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Mitochondrial targeted doxorubicin-triphenylphosphonium delivered by hyaluronic acid modified and pH responsive nano-carriers to breast tumor: in vitro and in vivo studies Hui-Na Liu, NingNing Guo, Tian-Tian Wang, Wang-Wei Guo, Meng-Ting Lin, Ming-Yi Huang-Fu, Mohammad Reza Vakili, Wen-hong Xu, Jiejian Chen, Qi-Chun Wei, Min Han, Afsaneh Lavasanifar, and Jianqing Gao Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00793 • Publication Date (Web): 22 Jan 2018 Downloaded from http://pubs.acs.org on January 23, 2018

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Molecular Pharmaceutics

Mitochondrial targeted doxorubicin-triphenylphosphonium delivered by hyaluronic acid modified and pH responsive nano-carriers to breast tumor: in vitro and in vivo studies Hui-Na Liu1,#, Ning-Ning Guo1,#, Tian-Tian Wang1, Wang-Wei Guo1, Meng-Ting Lin1, Ming-Yi Huang-Fu1, Mohammad Reza Vakili2, Wen-Hong Xu3, Jie-Jian Chen3, Qi-Chun Wei3, Min Han1,*, Afsaneh Lavasanifar2,*, Jian-Qing Gao1,*.

1

Institute of Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou,

310058 China 2

Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta

T6G 2E1, Canada 3

Department of Radiation Oncology, Cancer Institute, Key Laboratory of Cancer Prevention and

Intervention, the Second Affiliated Hospital, Zhejiang University, College of Medicine, Hangzhou, Zhejiang, China.

Corresponding author: Min Han Institute of Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, People’s Republic of China, Tel +86-571-88208437, Email: [email protected] Afsaneh Lavasanifar Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta T6G 2E1, Canada, Email [email protected] Jian-Qing Gao Institute of Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, People’s Republic of China, Tel +86-571-88208436, Email: [email protected]

First author #: These two authors contributed equally

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ABSTRACT Multidrug resistance (MDR) is the major obstacle for chemotherapy. In a previous study, we have

successfully synthesized

a

novel doxorubicin

(DOX)

derivative modified

by

triphenylphosphonium (TPP) to realize mitochondrial delivery of DOX and showed the potential of this compound to overcome DOX resistance in MDA-MB-435/DOX cells.1 To introduce specificity for DOX-TPP to cancer cells, here we report on the conjugation of DOX-TPP to hyaluronic acid (HA) by hydrazone bond with adipic acid dihydrazide (ADH) as the acid-responsive linker, producing HA-hydra-DOX-TPP nanoparticles. Hyaluronic acid (HA) is a natural water-soluble linear glycosaminoglycan, which was hypothesized to increase the accumulation of nanoparticles containing DOX-TPP in the mitochondria of tumor cells upon systemic administration, overcoming DOX resistance, in vivo. Our results showed HA-hydra-DOX-TPP to self-assemble to core/shell nanoparticles of good dispersibility and effective release of DOX-TPP from the HA-hydra-DOX-TPP conjugate in cancer cells which was followed by enhanced DOX mitochondria accumulation. The HA-hydra-DOX-TPP nanoparticles also showed improved anti-cancer effects, induced better tumor cell apoptosis and a better safety profile compared to free DOX in MCF-7/ADR bearing mice. KEYWORDS: MDR, mitochondrial delivery, DOX-TPP, hydrazone, hyaluronic acid

INTRODUCTION Multi-drug resistance (MDR) is one of the main obstacles for cancer therapy.2 The mechanisms involved in MDR include overexpression of efflux pumps (i.e. P-glycoprotein, P-gp), decreasing drug uptake, altering intracellular drug localization, enhancing repair of drug induced DNA damage and hinting apoptotic pathways.3 There are many solutions to overcome MDR and one of the most popular ways is to inhibit the overexpression or activity of efflux proteins that

can

reduce the intracellular concentration of anti-cancer drugs in tumor cells.4 Various nanocarriers aiming at overcoming efflux-mediated resistance has been studied recently.5-7 In addition, the inhibition of MDR-related genes by small interfering RNA (siRNA8) has been of particular interest. In this context, delivering siRNA and anti-cancer agents simultaneously is also becoming a promising approach to overcome drug resistance.9-11 Mitochondria are intracellular organelles that play key roles both in the regulation of survival and


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in the adjustment of cell apoptosis. On one hand, they are the site of energy production within the cell. On the other hand, mitochondria are also a vital center for cell apoptosis and trigger cell death via several mechanisms that included disrupting electron transport and energy metabolism, releasing or activating proteins that mediate apoptosis and altering cellular redox potential.12, 13 In general, the cell apoptosis pathway involves permeabilization of the outer mitochondrial membrane followed by the release of cytochrome c and other proteins from the inter-membrane of mitochondria. Once in the cytosol, cytochrome c interacts with its adaptor molecule, apoptotic protease activating factor-1 (Apaf-1), resulting in the recruitment and activation of pro-caspase-9, then active caspase-9 cleaves and activates pro-caspase-3 and pro-caspase-7, these caspase family are responsible for the cleavage of a variety of cellular proteins, leading to cell death.14 In addition, studies have shown a close relationship between mitochondrial dysfunction and cancer, Alzheimer's disease15, diabetes16, Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes(MELAS) and other diseases17,

18

. Thus, mitochondria used as a target

organelle for the treatment of many diseases have attracted more attention19. The high membrane potential20,

21

and protein import machinery of mitochondria-mitochondrial targeting sequence

(MTS) 22, 23 can be used to achieve delivery of chemotherapy drug to mitochondria effectively. The mitochondria targeting are usually achieved by means of anchoring mitochondrial targeting groups, including rhodamine, methyl- triphenyl phosphonium (TPP) and dequalinium chloride to nanocomposites or drugs. 24, 25Triphenyl phosphonium (TPP) is a mitochondriotropic ligand that is uptake by mitochondrial membrane due to its high lipophilicity and cationic charge.26, 27

TPP-modified nanoparticles, such as CoQ10/PEG-PCL-TPPB,28 LND/PLGA-b-PEG-TPP,

29

DOX/TPP-PEI,30 G4-PAMAM–poly(ethylene glycol)-triphenylphosphonium31,

32

have been

reported in the literature before. This approach poses some drawbacks, however. For instance, the positive surface charge of nanoparticles that are modified by TPP may make them prone to off-target in vivo capture and side effects. Besides, the large size of the particles may not be favorable for intracellular mitochondrial targeting. DOX can enter the nucleus to damage DNA or inhibit topoisomerase resulting in apoptosis of tumor cells.33 The anti-tumor effects of DOX may be deminished by MDR by several different mechanisms. Altering the intracellular distribution of DOX is, however, shown to be a promising approach leading to drug activity in some DOX resistant phenotypes of cancer cells. We have



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previously reported a small molecule mitochondrial targeted doxorubicin derivative (DOX-TPP), 1

in which TPP was conjugated directly to DOX by amide bond to achieve mitochondrial targeting.

The results of our study illustrated that DOX-TPP to enhance the cytotoxicity of DOX as a result of mitochondrial targeting, in MDA-MB-435/DOX cancer cells. However, due to its amphiphilic nature, it is difficult to physically embed DOX-TPP into a nano-formulation. Hyaluronic acid (HA) is a linear polysaccharide formed from disaccharide units containing N-acetyl-d-glucosamine and glucuronic acid,34 widely distributed in mammalian bone marrow extracellular matrix and loose connective tissue. As a biomaterial, it has many advantages, such as water solubility and good biocompatibility, biodegradability, nonimmune antigenicity and so on. In addition, hyaluronic acid-specific receptor, CD44, is overexpressed on the surface of many malignant tumor cells.35 In recent years, HA has emerged as a promising candidate for intracellular delivery of various therapeutic and imaging agents because of its ability to recognize specific cellular receptors that overexpressed on cancer cells.36, 37 The pH in tumor tissue is slightly acidic pH (6.5-6.8). By exploiting the acidic microenvironments in the tumor, pH sensitive delivery systems can be designed for intratumoral drug release. In this study, we report on the synthesis of HA-hydra-DOX-TPP by conjugating DOX-TPP to water-soluble HA using an acid cleavable hydrazone bond. Hydrazone linkage has been successfully utilized to conjugate many anti-tumor drugs such as paclitaxel,38 DOX39, 40 to nanocarriers in order to achieve pH responsive selective targeting to the tumors. HA-hydra-DOX-TPP self-assembly formed nanostructure with hydrophilic HA as shell and hydrophobic DOX as core. This nanostructure showed

good

dispensability and fully released the conjugated DOX-TPP in the lysosomes of cells. This was followed by targeting of mitochondria by released DOX-TPP and led to tumor cell apoptosis (Figure 1).

MATERIALS AND METHODS Materials. Doxorubicin Hydrochloride (DOX) was purchased from Zhejiang Hisun Pharmaceutical

Co.

Ltd.

N-Hydroxysuccinimide(NHS)

(Zhejiang, and

China),

andthe

triphenylphosphine

(TPP),

1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide

hydrochloride (EDC.HCL), Adipic dihydrazide (ADH) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The andydrous DMF was purchased from Shanghai Aladdin Reagent Co,


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Ltd.(Shanghai,China). RPMI-1640 media was purchased from Keno (Hangzhou, China). Fetal bovine serum was purchased from Gibco (USA) and the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Annexin V-FITC/PI apoptosis detection kit, active oxygen detection kit, mitochondrial membrane potential detection kit and caspase 3 activity detection kit were purchased from Beyotime Biotechnology Co (Shanghai, China). Hoechst 33342, Mito-tracker Green FM were purchased from Thermo Fisher Scientific, and the MCF-7/ADR cells were purchased from KeyGen Biotech Co, Ltd (Nanjing, China) . All other chemicals were analytical grade. Synthesis and Characterization of HA-hydra-DOX-TPP. DOX-TPP (80 mg) and Adipic acid dihydrazide (ADH, 72 mg) dissolved in 15 mL anhydrous methanol formed a suspension. After reacting for 30 min, 100 µL trifluoroacetic acid was added, then stirred with nitrogen protection overnight. The crude product was obtained by removing methanol under vacuum. Unreacted ADH was removed by centrifugation using ethanol because ADH is insoluble in ethanol. Finally, the ethanol layer was steamed again to obtain the red product ADH-DOX-TPP. HA

(45

mg)

was

dissolved

1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide

in

distilled

hydrochloride

water (EDC,

(15 23

mg)

mL). and

N-hydroxysuccinimide (NHS, 14 mg) were dissolved in distilled water (1 mL), which was added to HA solution dropwise, then pH was adjusted to 5.0 and reacted for 2 h to activate the carboxyl groups of HA. DOX-TPP-ADH (62 mg) in methanol (45 mL) was added to the above solution dropwise and reacted under pH 7.4. After reacting overnight, the reaction solution was poured into dialysis tube (MWCO 8000-14000 Da) and dialyzed against a large excess amount of deionized water overnight. Then it was lyophilized and stored at 4°C. The structural characterization of the freeze-dried product was confirmed by MASS spectroscopy(Agilent 1290 HPLC-6224 Time of Flight Mass Spectrometer), 1H NMR in D2O, and FTIR (FIIR-4100, JASCO) in the frequency range of 4000 to 500 cm−1 (KBr pellet). The particle size and zeta potential were analyzed in deionized water by dynamic light scanning (DLS) analysis using Zetaszier Nano-S90 (Malvern instruments, Ltd, UK). Morphologies of HA-hydra-DOX-TPP was observed under transmission electron microscopy (JEM1200EX, Japan). For

blue

fluorescence

labelling

of

HA-hydra-DOX-TPP,

HA-hydra-DOX-TPP and

2-Butyl(4-aminohexyl)-6-(dimethylamino)-1H-benz[de]isoquinoline-1,3(2H)-dione


(kindly


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provided by Dr. Xin Li, Zhejiang university, China) were dissolved in water, and EDC/NHS was then added at pH 7.0 with stirring overnight away from light. Blue-HA was then obtained by dialysis against deionised water, frozen and lyophilised. In vitro release. In this study, the release of DOX-TPP from HA-hydra-DOX-TPP nanoparticles was determined by dialysis bag method. Because of the pH sensitive characteristics of the hydrazine bond, the release experiments were investigated at pH 7.4 and pH 5.0. For each release study, 1 mL of HA-hydra-DOX-TPP solution at a concentration of 200 µg/mL was put into a dialysis tube (MWCO:8000-14000 Da) which was immersed into 25 mL PBS solution (pH=7.4 or 5.0) directly. Then, it was shaken at 37 °C in thermostatic oscillator. Samples from the dialysis solution (1 mL) were taken at different time points and replaced with fresh media (1 mL). The cumulative

release

of DOX-TPP was

subjected

for

determination

by fluorescence

spectrophotometer with the excitation wave of 480 nm and emission wave of 550 nm. Intracellular distribution. MCF-7/ADR cells were seeded in the chamber of the coverslips at a density of 2×104 cells/well and cultured for 24 h at 37 °C in 5% CO2. After that, the medium was removed and the fresh culture medium containing 8.6 µM drug was added. After incubation for 1, 4 and 16 h, Hoechst 33342 was added to cells for 30 min. Then phenol red free RPMI-1640 medium was used to clean the cells 3 times. After this, 140 nM Mito-tracker Green FM solution was added to stain cells for 40 min followed by rinsing with phenol red free RPMI-1640 medium, and addition of PBS buffer containing 10% fetal bovine serum. Finally, the distribution of drugs in MCF-7/ADR cells was observed under confocal microscopy. The extent of colocalization of two labels was measured by "Manders' overlap coeffecient (R)", which were calculated using image-Pro plus (Version 6.0.0.260; Media cybernetics, Inc. USA). R is proportional to the amount of fluorescence of the co-localizing pixels or voxels in each color channel, indicating the true degree of co-localization41-44. Furthermore, intracellular distribution of DOX in drug-sensitive MCF-7 cells was also evaluated to confirm the organelle targeting of HA-hydra-DOX-TPP. Cellular uptake studies. MCF-7/ADR cells were seeded in 6 well plates at a density of 105 cells/well and cultured for 48 h at 37 °C in 5% CO2. Then, the cells were further treated with free DOX solution or HA-hydra-DOX-TPP for 4 h. After incubation in the incubator, the cells were washed with ice PBS for three times. Then cell lysis buffer containing 1% Triton-100 was added overnight, and the centrifuged supernatant was taken to determine intracellular drug uptake


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measured by a microplate reader. Cytotoxicity assay in vitro. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate the in vitro cytotoxicity. First, MCF-7/ADR cells were seeded at a density of 104 cells/well into 96-well culture plates and cultured for 24 h at 37 °C in 5% CO2. Then the medium was discarded and the culture medium (150 µL) was added, and the final drug concentration was 86, 43, 22, 11, 5 and 0.7 µM, respectively. After incubation for 48 h, the medium were changed with fresh culture medium containing MTT solution (5 mg/mL). The incubation was continued at 37°C for 4 h. Then, the medium was discarded and DMSO was added to dissolve the formazan crystal formed by living cells. The absorbance was measured at 570 nm using a microplate reader. Finally, the relative cell viability was calculated as follows: cell viability (%) = OD570samples/ OD 570control×100. In vivo anti-tumour assays in mice model. Nude female BALB/c mice (4 weeks old) were fed water containing 0.3 mg/ml estradiol for one week, and then received a subcutaneous injection of 0.1 mL of MCF-7/ADR cancer cell suspension containing 107 cells on the right forelimb. When the tumor volume was bigger than 50 mm3, the tumour-bearing mice were intravenously administered either physiological saline, DOX solution, DOX-TPP or HA-hydra-DOX-TPP (equivalent DOX concentration, 4 mg/kg) every 2 days with six mice in each group. Eighteen days after initiation of treatment, the body weight and tumor dimensions were recorded. The tumor volume was calculated using volume = (width2 × length)/2. After the end of the experiment, the tumor tissue of nude mice were taken and fixed for 48 h in 4% formaldehyde solution in paraffin sections, and were detected by TUNEL immunohistochemical staining. In a separate experiment, the tumour-bearing mice were given PBS and equivalent 4 mg/kg DOX of free DOX solution or HA-hydra-DOX-TPP by intraperitoneal injection once every 2 days for 10 times. At the end of treatment, the mice were euthanized, and the DOX accumulation in the tumor site was observed by confocal microscopy. The heart, liver, spleen, lung, kidney and tumor tissue of nude mice were taken and fixed for 48 h in 4% formaldehyde solution for paraffin sections followed by H&E analysis.

Results and discussion Synthesis and characterization of HA-hydra-DOX-TPP. The HA-hydra-DOX-TPP was



synthesized as depicted in Figure 2. Briefly, the carbonyl group (C=O) of DOX is condensed with the amino group (-NH2) of hydrazine on ADH and formed the hydrazone bond (-C=N-) in DOX-TPP firstly. With the addition of the activated EDC and NHS, the amino group (-NH2) in ADH-DOX-TPP is condensed into carboxyl (-COOH) of hyaluronic acid to form amide bond (-CO-NH-), and finally HA-hydra-DOX-TPP is synthesized. The structural characterization of DOX-TPP and HA-hydra-DOX-TPP were confirmed by 1H NMR in D2O (Figure 3). The characteristic peaks at 7.5 to 7.8 ppm belong to protons of phenyl rings of TPP, the signals ranging from 3.0-4.0 ppm belong to HA, and the peaks at 1.0 ppm was attributed to the protons of CH3 of the DOX. The peaks of TPP and DOX spectra were also observed in DOX-TPP 1H NMR, which proved the formation of DOX-TPP conjugate. Besides, the peaks of TPP, DOX and HA spectra were all observed in HA-hydra-DOX-TPP 1H NMR, which confirmed HA-hydra-DOX-TPP was synthesized successfully. Compared to DOX-TPP, ADH-DOX-TPP with more saturated alkyl groups demonstrated stronerg absorption at 3000cm-1 in IR spectrum (Figure 4). In addition, the strong vibration peak at 1680cm-1 confirmed the newly formed amido bond and hydrazone bond (Figure 4). These data together with MASS characteristics of DOX [M+1: 544.1211], DOX-TPP [M+1: 874.2932] and ADH-DOX-TPP [M+1:1030.40] (Figure 5), confirmed the successful synthesis of ADH-DOX-TPP. Particle size, charge and TEM analysis of HA-hydra-DOX-TPP nanoparticles. HA-hydra-DOX-TPP was well dispersed with particle size around 192 nm determined by DLS (Figure 6 a). The experiment indicated that the zeta potential of HA-hydra-DOX-TPP was -28.8 mV (Figure 6 b). The negative charge on the surface of the nanoparticles is favorable for the long circulation in vivo and accumulation into the tumors via EPR effect. As observed under TEM (Figure 6 c), HA-hydra-DOX-TPP is spherical, perhaps with DOX-TPP self-assembly as hydrophobic core and HA as hydrophilic outer layer spontaneously. After DOX-TPP was removed, the spherical nanoparticle dis-assembled (Figure 6 d). The positive charge of TPP can encourage electrostatic interaction with the negative charge carried by HA, which makes the nanoparticles closely intertwined and improves the stability of the nano preparation. In vitro release. DOX-TPP release profile at different pHs was evaluated (Figure 6 e). pH 5.0 and 7.4 were used to simulate the intracellular acidic and physiological conditions. The result showed that the DOX-TPP released faster at pH 5.0 than pH 7.4. At 10 h, 70% DOX-TPP released


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at pH 5.0, while only 35% DOX-TPP released at pH 7.4. At 48h, 80% of the DOX-TPP released at pH 5.0,. The incomplete release of DOX-TPP may be attributed to the electrostatic interactions between the positive charge of DOX-TPP and the negative charge of HA group. Therefore, HA-hydra-DOX-TPP is expected to be relatively stable under physiological conditions (pH 7.4). However, when nanoparticles entered the acidic lysosome (pH 5.0), the hydrazone bond which links DOX-TPP with HA can be hydrolyzed causing the release of DOX-TPP rapidly. Many studies with pH microelectrodes have demonstrated lower pH values in tumors relative to normal tissues. pH triggered delivery is regarded as the most general strategy, targeting the acidic extracellular microenvironment and intracellular organelles of solid tumors. These Characteristic contribute to decreasing side effect in normal tissue and increased drug release in tumor cells. Intracellular distribution in vitro. We have demonstrated that DOX-TPP has the function of mitochondrial targeting before.1 In this study, we further explored the distribution of HA-hydra-DOX-TPP in cells and the intracellular release of DOX-TPP from this structure. The distribution of HA-hydra-DOX-TPP in MCF-7/ADR cells is shown in Figure 7. At 1 h, most of the red fluorescence related to HA-hydro-DOX-TPP was distributed in the cytoplasm, and some yellow fluorescence was observed, indicating that most of the HA-hydra-DOX-TPP had not yet achieved lysosome escape, and only a small amount of DOX-TPP was released from the HA-hydra-DOX-TPP and accumulated in the mitochondria. After 4 h, significant changes in yellow fluorescence were observed, indicating that more drug released from lysosomes and targeted mitochondria. DOX-TPP was also observed to show the same phenomenon. At 1 h, significant yellow fluorescent spots were observed in the picture. As the incubation time prolonged, the number of yellow fluorescence increased, indicating that DOX-TPP was targeted to mitochondria. Neither mitochondria nor nucleus was co-localized with the fluorescence of free DOX, indicating that free DOX did not enter into mitochondria and nucleus in MCF-7/ADR cells. In addition, little free DOX located in mitochondria of MCF-7 cells, and most of DOX entered into the nucleus of MCF-7 cells, which is different from that in MCF-7/ADR cells as drug resistant MCF-7/ADR cells can't allow access of free DOX to nucleus. In contrast, HA-hydra-DOX-TPP could hardly distribute into nucleus, but mainly transported to mitochondria, which is similar to HA-hydra-DOX-TPP in MCF-7/ADR, further confirming the mitochondria targeting of HA-hydra-DOX-TPP (Figure S1).



Furthermore, in order to investigate the release of DOX-TPP from HA-hydra-DOX-TPP nanocarriers, HA-hydra-DOX-TPP (Blue-HA) was labeled with blue fluorescent small molecule and then incubated with cells followed by observation under confocal microscopy. At 4 h, most of blue fluorescence and red fluorescence co-localized to show magenta fluorescence, implying co-localization of DOX and HA. A part of red fluorescence and green fluorescence were observed to show yellow fluorescence indicating that a portion of DOX-TPP is released from the vector and targeted to mitochondria. At 12h, most of green fluorescence and red fluorescence coincided shoing higher yellow fluorescence, indicating release of DOX-TPP and its localization in the mitochondria. Moreover, at 12 h much more free blue fluorescence was observed, which again implied the release of DOX from its HA conjugate (Figure 8). Cellular Uptake Study. HA-hydra-DOX-TPP was incubated with MCF-7/ADR cells for 4 h, and cell uptake was measured by a fluorescence microplate reader (Figure 9 a). When the drug concentration was 8.6 µM, the cell accumulation of HA-hydra-DOX-TPP was significantly higher than that of free DOX. At this concentration, the cellular level of free DOX was too low to be detected, As the drug concentration increased to 17.2 µM, cellular level of HA-hydra-DOX-TPP increased significantly and was 7 times that of the DOX. The results showed that HA-hydra-DOX-TPP can accumulate more in the cells when compared to free DOX. Due to the modification of DOX with TPP, P-gp may be avoided and the efflux of the DOX can be reduced. Meanwhile, HA (4mg/mL) pre-incubation inhibit uptake of HA nanoparticle and implied the role of CD44-mediated uptake in MCF-7/ADR cells for HA-hydra-DOX-TPP (Figure 10). No cytotoxicity was observed for HA in MCF-7/ADR cells at the 4 mg/mL concentration (Figure S2). Cytotoxicity assay in vitro. The HA-hydra-DOX-TPP showed concentration-dependent cytotoxicity in MCF-7/ADR cells (Figure 9 b).The cytotoxicity of HA-hydra-DOX-TPP was higher than that of DOX group, especially at the concentration of 86 µM, whose cell survival rate was 28%, while cell survival rate was 50% for DOX treatment, which suggested that mitochondrial targeting could induce apoptosis and enhance cytotoxicity. Anti-tumor effect in vivo. The tumor tissue was frozen and sliced to observe the accumulation of drugs in the tumor site after nude mice were treated with PBS, DOX or HA-hydra-DOX-TPP, and the red fluorescence of tumor tissue treated with HA-hydra-DOX-TPP was significantly stronger than that in free DOX group, indicating a prolonged the half life in the blood led to


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increased accumulation of DOX in tumor tissue by EPR effect (Figure 11). The tumour volumes and body weights were measured every 2 days up to 18 days to evaluate the in vivo anti-tumour efficacy of HA-hydra-DOX-TPP in nude mice bearing MCF-7/ADR tumours (Figure 12 a, b). Tumours in mice receiving HA-hydra-DOX-TPP grew up to 107.4 mm3 and were significantly smaller than those in mice that received free DOX solution (181.3 mm3) or DOX-TPP solution (180.9 mm3), while the tumour volume was as large as 263.5 mm3 for the control group (physiological saline treatment), demonstrating that HA-hydra-DOX-TPP exhibited a favourable anti-tumour effect in vivo. In addition, the body weights decreased gradually during the injection with free DOX due to the serious side effects of DOX. In addition, immunohistochemical staining was performed by Tunel assay to investigate the apoptosis of the tumor site, which is widely used in the detection of apoptosis by reflecting the breakdown of DNA in cells. In the Tunel immunohistochemistry, the nucleus of the cells that were apoptotic were stained brown, as shown in Figure 12 c. The result was that HA-hydra-DOX-TPP group had more apoptotic cells than DOX group. Heart, liver, spleen, lung, kidney, tumor and other tissue morphological changes was evaluated by H&E staining as shown in Figure 13, and the results showed that nude mice treated with DOX group suffered from heart muscle cracks, myocardial distortions, and the emergence of empty structure. Nude mice liver showed significant necrosis and inflammation (the Figure part of the dashed line of the liver), indicating that DOX group caused severe heart and liver damage. The tissue morphology of HA-hydra-DOX-TPP group was normal, and there was no significant difference with the blank control group. It was proved that hyaluronic acid nano-preparation was safe in vivo when compared to free DOX. For tumor tissue, in the animals that received DOX, HA-hydra-DOX-TPP nuclear condensation and dissolution of tumor cells was observed, indicating tumor cell apoptosis. Conclusions In this study, the water-soluble polysaccharide HA and DOX-TPP were conjugated by hydrazone bond, then self-assembled to form spherical nanoparticles. The prepared nanoparticles showed to have good biocompatibility and tumor target ability. In addition, the hydrazone bond was pH-sensitive, so the in vitro release of HA-hydra-DOX-TPP was acid triggered which had led to



gradual release of DOX-TPP and its accumulation in the mitochondria of MCF/ADR cells within 12h. Furthermore, HA-hydra-DOX-TPP showed increase DOX accumulation and cytotoxicity than that of free DOX, in MCF7/ADR cells, in vitro, which was coincided with its improved tumor accumulation, anti-cancer activity and safety profile compared to free DOX MCF-7/ADR bearing mice, in vivo. Acknowledgements This study was supported by National Basic Research Program of China (No. 2014CB931901), National Natural Science Foundation of China (No. 81373346, 81572952, 81673022), Zhejiang Provincial Natural Science Foundation of China (No. LY15H300001, LY17H160013), Fundamental Research Funds for the Central Universities (2017XZZX011-04), and Zhejiang Medical and Health Science and Technology Plan Project (2016KYB109).

Supporting Information Intracellular distribution of DOX in MCF-7 cells after being incubated with 8.6µM DOX or HA-hydra-DOX-TPP for 3h, and in vitro cytotoxicity of hyaluronic acid (HA) against MCF-7/ADR cells during 48 h.


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Han, M.; Vakili, M. R.; Soleymani Abyaneh, H.; Molavi, O.; Lai, R.; Lavasanifar, A.

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Figure Legends Figure 1. Schematic illustration to show the transport of HA-hydra-DOX-TPP in tumour cells. Figure 2. The synthesis path of HA-hydra-DOX-TPP. Figure 3. 1H nuclear magnetic resonance (NMR) spectra of ADH, DOX, ADH-DOX-TPP, HA, DOX-TPP and HA-hydra-DOX-TPP in D2O. Figure 4. Fourier Transform Infrared Spectrometer (FTIR) spectra of DOX-TPP and ADH-DOX-TPP. Figure 5. MASS spectra of DOX, DOX-TPP and ADH-DOX-TPP. Figure 6. Characterisation of HA-hydra-DOX-TPP. a) The size distribution of HA-hydra-DOX-TPP measured by dynamic light scattering (DLS). b) Zeta potential of HA-hydra-DOX-TPP. c) Morphology of HA-hydra-DOX-TPP observed under Transmission Electron Microscope (TEM). d) Morphology of HA-hydra-DOX-TPP after it was dialyzed against excess PBS (pH 5.0) to remove DOX-TPP. e) The release profiles of DOX-TPP from HA-hydra-DOX-TPP in PBS (pH 5.0 or pH 7.4) at 37°C during a 48 h period in vitro. Figure 7. Intracellular distribution of DOX in MCF-7/ADR cells after being incubated with 8.6µM DOX or HA-hydra-DOX-TPP at 37 °C for 1h(a) or 4h(b), and the intracellular mitochondria were stained by MitoTracker Green followed by nucleus staining with Hoechst 33342. The cells were then observed under confocal laser scanning microscope. The overlap between the fluorescence of DOX (red) and MitoTracker (green) appears as yellow and shows distribution of DOX in the mitochondria of cells, the scale bar represents 25 µm. The colocalization in MCF-7/ADR cells of mitochondria (green) and DOX fluorescence (red) is shown as Manders' overlap coeffecient (R) in the right-hand column, which indicates an actual overlap of the signals that is considered to represent the true degree of colocalization. Figure 8. Intracellular release of DOX-TPP after cells being exposed to blue fluorescence labeled HA-hydra-DOX-TPP (8.6µM) at 37 °C for 4h or 12h. And the intracellular mitochondria were stained by MitoTracker Green. Figure 9. a) Cellular uptake of DOX and HA-hydra-DOX-TPP in MCF-7/ADR cells. BDL means Below determination limit, **p < 0.01(n=3). b) In vitro cytotoxicity of DOX and HA-hydra-DOX-TPP against MCF-7/ADR cells during 48 h as determined by the MTT assay, Each value represents mean±SD (n=3). *p < 0.05, **p < 0.01 (n=3). Figure 10. Cellular uptake of HA-hydra-DOX-TPP in MCF-7/ADR cells. MCF-7/ADR cells were seeded into 6-well plates at a density of 1×105 cells/well and cultured for 24 h. And was then incubated with HA-hydra-DOX-TPP (8.6 µM) (A), and HA-hydra-DOX-TPP (8.6 µM) together with the cells pre-incubated with HA solution (4mg/mL) overnight (B). The cells were then washed with ice-cold PBS twice, and trypsinised and finally examined by flow cytometric


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analyses (FC500MCL, Beckman Coulter, USA). 1.2% and 0.2% in the figure indicate the percentage of fluorescence incidence, i.e. the percentage of tumor cells positive for DOX. Figure 11. Accumulation of DOX and HA-hydra-DOX-TPP in tumor tissue after injection of DOX solution and HA-hydra-DOX-TPP on nude mice bearing MCF-7/ADR tumors every 2 days for 9 times at a dosage of 4.0mg/kg DOX (n=6). Figure 12. Anti-tumour effects in nude mice bearing MCF-7/ADR tumours after being treated with DOX, DOX-TPP and HA-hydra-DOX-TPP. Tumour growth (a) and body weight (b) curves after intravenous administration of DOX, DOX-TPP and HA-hydra-DOX-TPP on nude mice bearing MCF-7/ADR tumours every 2 days for 9 times at a dosage of 4.0 mg/kg of DOX (n=6) when the tumour reached a size of 50 mm3 (n=5), and representative images of TUNEL assay xenografted tumours (×20) (C). Figure 13. Histopathological analysis of tissue sections stained with hematoxylin and eosin (H&E, ×20) after mice were administrated with DOX or HA-hydra-DOX-TPP.

For Table of Contents Use Only

Mitochondrial targeted doxorubicin-triphenylphosphonium delivered by hyaluronic acid modified and pH responsive nano-carriers to breast tumor: in vitro and in vivo studies Hui-Na Liu1,#, Ning-Ning Guo1,#, Tian-Tian Wang1, Wang-Wei Guo1, Meng-Ting Lin1, Ming-Yi Huang-Fu1, Mohammad Reza Vakili2, Wen-Hong Xu3, Jie-Jian Chen3, Qi-Chun Wei3, Min Han1,*, Afsaneh Lavasanifar2,*, Jian-Qing Gao1,*.

Table of Contents Graphic




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Figure 1. Schematic illustration to show the transport of HA-hydra-DOX-TPP in tumour cells. 193x135mm (96 x 96 DPI)



Figure 2. The synthesis path of HA-hydra-DOX-TPP. 503x329mm (96 x 96 DPI)


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Figure 3. 1H nuclear magnetic resonance (NMR) spectra of ADH, DOX, ADH-DOX-TPP, HA, DOX-TPP and HAhydra-DOX-TPP in D2O. 352x289mm (96 x 96 DPI)



Figure 4. Fourier Transform Infrared Spectrometer (FTIR) spectra of DOX-TPP and ADH-DOX-TPP. 269x189mm (96 x 96 DPI)


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Figure 5. MASS spectra of DOX, DOX-TPP and ADH-DOX-TPP. 275x322mm (96 x 96 DPI)



Figure 6. Characterisation of HA-hydra-DOX-TPP. a) The size distribution of HA-hydra-DOX-TPP measured by dynamic light scattering (DLS). b) Zeta potential of HA-hydra-DOX-TPP. c) Morphology of HA-hydra-DOXTPP observed under Transmission Electron Microscope (TEM). d) Morphology of HA-hydra-DOX-TPP after it was dialyzed against excess PBS (pH 5.0) to remove DOX-TPP. e) The release profiles of DOX-TPP from HAhydra-DOX-TPP in PBS (pH 5.0 or pH 7.4) at 37°C during a 48 h period in vitro. 336x126mm (96 x 96 DPI)


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Figure 7. Intracellular distribution of DOX in MCF-7/ADR cells after being incubated with 8.6µM DOX or HAhydra-DOX-TPP at 37 °C for 1h(a) or 4h(b), and the intracellular mitochondria were stained by MitoTracker Green followed by nucleus staining with Hoechst 33342. The cells were then observed under confocal laser scanning microscope. The overlap between the fluorescence of DOX (red) and MitoTracker (green) appears as yellow and shows distribution of DOX in the mitochondria of cells, the scale bar represents 25 µm. The colocalization in MCF-7/ADR cells of mitochondria (green) and DOX fluorescence (red) is shown as Manders' overlap coeffecient (R) in the right-hand column, which indicates an actual overlap of the signals that is considered to represent the true degree of colocalization. 279x190mm (96 x 96 DPI)



Figure 8. Intracellular release of DOX-TPP after cells being exposed to blue fluorescence labeled HA-hydraDOX-TPP (8.6µM) at 37 °C for 4h or 12h. And the intracellular mitochondria were stained by MitoTracker Green. 421x218mm (96 x 96 DPI)


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Figure 9. a) Cellular uptake of DOX and HA-hydra-DOX-TPP in MCF-7/ADR cells. BDL means Below determination limit, **p < 0.01(n = 3). b) In vitro cytotoxicity of DOX and HA-hydra-DOX-TPP against MCF7/ADR cells during 48 h as determined by the MTT assay, Each value represents mean±SD (n = 3). *p < 0.05, **p < 0.01 (n = 3). 668x273mm (96 x 96 DPI)



Figure 10. Cellular uptake of HA-hydra-DOX-TPP in MCF-7/ADR cells. MCF-7/ADR cells were seeded into 6well plates at a density of 1×105 cells/well and cultured for 24 h. And was then incubated with HA-hydraDOX-TPP (8.6 µM) (A), and HA-hydra-DOX-TPP (8.6 µM) together with the cells pre-incubated with HA solution (4mg/mL) overnight (B). The cells were then washed with ice-cold PBS twice, and trypsinised and finally examined by flow cytometric analyses (FC500MCL, Beckman Coulter, USA). 1.2% and 0.2% in the figure indicate the percentage of fluorescence incidence, i.e. the percentage of tumor cells positive for DOX. 119x54mm (96 x 96 DPI)


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Figure 11. Accumulation of DOX and HA-hydra-DOX-TPP in tumor tissue after injection of DOX solution and HA-hydra-DOX-TPP on nude mice bearing MCF-7/ADR tumors every 2 days for 9 times at a dosage of 4.0mg/kg DOX (n=6). 179x94mm (96 x 96 DPI)



Figure 12. Anti-tumour effects in nude mice bearing MCF-7/ADR tumours after being treated with DOX, DOX-TPP and HA-hydra-DOX-TPP. Tumour growth (a) and body weight (b) curves after intravenous administration of DOX, DOX-TPP and HA-hydra-DOX-TPP on nude mice bearing MCF-7/ADR tumours every 2 days for 9 times at a dosage of 4.0 mg/kg of DOX (n=6) when the tumour reached a size of 50 mm3 (n=5), and representative images of TUNEL assay xenografted tumours (×20) (C). 329x190mm (96 x 96 DPI)


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Figure 13. Histopathological analysis of tissue sections stained with hematoxylin and eosin (H&E, ×20) after mice were administrated with DOX or HA-hydra-DOX-TPP. 678x284mm (96 x 96 DPI)



219x150mm (96 x 96 DPI)


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Mitochondrial Targeted Doxorubicin ... - ACS Publications

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