Self-Assembled Indomethacin Dimer Nanoparticles Loaded with

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Biological and Medical Applications of Materials and Interfaces

Self-Assembled Indomethacin Dimer Nanoparticles Loaded with Doxorubicin for Combination Therapy in Resistant Breast Cancer Xin Wang, Xu Cheng, Le He, Xiaoli Zeng, Yan Zheng, and Rupei Tang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b05855 • Publication Date (Web): 17 Jul 2019 Downloaded from pubs.acs.org on July 17, 2019

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ACS Applied Materials & Interfaces

Self-Assembled Indomethacin Dimer Nanoparticles Loaded with Doxorubicin for Combination Therapy in Resistant Breast Cancer

Xin Wang#, Xu Cheng#, Le He, Xiaoli Zeng, Yan Zheng, Rupei Tang*

Engineering Research Center for Biomedical Materials, Anhui Key Laboratory of Modern Biomanufacturing, School of Life Sciences, Anhui University, 111 Jiulong Road, Hefei, Anhui Province, 230601, P. R. China

* Corresponding author. Email: [email protected] (R. Tang) # Authors

contributed equally to this work.

KEYWORDS: indomethacin, ortho ester, synergistic effect, multidrug resistance

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ABSTRACT An ortho ester-linked indomethacin (IND) dimer-based nanodrug delivery system was prepared to improve the therapeutic effect of doxorubicin (DOX) by reversing the multidrug resistance. The synthesized dimer (IND-OE) could form stable nanoparticles (IND-OE/DOX) loaded with DOX via single emulsion method. Compare to non-sensitive nanoparticles (IND-C12/DOX), IND-OE/DOX showed a rapid degradation behavior and accelerated drug release at mildly acidic environments. In vitro cell experiments verified that IND-OE nanoparticles could increase DOX concentration due to the efficient intracellular drug release by the degradation of ortho ester as well as reduced DOX efflux by IND-mediated P-gp down-regulating. In vivo studies further demonstrated that IND-OE/DOX displayed the maximized synergetic antitumor efficacy than free DOX or IND-C12/DOX, and the tumor inhibition rate versus saline was 46.78% (free DOX), 60.23% (IND-C12/DOX), and 80.62% (IND-OE/DOX),

respectively.

Overall,

this

strategy

of

combination

with

chemosensitizers and ortho ester linkage has great potentials to serve as an amplifying chemotherapy platform against various drug-resistant tumors.

1. INTRODUCTION Chemotherapeutic efficacy are often hindered by the multidrug resistance (MDR) in solid tumor.1,2 MDR can be either generated during drug treatment or induced by the tumor endogenous micro-environments such as hypoxia, acidity, and up-regulation glutathione S-transferases.3,4 Based on the development of nanotechnology, various


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nano-drug delivery systems (nDDS) provide alternative strategies to reverse chemo/thermotherapy resistance by co-encapsulating anticancer drugs and MDR inhibitors or attaching multifunctional components.5–9 However, their clinical applications were greatly limited by the low drug loading efficiency, uncontrolled drug release and potential immunogenic effects.10,11 Besides, some MDR inhibitors (such as verapamil and cyclosporin A) possess high inherent toxicity, which may cause severe side effects in vivo.12–14 Moreover, most intelligent nano-carriers usually involve complex fabrication process, such as materials synthesis, self-assembly, separation, purification and modification, leading to batch-to-batch differentiation and quality challegen.15,16 Thus, it is necessary to design a simple-feasible and stable-efficient platform for reversing MDR and specific tumor treatment. Recently, much attention has been devoted to prepare carrier-free nDDS on the basis of small molecule drug dimer, which directly used the amphiphilic conjugates,17,18 hydrophobic conjugates,19 or anticancer agents themselves as carriers for drug delivery.20,21 Compared with traditional nano-carriers, these self-assembled dimers-based nanoparticles possessed very high drug loading efficiency, since they avoided using excessively high amounts of carrier materials. Furthermore, it is propitious to scale up production and promote their clinical translation because of the well-defined molecular structure and simple purification process.22–24 Xie et al prepared various paclitaxel (PTX) dimer that contained different linkers, which could self-assembled into stable nanoparticles through a nanoprecipitation method.25–27 Compared with free PTX, these dimer formulations exhibited enhanced antitumor



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efficacy toward tumor cells and reduced systemic toxicity to normal cells. Inspired by these works, we tried to develop a new type of small molecule dimer-based nDDS composed of the hydrophobic MDR reversal agents and the bio-responsive linkage to reach rapidly drug release and high drug concentration in drug-resistant tumor cells. Indomethacin (IND), as a non-steroidal anti-inflammatory drug, has been applied as analgesic, anti-inflammatory and antipyretic agents for many years.28,29 In addition, recent studies have demonstrated that IND could reduce cell motility and invasion, suppress cell growth and accelerate cell apoptosis in various types of tumor.30,31 More importantly, IND could reduce the expression level of multidrug resistance protein 1 (MRP1) by altering the activity of their promoter, and then sensitize the drug-resistant cells by reversing MDR.32 The enhanced efficiency of doxorubicin (DOX) in leukemia cells by IND-mediated down-regulating of MRP1 was proved by Seita and Arisa.33 Furthermore, indomethacin analogues or conjugates have also the similar effect against various cancer resistance.34–36 In other words, IND has great potential as an amplifier of chemotherapeutical toxicity for the combination therapy. In this context, we designed and developed an acid-sensitive indomethacin dimer (IND-OE) by ortho ester linkage, which subsequently self-assembled with DOX to give a complex nDDS. IND-OE was used for the combination therapy against MCF-7/ADR tumor. Ortho ester as an acid-responsive linkage possessed excellent stability at neutral conditions and fast hydrolyze ability at weak acidic environment,37,38 which was beneficial to the stability of circulation in blood and the rapid

drug

release

in

the

endo/lysosomes


(pH

4.0~5.5).39

Besides,

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1,12-dodecanediamine (C12)-linked IND dimer (IND-C12) was also prepared as the acid-insensitive control. Their characterization including size, morphology, stability and acid-degradation behaviors, drug release were investigated. A series of single-layer cell (2D) and tumor-like spheroids (3D) experiments were performed to evaluate the anti-MDR activities in vitro. Finally, antitumor ability and biosafety were studied using the MCF-7/ADR tumor-bearing nude mice. As an expectation, the pH-triggered and synergetic nano-drug delivery system displayed the enhanced combination therapeutic activity by reversing tumor MDR and controlling drug release in cancer cells.

2. EXPERIMENTAL SECTION 2.1. Materials. Acid-labile ortho-ester monomer (OE) was prepared according our previous work.38 Indomethacin (IND) and 1,12-Dodecanediamine (C12) were bought from

Energy

Chemical

Co.,

1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide

Ltd.

(Shanghai,

hydrochloride

China). (EDC·HCL),

N-hydroxysuccinimide (NHS), 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenyl-formazan (MTT) was bought from Mackin Biochemical Co., Ltd. Doxorubicin hydrochloride was obtained from Meilun Biological Technology (Dalian, China). MRP1 antibody and Cy3 conjugated donkey anti-human IgG was bought from Sangon Biotech Co., ltd (Shanghai, China). MCF-7 and MCF-7/ADR were bought from NanJing KeyGen Biotech (Nanjing, China). BALB/c nude mice (female, 5-6 week) were brought from the Cavens Laboratory Animal Limited Company (Changzhou, China).



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2.2. Synthesis of IND-OE and IND-C12. IND was mixed with EDC (1.5 mmol) and NHS in 1 mL of anhydrous dimethyl sulfoxide (DMSO) at a ratio of 1:1.5:1.5, and then activated for 30 min. After that, 0.16 g OE (0.52 mmol) and 20 μL triethylamine (TEA) was dropwised into the system, and reacted for 1 day. Crude product (IND-OE) was obtained by dialyzing for 1 day and subsequently freeze-drying. Furthermore, the crude product was purified with silica gel column chromatography (CH2Cl2/CH3OH, v/v) = 2:1 as the eluent) to yield 0.37 g (72.1%). The chemical structure of IND-OE was detected by 1H and

13C

NMR (Bruker

Aavance 400 NMR spectrometer) and Fourier transform infrared (FT-IR, NEXUS-870, Nicolet, USA). IND-C12 was synthesized using the same method, and the yellowish powder was obtained and the yield was 64.7 %. 2.3. Preparation and Characterization of NPs. IND-OE- or IND-C12-based nanoparticles were prepared via an oil-in-water (o/w) single emulsion solvent volatilize method.40 30 mg IND-OE or IND-C12 was dissolved in 1 mL dichloromethane (DCM), and then slowly added into 2 mL PVA aqueous solutin (5%, w/v) at pH 8.0. After that, the solution was sonicated three time (10 s/time) under ice bath, followed poured into 20 mL of PVA aqueous solution (0.3%, w/v). Finally, DCM was volatilized and the nanoparticles were collected by centrifugation at 1×104 rpm. The obtained nanoparticles were named as IND-OE NPs and IND-C12 NPs. The size, zeta potential and micromorphology of IND-OE NPs or IND-C12 NPs were observed by DLS, TEM and SEM as previously work.38


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2.4. pH-Dependent Degradation. pH-sensitive ability of these two NPs were measured at two pH values. After incubated at pH 7.4 or 5.0 for various time, nanoparticles were freeze-dried and analyzed by

1H

NMR (DMSO-d6). The

hydrolysis rate of ortho-ester was measured using the following formula: IA δ 8.23 / (IA δ 8.23 + IA δ 5.81), and IA meant integral area.38 Meanwhile, the changes of hydrodynamic diameter and count rate were monitored by DLS. Furthermore, the micromorphology of NPs after incubating at pH 5.0 for various time were also measured by TEM. 2.5. Drug Loading and Releasing. In order to prepare DOX-loaded nanoparticles, DOX·HCL was firstly desalted to obtain hydrophobic DOX and the method was used as reported in the previous literature.41 DOX-loaded IND-OE or IND-C12 nanoparticles were centrifuged, collected and re-dispersed, and named IND-OE/DOX and IND-C12/DOX, respectively. Finally, the released DOX content in the supernatant was detected and calculated as previous work:42

DLC (%) =

DLE (%) =

Weight of DOX in NPs × 100% Weight of DOX - loaded NPs

(1)

Weight of DOX in NPs × 100% Total of the feeding DOX

(2)

The stability of DOX-loaded nanoparticles was assessed via monitoring the change of particles' diameter. Briefly, IND-OE/DOX or IND-C12/DOX was dispersed in 0.01 M PB buffer solution with pH 7.4, and then measured during a week. After that, the drug release was performed as described.42 1.0 mL DOX-loaded NPs (450 μg/mL) was added into a dialysis bag, immersed with PB solution (5 mL), and shaken at 100



rpm. The pH of system were set at 5.0, 6.0 and 7.4. At the selecting time, the old released medium was completely replaced with 5 mL new PB solution. DOX concentration in medium was measured by a microplate system (Molecule Devices, USA) at 480 nm by UV absorption. 2.6. Cellular Uptake. Cells were cultured as reported work.6 MCF-7 or MCF-7/ADR cells were co-cultured with free DOX, IND-OE/DOX and IND-C12/DOX for 2 h. DOX concentration was 4 μg/mL. After that, the old medium was replaced with 2 mL fresh medium and cultured for another 4 h. Finally. the cellular uptake profiles of IND-OE/DOX and IND-C12/DOX were observed by confocal laser scanning microscopy (CLSM, FluoView TM FV1000, Olympus) as previous reports.42 2.7. Subcellular Co-localization. MCF-7 or MCF-7/ADR cells were seeded onto a 6-well plate containing a cover glass and cultured for 24 h. Then, cells were incubated with Lyso-Tracker Green for 0.5 h and co-cultured with IND-OE/DOX or IND-C12/DOX at a dose of 4 μg/mL for 2 h, 4 h and 8 h. Finally, cells co-lacalization were then observed and recorded by CLSM. 2.8. Flow Cytometry. Intracellular DOX level was carried out using the Flow cytometry.38 Two cell lines were co-incubated with free DOX, IND-OE/DOX and IND-C12/DOX (4 μg/mL) for 2 h and then cultured with fresh medium. At selected time intervals, the fluorescence intensity of DOX in each group was observed by flow cytometry (FCM, Becton Dickinson,USA). 2.9. Cytotoxicity. The cytotoxicity was tested via MTT assay.36 Briefly, against


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two cell lines were incubated with IND, DOX, IND+DOX, IND-C12 NPs, IND-OE NPs, IND-C12/DOX and IND-OE/DOX at different concentrations for 2h, and cultured with fresh medium for another 24 h. Then, each well cells were incubated with 20 μL MTT with 5 mg/mL for 4 h. Finally, the formazan crystals were dissolved with DMSO, and then the absorbance was detected via SpectraMax M2e Molecular devices at 570 nm. Beisdes, the change of cell morphology was also observed by a fluorescent microscope (BX51, Olympus, Japan). And RPMI 1640 was used as the control. 2.10. Apoptosis Analysis. Apoptosis after co-incubated with each sample was detected by FCM. Briefly, two cell lines were cultured with DOX (10 μg/mL), DOX-loaded NPs (10 μg/mL), free IND (IND = 1 mg/mL) and blank NPs (IND = 1 mg/mL). After incubation of 2 h, the old medium was removed, 1 mL fresh medium was added and cultured for another 24 h before measured by FCM. Besides, to further verify the cell-killing capacity of four DOX formulations, the apoptosis testing were performed under their IC50 concentration, respectively. 2.11. MRP1 Expression. First, MRP1 level in two cancer cells was observed via western blot as previous work.32 Then, the change of MRP1 expression after treatment with different samples were assessed via fluorescent immunostaining.43 Besides, the fluorescence intensity of MRP1 was quantified by a software (Image-Pro Plus 6.0). 2.12. Penetration and Growth Inhibition in multicellular spheroids (MCs). MCs were cultured as described previously.38 MCF-7 and MCF-7/ADR MCs were cultured with free DOX, IND-OE/DOX and IND-C12/DOX at a dose of 10 μg/mL



(DOX) for 2 h, then the medium was replaced with 200 μL fresh medium. At predetermined time (0.5, 2, 6, 12 or 24 h), DOX distribution in MCs were investigated by CLSM and the intensity was calculated by Image J. In order to evaluate the growth inhibition on MCs, the spheroids were co-cultured with IND, IND-C12 NPs, IND-OE NPs, DOX, IND-C12/DOX and IND-OE/DOX for 12 h, respectively. Next, the growth of MCs was investigated by an optical microscope every other day. The average diameter of MCs in all groups were calculated during a week. 2.13. Drug Accumulation at Tumor Sites. Tumor-bearing female BALB/c nude mice was established as previous report.6 These mice were divided into 3 groups and administrated with free DOX, IND-C12/DOX and IND-OE/DOX at a dosage of 5 mg/kg via tail vein. At set times, tumor mass were picked, embedded, sectioned and stained, then imaged by CLSM. Besides, the DOX content in the heart of treated mice was also assessed. 2.14. Antitumor Efficacy. Antitumor ability of DOX, IND-OE/DOX and IND-C12/DOX (5 mg/kg DOX eq.) were investigated as previous work, and saline was used as control.42 Tumor size was recorded every day with a vernier caliper, and mice body weight was obtained by mini digital scale. At day 7, tumor tissues were collected, imaged and weighted. Besides, tumor volume was calculated by the formula:

Tumor volume = d 2 × D / 2

(3)

d represent the short diameter and D represent the long diameter.


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2.15. Histological Analysis. After treating with free DOX, IND-OE/DOX and IND-C12/DOX for 72 h, major organ sections were stained by hematoxylin and eosin (H&E),38 and then observed by a fluorescent microscope. Besides, apoptotic cells in solid tumors were also evaluated by the TUNEL method accordingly (Solarbio, T2190, Beijing). 2.16. Statistical Analysis. Statistical comparisons were performed by one-way ANOVA analysis. P values less than 0.05 were considered statistically significant.

3. RESULTS AND DISCUSSION 3.1. Synthesis and characterization. Chemical structure of the synthesized IND-OE and IND-C12 were determined by 1H NMR, 13C NMR and the electro spray interface mass spectroscopy (ESI-MS). From figure 1a, the characteristic peak of benzene ring was found at 6.6-6.9 and 7.4-7.7 ppm, which belonged to the IND. The peak of ′a′ in 1H NMR was assigned to methene protons (-CH2) from C12, and the actual integral ratio of a:f:g:e= 1:0.99:1.43:1.44, which was close to the theoretical value (a:f:g:e=1:1:1.5:1.5). Similarly, the characteristic peak of ortho ester from OE was also observed at 5.79-5.84 ppm (Figure 1b), and the integral ratios against benzene ring was 1:3.05:4.11, which agreed well with the theoretical values (1:3:4). These results indicated that the synthesis of IND-C12 and IND-OE was successful. Besides,

13C

NMR spectra and ESI-MS further proved the structure of the product

(Figure S1 and S2). FT-IR was also used to determine the amidation reaction between IND and C12 or OE. As shown in Figure S3, the C=O stretching vibration of IND



was found at 1714 and 1683 cm-1, while the new adsorption peak of the C=O stretching vibration (amide I) and N-H bending vibration (amide II) was observed at 1582 and 1638 cm-1 for IND-C12, and at 1580 and 1647 cm-1 for IND-OE. These results further demonstrated that IND-C12 and IND-OE had been successfully synthesized. 3.2. Size and Morphology of NPs. IND dimer NPs was prepared via O/W single emulsion solvent volatilize method.40 DLS measurements showed that IND-OE NPs and IND-C12 NPs had a similar size in the range of 160~170 nm and the PDI of 0.12~0.14 (Figure 2a and 2b). TEM and SEM measurements indicated that IND-OE NPs and IND-C12 NPs had a spherical shapes with a mean size around 70~100 nm (Figure 2c~2f). 3.3. pH-triggered Degradation. As well known, ortho ester bonds are highly sensitive to acidic conditions, and their hydrolysis rate is closely related to the hydrophobicity/hydrophilicity of its surroundings environments.38,44 In this work, the acid-degradation kinetics of ortho ester bonds in IND-OE NPs was tracked at pH 7.4 or 5.0 by 1H NMR analysis. From figure 3a, the proton peak of ortho ester at δ 5.80 ppm (red labeled) constantly decreased when IND-OE NPs were incubated at pH 5.0 buffer, while new proton peaks of formate (degradation product, blue labeled) appeared at δ 8.23 ppm and the intensity enhanced with the prolonging time. On the contrary, there was few degradation at pH 7.4, which meant these nanoparticles were highly stable (Figure 3b). The hydrolysis mechanism of IND-OE NPs also followed the exocyclic pathway exclusively and the degradation pathways were presented in


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Figure S4. Finally, the hydrolysis rate were calculated, the degradation of ortho esters in IND-OE NPs reached 100% at pH 5.0 within 48 h while only less 10% at pH 7.4 (Figure 3c). Besides, the degradation of IND-OE NPs under pH 6.0 is slower than pH 5.0, and need more than 72 h to completely degrade, which was attributed to the strong hydrophobicity of IND dimer (Figure S5a and S5b). All findings indicated that ortho ester-linked nanoparticles could easily respond to intracellular pH gradients, which could efficiently trigger drug release at tumor sites. Then, pH-responsiveness of IND-OE NPs was further determined via observing the change of NPs' average diameter and light scattering intensity at two pH values by DLS. Figure 3d and 3e showed that the sizes of these particles remained stable at around 175-190 nm in pH 7.4, and the intensity had only slight fluctuation (383-367 kcps), which meant these nanoparticles possessed good stability in neutral condition. However, when incubation with pH 5.0 buffer, the NPs' size gradually increased and reached 335 nm within 12 h, and then rapidly decreased to 141 nm for 48 h. Meanwhile, the NPs' intensity continued to decline and the final value was just 149 kcps compared to the initial value of 310 kcps. Besides, the similar phenomenon was also observed by TEM images, and the degradation process included two main stages (Figure 3f). At the starting time, the acid-treated NPs became loose and their diameter increased. After treatment for 12 h, these particles disintegrated into fragments, the diameter and count rate decreased. This result was probably because the internal linkage of nanoparticles was broken on the basis of the hydrolysis of ortho-esters at weak acidic conditions.



3.4. Drug Loading and Release. DOX was encapsulated into IND-OE and IND-C12 nanoparticles to give IND-OE/DOX and IND-C12/DOX, respectively. Drug loading content (DLC) of IND-OE/DOX and IND-C12/DOX were 11.39% and 9.43%, and the drug loading efficiency (DLE) were 58.93% and 54.65% (Table S1). The desirable loading capacity was probably based on the hydrophobicity of dimer and the intermolecular π-π stacking of IND and DOX.25,27 DOX encapsulation did not display distinct influence to the nanoparticles, and the mean size of these drug-loaded NPs remained at around 180 nm. In addition, surface zeta potential showed that either blank particle or drug-loaded particle both had a negative charge (Figure 4a), which was beneficial to the long-term blood circulation of them by declining protein adsorption. In addition, the stability of DOX-loaded IND-OE and IND-C12 nanoparticles in several physiological environments was observed in detail and the result was presented in Figure 4b and 4c. It could be seen that two particles showed excellent storage stability, and the size had almost unchanged during 7 days. Also, there was just slight fluctuation when these particles were incubated with different physiological environments. Drug release results of IND-OE/DOX and IND-C12/DOX at pH 5.0 and pH 7.4 were shown in Figure 4d. DOX release rate of IND-C12/DOX was obviously suppressed and only a few DOX (~20%) was released within 4 days after incubation with pH 5.0 or 7.4, indicating that C12-linked nanoparticles lacked a stimuli-responsive drug release. However, IND-OE/DOX displayed an obviously pH-dependent drug release profile. More than 85% DOX was released at pH 5.0


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within 48 h, and only 15% of drug was discharged at pH 7.4. Besides, when treatment with pH 6.0, the DOX release rate reached to 70% at the same time, which was consistent with the degradation results (Figure S5c). These findings demonstrated that ortho ester-linked indomethacin dimers nanoparticles might be potentially promising as nanocarrier, since it could remain stable in neutral environment while promptly hydrolyzed in acidic environment, leading to rapid intracellular DOX release in tumor site. 3.5. Cellular Uptake and Co-localization. DOX accumulation of IND-OE/DOX and IND-C12/DOX in tumor cells were investigated by CLSM. From Figure 5, DOX signal appeared in the cytoplasm and nucleus area, which meant that DOX-loaded nanoparticles could be internalized by tumor cells. For MCF-7 cells, most of DOX was located in nucleus after incubated with free DOX or IND+DOX, which was because the diffusion of DOX and rapidly moving into cell nucleus (Figure 5a). When co-incubated with DOX-encapsulated NPs, the red signal from IND-C12/DOX mainly appeared in cytoplasm region, while DOX staining from IND-OE/DOX was observed in cell cytoplasm and nucleus. This result was probably because IND-C12/DOX had little drug release in endo/lysosomal, while a large number of drug was released from IND-OE/DOX owing to the cleavage of ortho ester at low pH after cell internalization by endocytosis pathway. Besides, there was great difference in these formulations after incubation with MCF-7/ADR (Figure 5b). Notably, the red fluorescence intensity in IND+DOX and IND-OE/DOX groups was much higher than free DOX group, which was probably due to the MRP1-mediated efflux effects caused a large



number of DOX were removed from drug-resistant cells, but IND could efficiently suppress this effect. Besides, we also found that IND-C12/DOX were mainly accumulated in cytoplasm rather than cell nucleus in MCF-7/ADR cells, as similar to the results of

MCF-7 cells.

To further track the intracellular DOX release and distribution in tumor cells, subcellular co-localization were performed (Figure S6). After 2 h, IND-C12 NPs and IND-OE NPs were mainly located in lysosome (seen Merge). However, DOX signal was observed throughout intracellular areas after incubated with IND-OE NPs for 4 and 8 h, this meant DOX could be rapidly release from acid-sensitive IND-OE NPs and then diffuse into nucleus to exert its antitumor activity. However, IND-C12/DOX just appeared at cytoplasm region rather than nucleus. It suggested that IND-C12/DOX remained in the particle state and less drug was released, even if treatment with acidic lysosome. Compared to some degraded polymers (like PLGA) or sensitive chemical bonds, the amide bonds were more stable in vivo, thus leading to a higher stability for IND-C12 [45]. In other word, ortho ester linked IND dimer (IND-OE) had significant advantages for in vivo using, because it could keep stale in blood circulation while accelerate drug release at the acidic organelles. Finally, the DOX retention in two tumor cells was further measured by FCM. From Figure S7a and S7b, DOX accumulation in tumor cells followed a time-dependent trend, and the intracellular DOX level in all samples gradually decreased with the prolonging time. After treatment for 4 h, the mean fluorescence intensity of free DOX, IND+DOX, IND-C12/DOX and IND-OE/DOX in MCF-7 cells remained at 78.1, 79.7,


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97.5 and 139, respectively (Figure S7c). In MCF-7/ADR cells, these groups containing IND had also relatively high DOX concentration, but the lowest DOX intensity appeared in the absence IND group (free DOX) (Figure S7d). Besides, the drug efflux rate ((t4-t0.5)/t4) was also calculated (Figure S7e). The efflux ratio of free DOX and IND+DOX were stronger than DOX-loaded NPs in MCF-7 cells, which was due to the controlled and sustained drug release. Besides, this ratio in MCF-7/ADR cells treated with free DOX reached 73.31, which were 1.82, 3.14 and 2.21 fold higher than IND+DOX, IND-C12/DOX and IND-OE/DOX, respectively. MCF-7/ADR cells had distinct resistance toward free DOX due to the high expression of MRP1. From the above data, we could draw the following conclusions: (1) Efficient intracellular drug release could be accomplished by introducing the acid stimulus-responsive ortho ester bonds; (2) High drug accumulation in MDR tumor cells could be achieved because IND could reverse MRP1 mediated drug efflux. 3.6. Determination of MRP1. MRP1 as an ATP-dependent membrane transporters could decline the intracellular drug concentration and limit the cytotoxicity.46 As shown in Figure S8, the MRP1 level was measured by western blot, and results suggested that MCF-7/ADR cells had more MRP1 expression than MCF-7 cells. Besides, IND as a cyclooxygenase-2 (COX-2) specific inhibitor can remarkably down-regulate the level of MRP1 in various drug-resistant tumor cells.33,47 Thus, in this section, we further evaluated the expression of MRP1 in two cell lines after treatment with free IND, IND-C12 NPs and IND-OE NPs by immunofluorescence. From Figure 6a and 6b, the red signal in MCF-7/ADR cells was stronger than in



MCF-7 cells, indicating that more expression of MRP1 in drug-resistant cells. But incubated with IND groups, the level of MRP1 was remarkably down-regulated. Particularly, IND-OE NPs displayed lower red MRP1 signal in MCF-7/ADR cells (Figure 6c), which was due to the rapid hydrolysis of ortho ester under mildly acidic environments, then IND could exert its activity in tumor cells. 3.7. Cytotoxicity study. Cytotoxicity of each sample was detected via MTT assay. First, IND groups exhibited little toxicity with all tested concentration (1-1000 µg/mL) in two cell lines (Figure 7a and 7b), which was similar to the previous reports.35,36 On the contrary, DOX-loaded NPs displayed a concentration-dependent cytotoxicity in two tumor cells. For MCF-7 cells (Figure 7c), cytotoxicity gradually increased with the increasing concentration of each sample, and cell viability was 26.28 % (DOX), 20.05 % (IND+DOX), 62.84 % (IND-C12/DOX) and 23.17 % (IND-OE/DOX), respectively. Besides, we also found that IND-OE/DOX showed higher cytotoxicity than IND-C12/DOX because of the rapid intracellular drug release of IND-OE/DOX. For MCF-7/ADR cells (Figure 7d), free DOX and IND-C12/DOX showed a relatively weak cytotoxicity, and cell viabilities were more than 75%. However, IND+DOX and IND-OE/DOX possessed stronger cell-killing ability, and less than 40% cells were alive at 10 µg/mL of DOX after incubation for 24 h. These results revealed that IND could sensitize drug-resistant tumor cells and increase DOX toxicity through down-regulating the expression of MRP1. Table 1 present the IC50 value, resistance index and reversal index of each sample.48 The IC50 in MCF-7 cells was 5.01 (DOX), 3.91 (IND+DOX), 36.47


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(IND-C12/DOX)

and

4.26

(IND-OE/DOX),

respectively.

Obviously,

these

formulations in MCF/ADR had higher IC50 values, particularly free DOX reached to 209.32. Thus, the resistance index of MCF-7/ADR cells against DOX was 43.88. Otherwise, the reversal index of IND-C12/DOX and IND-OE/DOX were 1.38 and 41.78, which meant ortho ester-linked nanoparticles had advanced reversing effects on tumor MDR. Besides, the change of cells morphology after treatment with different samples was also observed by fluorescent microscope. As shown in Figure 7e and 7f, there was little change in two cells after incubation with empty IND groups owing to them low toxicity. Conversely, DOX showed distinct cytotoxicity to MCF-7 cells, as its membrane shrinkage and size reduction. Besides, MCF-7/ADR cells had only a few changes after incubation with free DOX and IND-C12/DOX groups, while more cell necrosis or apoptosis appeared in IND+DOX and IND-OE/DOX groups. This difference in morphology was consistent with the MTT test. 3.8. Apoptosis Analysis. The apoptosis results measured by FCM were presented in Figure S9. In MCF-7 cells, the sum apoptosis was 57.2% (DOX), 63.0% (IND+DOX), 22.99% (IND-C12/DOX) and 58.3% (IND-OE/DOX), respectively. Notably, IND-C12/DOX had lower cytotoxicity than other groups, since the inefficient and slow DOX release mediated by the unbreakable carbon-carbon linkage. Besides, free DOX-induced apoptosis in MCF-7/ADR cells was also limited and the apoptosis rate was just 9.77%, which was probably attributed to the less DOX concentration in tumor cells. However, IND+DOX and IND-OE/DOX groups could



significantly enhance DOX cell-killing ability by inhibiting MRP1 level, and the apoptosis ratio reached to 56.0% and 55.7%, respectively. These result were consistent with MTT testing. Thus, in order to further verify the above conclusion, the apoptosis were performed using the IC50 dosage. IND+DOX and IND-OE/DOX could kill ~ 50% tumor cells at low drug concentration in MCF-7 and MCF-7/ADR cells, while IND-C12/DOX needed extremely high drug dosage (Figure S10). In other word, IND could promote DOX mediated apoposis, and IND-OE dimer had great potential for in vivo drug delivery and synergistic cancer treatment. 3.9. Accumulation and growth inhibition in MCs. MCs were used to evaluate the accumulation and permeation behavior of these DOX-encapsulated nanoparticles.49 Figure 8a and 8b showed the results captured by CLSM. Notably, a time-dependent DOX accumulation was observed, since the DOX signal arose in all MCs and the fluorescent intensity increased over time. For MCF-7 MCs, DOX accumulation in spheroids treated with free DOX or IND+DOX were relatively weak, and red signal mainly accumulated in the out-layer of MCs. Besides, IND-C12/DOX was also restricted in the periphery of spheroids with deepness of ~25 μm, and only a few red signals were penetrate into their cores because of the slow drug release. However, the fluorescence intensity of IND-OE/DOX gradually increased from outside to inside in spheroids, indicating that DOX-loaded NPs had higher accumulation in MCs via nano-particles' size effect than that of free drugs, and the efficient penetration and diffusion in spheroids could be achieved owing to the rapid DOX release caused by the hydrolysis of ortho-ester. Besides, the similar result was observed in MCF-7/ADR


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spheroids, but DOX fluorescence intensity in these MCs were less than that of MCF-7 MCs, which was due to the existence of MRP1 efflux pump. Figure 8c and 8d showed the quantitative statistics for the average DOX fluorescence intensity in two MCs. It could be seen that the average fluorescent intensity gradually increased with the prolong of incubation time in two MCs. Particularly, there was a distinct difference between IND-OE/DOX and free DOX in drug-resistant spheroids, which might be because IND could inhibit DOX efflux and increase DOX accumulation. The growth inhibitory effect on MCs treated with various formulations was further assessed, and the size of each MCs was measured every day. After treatment with free IND or IND-dimer NPs, there were no tumor growth inhibition in MCF-7 and MCF-7/ADR spheroids (Figure 9a and 9b). In contrast, four DOX formulations exhibited different antitumor effect in two spheroids. For MCF-7 spheroids, lots of dead cells appeared in the outer layer of spheroids cultured with free DOX, IND+DOX, IND-C12/DOX and IND-OE/DOX, and the diameter of these MCs continually declined with time prolonging. Besides, we also found that IND-OE/DOX had the strongest tumor-killing, spheroids' 3D structure was broken and final volume just was about 700 μm3 after treatment 5 days (Figure 9c). The similar result was observed in MCF-7/ADR spheroids. The volume of MDR-spheroids were 10298, 3399, 8320 and 1432 μm3 for DOX, IND+DOX, IND-C12/DOX and IND-OE/DOX, respectively (Figure 9d). Obviously, free DOX and IND-C12/DOX lacked efficient cell-killing ability in drug-resistant spheroids owing to the powerful drug efflux and inefficient drug release. In summary, the combination action between the



acid-triggered drug release and IND-medicated MDR reversal could improve DOX accumulation and penetration, resulting in the amplification of antitumor effects in tumor-like spheroids. 3.10. Drug accumulation in tumor tissues. To determine the retention ability of DOX-loaded NPs at tumor site, tumor-bearing mice were administrated with free DOX, IND-C12/DOX and IND-OE/DOX, and the DOX dosage was 5 mg/kg for all groups. After treatment with different time, tumor tissues slices were observed by the fluorescent microscopic. As expected, mice treated with IND-C12/DOX and IND-OD/DOX had higher drug accumulation than free DOX treated mice at all tested times (Figure 10a). DOX-loaded NPs showed the strongest red fluorescence signal at 2 h, and then the fluorescence intensity gradually decreased over time. Even treatment for 24 h, DOX staining was still visible. However, no obvious fluorescence signals were observed in free DOX group at the end time. Besides, DOX intensity in different formulations was also quantitatively analyzed (Figure 10b). It was obviously that two DOX-loaded NPs possessed higher drug retention at tumor tissues than that of free DOX, which was probably due to the enhance permeation and retention (EPR) effect.50 3.11. Antitumor ability. Antitumor ability of IND-OE/DOX and IND-C12/DOX were then investigated. Figure 11a and 11b showed the mean tumor volume and body weight variation during the study. For saline treated group, tumor grew continually and the mean volume reached to 225.51 mm3 at day 7. Meanwhile, the tumor volume of free DOX-treated mice continuously increased and the final volume was 120 mm3.


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Besides, after treatment with IND-C12/DOX, tumor growth was suppressed in the initial 3 days. After that, the tumor sharply increased and reached 89.69 mm3. However, IND-OE/DOX showed the highest tumor growth inhibition, and the tumor volume declined in day 1 to day 4, and the final volume just was 43.7 mm3. Thus, the tumor inhibition rate versus saline group was 46.78% (free DOX), 60.23% (IND-C12/DOX), and 80.62% (IND-OE/DOX), respectively. Otherwise, body weight of free DOX-treated mice displayed a significant decrease, which was related to the serious side effects caused by DOX heart retention (Figure S11). Figure 11c shows all tumor tissues at the end of the study. It could be seen that IND-OE/DOX displayed the minimum tumor than other groups, which was due to the combination therapy of chemosensitizers and anticancer drug. Figure 11d showed the average tumor weights of saline, DOX, IND-C12/DOX and IND-OE/DOX were 115.71 ± 15.32, 72.08 ± 8.94, 57.07 ± 11.19 and 25.45 ± 12.68 mg, respectively. All above results fully demonstrated that ortho ester-linked IND dimer nanoparticles could greatly improve antitumor effect than free DOX or IND-C12/DOX by the acid-triggered DOX release and IND-promoted DOX sensitivity in drug-resistant solid tumors. 3.12. Histological analysis. To further demonstrate that IND-OE/DOX could effectually inhibit tumor growth, TUNEL staining was used to detect cell apoptosis in tumor tissues.51,52 As shown in Figure 12a, few apoptotic signals (green color) were observed in saline or free DOX groups, indicating that there were no or weak therapeutic effect. However, we found that DOX-loaded NPs could induce a remarkably increase in the percentage of TUNEL-positive cells, and particularly,



IND-OE/DOX group produced the extensive tumor necrosis. H&E staining images confirmed the above result again. After treatment with IND-OE/DOX, tumor tissue was necrotic and fragmented, along with a decrease in the number of cells and shriveled cytoplasm (Figure 12b). These results demonstrated that IND-OE NPs were the most effective in inducing cell death while reducing cell proliferation. Otherwise, no noticeable injury in major organs after treating with IND-C12/DOX and IND-OE/DOX (Figure S12), indicating that these DOX-loaded NPs were safe in vivo.

4. CONCLUSION In summary, we had successfully developed the pH-sensitive indomethacin (IND) dimer nanoparticles loaded with doxorubicin for the combination therapy in drug resistant breast cancer. IND dimer was prepared by the ortho ester monomer (OE) linkage under the EDC/NHS catalysis and then self-assembled into nanoparticles (IND-OE NPs) with the diameter of ~160 nm. The prepared NPs displayed excellent stability in physiological conditions, while quickly degraded at low pH condition and subsequently accelerate drug release in tumor site. In vitro 2D and 3D cell experiments confirmed that IND-OE NPs could obviously improve the DOX accumulation and penetration, leading to enhanced cytotoxicity by inhibiting the MRP1 expression in MCF-7/ADR cells. Furthermore, in vivo results also verified that the complex nanoparticles could increase antitumor efficiency of DOX while reducing negative effects through the acid-triggered drug release and IND-mediated chemotherapy sensitizing. This strategy of designing pH-sensitive small agent


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conjugates is potentially useful, and then other chemosensitizers or functional molecule can be developed for more efficient combination therapy in future.

SUPPORTING INFORMATION Supplementary data to this article can be found online.

ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (No. 51503001, 51803001 and 51603001), the Research Foundation of Education Department of Anhui Province of China (No. KJ2018ZD003, and KJ2018A0006), and the Academic and Technology Introduction Project of Anhui University (AU02303203).

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] (R. Tang) Author Contributions # X.W.

and X.C. contributed equally to this work.

Notes The authors declare no competing financial interest.



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Figure 1. 1H NMR spectra of IND-C12 (a) and IND-OE (b).

Figure 2. Hydrodynamic diameter and distribution of IND-OE NPs (a) and IND-C12 NPs (b); SEM images of IND-OE NPs (c) and IND-C12 NPs (e); TEM images of IND-OE NPs (d) and IND-C12 NPs (f).


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Figure 3. The degradation kinetics of IND-OE NPs at pH 5.0 and 7.4: 1H NMR spectra of degradation products at pH 5.0 (a) and pH 7.4 (b); Hydrolysis rate (c); Time and pH-dependence change of diameter (d) and count rate (e); SEM images of IND-OE NPs after incubation with pH 5.0 bufffer at different times (f).



Figure 4. Zeta potential of blank and DOX-loaded nanoparticles (a); Stability of IND-OE/DOX and IND-C12/DOX during storage for 7 days (b); Stability of IND-OE/DOX and IND-C12/DOX against various environments (c); In vitro drug release from IND-OE/DOX and IND-C12/DOX at pH 5.0 and 7.4 (d); Data are represented as mean ± SD (n = 3).


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Figure 5. Cellular uptake of free DOX, IND+DOX, IND-C12/DOX and IND-OE/DOX via CLSM in MCF-7 cells (a) and MCF-7/ADR cells (b); Scale bar = 15 μm.



Figure 6. The expression of MRP1 after treatment with different samples in MCF-7 cells (a) and MCF-7/ADR cells (b); Intracellular MRP1 level was calculated (c); Data are represented as mean ± SD (n = 3). Scale bar = 10 μm.


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Figure 7. In vitro cytotoxicity of free IND, IND-C12, and IND-OE in MCF-7 cells (a) and MCF-7/ADR cells (b); In vitro cytotoxicity of free DOX, IND+DOX, IND-C12/DOX, and IND-OE/DOX in MCF-7 cells (c) and MCF-7/ADR cells (d); Data are represented as mean ± SD (n = 3); The change of cells morphology after



treatment with different samples for MCF-7 cells (e) and MCF-7/ADR cells (f).

Figure 8. CLSM images of MCs incubated with free DOX, IND+DOX, IND-C12/DOX and IND-OE/DOX for MCF-7 cells (a) and MCF-7/ADR cells (b); Scale bar = 150 μm; The fluorescence intensity of MCs was quantified at different time for MCF-7 cells (c) and MCF-7/ADR cells (d); Data are represented as mean ± SD (n = 3); * represents P < 0.05, ** represents P < 0.01 and *** represents P < 0.001, against free DOX at the same time.


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Figure 9. Growth inhibition of MCs after treatment with different samples for MCF-7 cells (a) and MCF-7/ADR cells (b); Scale bar = 150 μm; The volume of MCs was calculated at 7 day for MCF-7 cells (c) and MCF-7/ADR cells (d); Data are represented as mean ± SD (n = 3); * represents P < 0.05, ** represents P < 0.01 and *** represents P < 0.001.



Figure 10. Representative fluorescent images of tumor tissues from MCF-7/ADR tumor-bearing mice after intravenous administration of different DOX formulations at different times (a); DOX fluorescence intensity was quantified via Image J. Z-stack (b); Data are represented as mean ± SD (n = 3).


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Figure 11. In vivo tumor growth curves of MCF-7/ADR tumor-bearing mice after treated with saline, free DOX, IND-C12/DOX and IND-OE/DOX (a); The body weight change of mice every day (b); Images of tumor after 7 days treatment (c); The mean tumor weight was calculated after 7 days treatment (d); Data are represented as mean ± SD (n = 8), * represents P < 0.05, ** represents P < 0.01 and *** represents P < 0.001.



Figure 12. Histological sections of tumor tissues with TUNEL staining, nuclei were stained blue and apoptotic cells were stained green (a); Histological sections of tumor tissues with H&E staining (b). Scale bar = 5 μm.


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Table 1. IC50 values, resistance and reversal index of different formulations against MCF-7 cells and MCF-7/ADR cells. IC50 (µg/mL)

Resistance

Reversal

MCF-7

MCF-7/ADR

index

index

Free DOX

5.01

209.32

41.78

——

IND+DOX

3.91

4.77

1.22

43.88

IND-C12/DOX

36.47

151.43

4.15

1.38

IND-OE/DOX

4.26

5.01

1.18

41.78

Resistance index: the ratio of IC50MCF-7/ADR against IC50MCF-7. Reversal index: the ratio of IC50free drug to IC50drug-loaded nanoparticles against MCF-7/ADR cells.



Table of Contents Graphic (TOC)


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Self-Assembled Indomethacin Dimer Nanoparticles Loaded with

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