China) for immunohistochemical analysis, and finally observed by an optical microscope (Olympus, Japan). Western Blot for Apoptotic Related Protein In Vivo. Briefly, RIPA lysis buffer was added into the tumor tissues of each group, then lysed on ice and centrifuged to obtain the protein supernatant. Then, the total protein was quantified and electrophoresed in SDS-PAGE. A rabbit monoclonal antibody against Bax (Abcam, Cambridge), rabbit polyclonal antibodies against Bcl-2, caspase-9/3 (Abcam, Cambridge), p-H2A.X (CST, USA) and VDAC1 (Absin, Shanghai) were used in this detection, respectively. After washing with TBST buffer, the HRP-conjugated secondary antibody (Abcam, Cambridge) was utilized to treat the PVDF membrane. Eventually, the ECL kit ((Thermo Scientifc) was employed to observe the target protein. Statistical Analysis. The statistical data was analyzed by using GraphPad Prism 7.0 software. All tests were performed in triplicates. Data were presented as means ± standard deviation. One way single factorial analysis of variance (ANOVA) was performed to determine statistical significance of the data. The differences were considered significant for p values * < 0.05, **

Light-Activated ROS-Responsive Nanoplatform Co-delivering Apatinib

Recommend Documents