Sialic Acid-functionalized pH-triggered Micelles for Enhanced Tumor

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Biological and Medical Applications of Materials and Interfaces

Sialic Acid-functionalized pH-triggered Micelles for Enhanced Tumor Tissue Accumulation and Active Cellular Internalization of Orthotopic Hepatocarcinoma Xiao-Ling Xu, Kong-Jun Lu, Meng-Lu Zhu, Yang-Long Du, Ya-fang Zhu, NanNan Zhang, Xiao-Juan Wang, Xu-Qi Kang, De-Min Xu, Xiao-Ying Ying, Risheng Yu, Chen-Ying Lu, Jiansong Ji, Jian You, and Yong-Zhong Du ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 04 Sep 2018 Downloaded from http://pubs.acs.org on September 4, 2018

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ACS Applied Materials & Interfaces

Sialic Acid-functionalized pH-triggered Micelles for Enhanced Tumor Tissue Accumulation and Active Cellular Internalization of Orthotopic Hepatocarcinoma Xiao-Ling Xu1, Kong-Jun Lu1, Meng-Lu Zhu2, Yang-Long Du1, Ya-Fang Zhu1, Nan-Nan Zhang3, Xiao-Juan Wang1, Xu-Qi Kang1, De-Min Xu4, Xiao-Ying Ying1, Ri-Sheng Yu4, Chen-Ying Lu3,4, Jian-Song Ji3*, Jian You1, Yong-Zhong Du1* 1

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

Hangzhou 310058, PR China; 2

Department of Pharmacy, The Fourth Affiliated Hospital, Zhejiang University

School of Medicine, Yiwu 322000, PR China; 3

Lishui Hospital, Zhejiang University School of Medicine, Lishui 323000, PR China

4

Department of Radiology, The Second Affiliated Hospital, Zhejiang University

School of Medicine, Hangzhou 310009, PR China;

(1) Correspondence should be addressed to the following: Dr. Y. Z. Du (Institute of Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University), 866 Yu-Hang-Tang Road, Hangzhou, 310058, China. Tel: +86-571-88208435; Fax: +86-571-88208439. E-mail: [email protected] Dr. J.S. Ji (Department of Radiology, Lishui Hospital, Zhejiang University School of Medicine), Tel: +86-578-2285011; Fax: +86-578-2285011; E-mail:[email protected]

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ABSTRACT Both targeted and stimuli-sensitive drug delivery systems (DDSs) have been developed to augment anti-tumor effects. However, lack of knowledge regarding tumor tissue targeting and different effects of the stimuli-sensitive DDSs in the orthotropic and ectopic tumors have impeded further advances in their clinical applications. Herein, we first reported a pH-triggered micelle with sialic acid-driven targeting ability (Sialic acid-PEG-hydrazone linker-Doxorubicin, SPD). The SPD micelles encapsulated with DOX (SPDD) showed sustained drug release over 48h in response to the pH gradient in vivo–slow under physical conditions and accelerated in the acid tumor microenvironment. In addition, the SPD micelles showed 2.3-fold higher accumulation in tumors after 48h compared to the micelles lacking the SA moiety. The overexpression of E-selectin on the inflammatory vascular endothelial cells surrounding tumors increased the accumulation of SPD micelles in tumor tissues, while that on the tumor cells increased the internalization of micelles. Consequently, SPDD micelles exerted remarkable anti-tumor effects in both orthotopic and ectopic models. Application of SPDD micelles in situ model reduced tumor volume (77.57 mm3 versus 62.13 mm3) and metastasis after treatments for 25 days. These results suggest that SA-driven targeted DDS with a pH-responsive switch has the potential to treat hepatocarcinoma effectively both ectopically and orthotopically. Keywords: E-selectin, sialic acid, pH-responsive, Hepatocarcinoma, Orthotopic


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INTRODUCTION

Hepatocarcinoma is the most common cancer in humans, and ranks fifth in terms of worldwide occurrence. The incidence and mortality are significantly high and have been increasing annually1. In China, around 383,000 people die of hepatocellular carcinoma every year, accounting for 51% of all liver cancer related deaths in the world2. Doxorubicin (DOX) is commonly used in the treatment of liver cancer. However, due to lack of tumor-specific targeting, it often produces serious side effects which limits its clinical use. Therefore, it is essential to develop novel technologies for rapid delivery of high-dose DOX into hepatoma cells, so that tumor cell clearance can be efficiently improved. Drug delivery systems (DDSs) have been central to mainstream tumor therapy since the early 1990s3-5. Due the “leaky” vasculature in tumors6-8, DDSs often extravasate through the tumor capillaries which alters the bio-distribution of drugs and results in a higher therapeutic index. Furthermore, by surface modification of targeting moiety, the accumulation of DDSs can be increased in the tumor tissues9-10. However, contrary to expectations, DDSs have not remarkably improved the anti-tumor efficiency of drugs. The main limitation is that the accumulation of DDS, and therefore of drugs, in tumor tissues is still inadequate. The final drug concentration in tumor cells depends on two factors: its accumulation in tumor tissue, and internalization into tumor cells. The existing targeted DDSs were designed based on the recognition of specific surface receptors overexpressed on tumor cells. These targeted DDSs primarily accumulated in tumor tissue through enhanced permeability



and retention (EPR) effect, due to the incomplete and discontinuous distribution of endothelial cells in the blood vessels of tumors. Only after substantial accumulation in the tumor tissue would these targeted DDSs recognize the tumor cell specific receptors. The modified targeting moiety on DDSs would then accelerate the process of tumor cell internalization. Therefore, these targeted DDSs have only improved the transportation of DDSs into tumor cells, instead of tumor tissue accumulation. In addition, the drug released from DDSs is too slow and incomplete to reach the requisite concentration to kill tumor cells. Therefore, stimuli-responsive DDSs, which are sensitive to different endogenous and exogenous stimuli11, such as pH12-13, redox status14, and temperature15-16, were developed as a promising strategy to enhance drug release during tumor therapy. The pH triggered hydrazone bond (C=NNH2) has been widely incorporated in various stimuli-responsive DDSs17. Ondrej Sedlacek et al18 designed a novel DDS using the acid-sensitive hydrazone bond between poly (2-ethyl-2-oxazoline) and polymer backbone for the treatment of subcutaneous EL4 lymphoma. Similarly, Wei-liang Ye et al19 synthesized a pH sensitive multifunctional micelle for delivering DOX (DOX-hyd-PEG-ALN), which effectively delayed tumor growth, diminished bone loss and reduce cardiac toxicity in tumor-bearing nude mice compared to free DOX. However, stimuli-responsive DDSs have been intensively studied only in ectopic and not in orthotopic tumor models. Since the microenvironment of orthotopic and ectopic xenografts are different, the anti-tumor effect of stimuli-responsive DDSs need to be evaluated in both settings. Sialic acid (SA), an N-acetylneuraminic acid, is a negatively charged 9-carbon


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monosaccharide

presenting

in

the

outermost

layer

of

cell

membrane.

N-acetylgalactosamine or Lewis oligosaccharide X is always linked to SA, which can specifically bind to E-selectin and P-selectin20-21 to promote leukocytes migration and adhesion to the blood vessels. E-selectin, a transmembrane glycoprotein, is highly upregulated on inflammatory vascular endothelial cells22-23 (VECs) and tumor cells24-26. Therefore, DDSs targeting E-selectin can be highly specific nanocarriers for tumor tissues and cells and achieve greater tumor tissue accumulation on account of the inflammatory VECs around tumors. Attempts have been made to introduce SA onto the surface of DDSs25, 27 to enhance their E-selectin targeting properties. SA anchored nanoparticles specifically bind to E-selectin, reduce the inflammatory response28, inhibit phagocytosis of immune cells29, and have been shown to improve the therapeutic effects against acute kidney injury23. But across the SA-based DDSs, concerns about E-selectin-mediated large accumulation both in tumor tissues and tumor cells haven’t been studied. We

therefore

developed

VECs-

and

tumor

cell-dual

targeting

SA-PEG-hydrazone-DOX (SPDD) pH-responsive micelles for both ectopic and orthotropic hepatoma therapy. SA, would serve as a binding agent of E-selectin receptor and achieve large tumor tissue accumulation by the combination between SA and E-selectin on the surface of inflammatory VECs around tumors. After entering the tumor tissue, these micelles would be selectively internalized into the E-selectin overexpressing tumor cells instead of normal cells, resulting in only minor side effects. Furthermore, due to the acid-sensitive hydrazone linker between PEG and DOX, the



drug will be quickly released upon internalization and accumulation inside the tumor cells in a short period of time, thereby exerting a strong anti-tumor effect.

RESULTS AND DISCUSSION Synthesis and characterization of SA-PEG-hyd-DOX H2N-PEG-SA conjugates (0.2088g, 68.66% yield) were first synthesized via substitution and elimination reactions between the hydroxyl group of SA and the amino group of H2N-PEG-NH2 to form a C-N bond. DOX was then conjugated to H2N-PEG-SA via the hydrazone bone to obtain SA-PEG-hyd-DOX (SPD, 0.0846g, 35.4% yield). The synthesis steps are outlined in Scheme 1. The structure of SPD was confirmed by 1H NMR spectrum (Figure 1A, Supporting S1). The peaks at 3.58 ppm corresponding to H2N-PEG-NH2 (-O-CH2CH2-) were observed. The appearance of DOX was verified by the signals at peaks of 1.24 ppm, 1.76 ppm, 3.06 ppm and 4.08 ppm30, while those at 2.01 ppm, 3.73 ppm and 3.98-4.12 ppm confirmed the existence of SA27 in resulting SPD conjugates. Besides, owing to the conjugation of SA and DOX onto the terminal functional groups of H2N-PEG-NH2, peaks of 3.53 ppm in SA spectrum (-CH2-OH) and 4.70 ppm in DOX spectrum (-CH2-OH) were moved to 2.85 ppm and 4.40 ppm in SPD spectrum, respectively. The IR spectrum of SPD (Supporting S2) revealed the characteristic absorption band of the hydrazone bond at 1608 cm-119. During the synthesis of SPD conjugates, a series of options were taken to reduce by-products including controlling the ratio of H2N-PEG-NH2 and SA, dropwise dripping of SA-OTs and controlling reaction time. However, a small amount


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of DOX-PEG-DOX was inevitably obtained in the resultant product. The DOX coupling degree to SPD was 19.5% (calculated by the proton peak areas of H2N-PEG-NH2 (at about 3.58 ppm) and DOX (at about 4.08 ppm) in the 1H NMR spectrum), which was close to the theoretical content of DOX (19.0%). Besides, the polydispersity of the synthesized SPD was 1.36, indicating a relatively uniform distribution of polymers (Supporting S3).

Scheme 1. Synthetic scheme of SA-PEG-hyd-DOX As shown in Figure 1B, SPD and PD could assemble to form micelles at the low critical micelle concentrations (CMC) of 26.52 µg/mL and 21.64 µg/mL respectively.



DOX loaded SPD micelles (SPDD) and PD micelles (PDD) were then prepared by dialysis, and their drug-loading efficiencies (DL %) were estimated at 3.88 % and 4.11 % respectively (Supporting Table S1), respectively. The diameters of SPD (Figure 1C) and PD (Figure 1E) micelles were ~40 nm, smaller than those of DOX-loaded micelles (Figure 1D and 1F). Furthermore, TEM images showed micelles with a sub-100 nm diameter and a uniform, round morphology. The smaller diameter observed by TEM compared to DLS was likely due to the drying and constriction of the micelles during TEM sample preparation. Besides, no micellar structures of SPD and PD conjugates were observed when the pH is no more than 5.0 (Figure S4). It indicated that with the decrease of pH value, the micelles disassembled. The stability of SPD and PD micelles dissolved in 10 % fetal bovine serum (FBS)/phosphate buffer solution (PBS, pH7.4) was explored as a function of time. The time-dependent size and PDI curves of the micelles were shown in Figure S5. The results showed that SPD and PD micelles exhibited excellent stability in a 7-day incubation with 10 % FBS/PBS (pH7.4), as reﬂected by the small changes in size and PDI. The results indicated that SPD had good stability at pH 7.4.


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Figure 1. Preparation and characterization of SPD micelles. (A) 1H-NMR of H2N-PEG-NH2, SA, DOX and SPD. (B) The variations of fluorescence intensity ratio (I1/I3) as a function of the concentration of SPD and PD micelles. Size distribution of blank SPD micelles (C), DOX loaded SPD micelles (D), blank PD micelles (E), and DOX loaded PD micelles (F) obtained by DLS and TEM images (inserted images). (G) In vitro drug release profiles of DOX loaded SPD micelles (Full line) and DOX loaded PD micelles (Dotted line) micelles in pH 7.4 (Blue), pH 6.5(Purple) and pH 5.0 (Red) PBS at 37 ºC.



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The drug release behaviors of SPDD and PDD micelles were analyzed under simulated physiological and pathological conditions (pH 7.4 of blood, pH 6.5 of tumor microenvironment and pH 5.0 of tumor cell lysosomes) at 37 °C. As shown in Figure 1G, DOX released from the micelles was strongly pH-dependent. At pH 7.4, only 34-38% of DOX was released after 48h, indicating the stability of micelles in circulatory process. DOX release was considerably faster in the acidic environments (pH 6.5 and 5.0), and almost 92% of the DOX was released at pH 5.0 in 48 h. The release kinetics of SPDD and PDD micelles were similar, implying that SA modification did not affect drug release.

In vitro anti-tumor activity The cytotoxicity of DOX-loaded micelles and free DOX towards the hepatoma carcinoma

cell

line

Bel-7402

were

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

compared bromide

by

(MTT)

the assay.

Compared to PDD micelles and the commercially available PEG-PLGA/DOX (PPD) micelles, the anti-tumor activity of SPDD micelles was remarkably enhanced as suggested in Figure 2A, 2B. The 50% inhibitory concentration (IC50) value of SPDD micelles was also significantly lower (0.805 µg/mL) compared to that of PDD micelles (2.357 µg/mL), which may be due to SA-driven targeting in the former. In addition, pH-responsive switch enhanced anti-tumor activity of PDD micelles (2.357 µg/mL) in comparison to PPD micelles (5.5549 µg/mL).


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Figure 2. Anti-tumor effects of the DOX-loaded micelles in vitro. (A) Cell viability towards Bel-7402 as different concentrations of different preparations. (B) The 50% cellular inhibition (IC50) value of different preparations. (C) Fluorescence images of alive Bel-7402 cells after incubation with different preparations for 48 h. (D) The semi-quantitative analysis of cell counts of Bel-7402 cells in Figure 2B. (E) Fluorescence images of Bel-7402 cells treated with Nile Red-loaded micelles at 0.5 h,1 h, 2 h, 4 h, 8 h and 12 h, respectively. (F) The semi-quantitative analysis of florescence intensity of Bel-7402 cells in Figure 2E. (G) Fluorescence images of Bel-7402 cells treated with different micelles for 0.5 h and stained with lysotracker and DAPI. (H) Analysis of fluorescence intensity of Bel-7402 cells in Figure 2G.



To visualize the anti-tumor effects of different treatments, viable Bel-7402 cells were stained with Calcein-AM after the respective treatments. As shown in Figure 2C and 2D, the SPDD-treated group had the fewest viable cells, indicating a higher anti-tumor effect than PDD micelles. In addition, presence of the hydrazone bond in PDD micelles decreased the viability of cells compared to ordinary micelles (PPD), which further confirmed the pH switch function of the hydrazone linker. Nile red (NR) is a lipophilic stain. When capsulated in the core of micelles, Nile red will not fluoresce; however, when released from micelles, it can be intensely fluorescent in the lipid-rich environment31. Therefore, the pH-triggered release behavior in Bel-7402 was visualized using the NR loaded micelles. As shown in Figure 2E and 2F, compared to PP/NR micelles, a stronger fluorescence signal was observed following exposure to PD/NR micelles for different durations, suggesting that PD micelles had undergone acid-catalyzed breakage at the hydrazone site. Furthermore, SPD micelles displayed the strongest intracellular DOX fluorescence intensity at each time point, which maybe result from the modification of SA. Micelles are transported to the lysosomes by endocytosis, which makes the acid-labile linker broken possible in the low pH environment and the DOX is released into the cytosol and then trafficked to the nuclei32. Figure 2G showed that SPD micelles were largely accumulated in and co-located with lysosomes in comparison to PD micelles (Figure 2H), thus ensuring the release of DOX from micelles. To elucidate the targeting ability of SPD micelles in vitro, SPD and PD micelles were incubated with the human hepatic cell line LO2 and human hepatic tumor cell


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line Bel-7402 for 0.5 h, 1 h, 2 h, 4 h. Untreated cells were used as negative controls. The internalization of micelles was observed by confocal laser scanning microscopy. A stronger fluorescence signal was detected in Bel-7402 cells incubated with SPD micelles than those exposed to PD micelles (Figure 3A). However, the fluorescence intensities of SPD and PD micelles co-cultured with LO2 cells were similar. Besides, Bel-7402 cells incubated with SPD micelles showed stronger fluorescence signal than LO2 cells incubated with SPD micelles. Whereas the fluorescence intensities of PD micelles co-cultured with Bel-7402 and LO2 cells were similar. These findings were also confirmed by quantitative flow cytometric analysis. (Figure 3B). The fluorescence intensity of the negative control was 4.42 and was considered background auto-fluorescence. The mean fluorescence intensities of the Bel-7402 cells incubated with SPD micelles were 27, 35.4, 50.2 and 58.13 at the four respective time points, and higher than those of the other three groups at each time point. Taken together, we observed a strong internalization of SPD micelles in Bel-7402 cells. To determine whether SA mediated targeting improved the cellular uptake of micelles, Bel-7402 cells were pre-incubated with different concentrations of free SA which competed with SPD micelles. Untreated cells were used as negative control and those incubated with SPD micelles without free SA pre-treatment as the positive control. Compared to the positive control, the fluorescence intensity indicating cellular uptake decreased by 11.9% and 38.9% respectively after pre-incubation with 2 mg/ml and 6 mg/ml free SA. This suggested that the greater internalization of SPD micelles could be attributed to SA incorporation.



Figure 3. Evaluation on the targeting capability of SPD micelles in vitro. (A) Fluorescence images of the human hepatic cell LO2 and human hepatic tumor cell Bel-7402 incubated with SPD and PD micelles for 0.5 h, 1 h, 2 h, 4 h, respectively. (B) Fluorescence intensity measured by flow cytometry when LO2 and Bel-7402 cells were treated with SPD and PD micelles for 0.5 h, 1 h, 2 h, 4 h, respectively. (C) Fluorescence intensity obtained by flow cytometry when Bel-7402 cells were incubated with free SA (2.0 mg/mL, 6.0 mg/mL) beforehand and then treated with SPD and PD micelles for 1 h, respectively. (D) Fluorescence images of Bel-7402 cells exposed to SPD and PD micelles. The red staining showed micelles. The green channel showed E-selectin receptor localization. (E) The process of E-selectin docking with SA. To further verify the specificity of SA moiety to the surface E-selectin on


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Bel-7402 cells, we demonstrated the co-localization of E-selectin and SPD micelles in the cells. As shown in Figure 3D, in addition to the higher cellular uptake compared to the PD micelles, SPD micelles also co-localized with the E-selectin. Taken together, SA improved the cellular uptake of micelles via the E-selectin receptors. The process of E-selectin docking with SA is shown in Figure 3E.

In vivo targeting and pH-triggering The in vivo targeting efficiency of SPD was evaluated in a Bel-7402 tumor-bearing nude mouse model. As shown in Figure 4A, the fluorescent intensities of the tumors increased following injection of the different indocyanin green (ICG)-loaded micellar preparations and peaked at 24 h. However, a significantly stronger fluorescent signal was observed in the SPD/ICG treated group compared to the PD/ICG treated group, reflecting more accumulation of SPD/ICG micelles in the tumor tissues. The mice were sacrificed 48h after injection, and the fluorescent images of the major organs were displayed in Figure 4B. While the fluorescence signal of the tumors in SPD/ICG group were 2.3-fold stronger than that in the PD/ICG group, the signal in the liver was 0.52-fold weaker, indicating the tumor-selective accumulation of SPD/ICG micelles.

The quantitative analysis of fluorescence

intensity in collected tissues further demonstrated the enhanced accumulation of the SPD/ICG micelles into tumors (Figure 4C). In addition, the DOX concentration in the tumors peaked at 24h and then declined, being consistent with that of the ICG loaded micelles. (Figure 4D, 4E). Various pro-inflammatory factors trigger the overexpression of E-selectin on the surface of VECs surrounding the tumors33, which



are then released into the tumor tissues in the form of soluble E-selectins34. As shown in Figure 4F, E-selectin expression was detected in both the round tumor VECs (white arrow) as well as the tumor tissue (red arrow) to confirm E-selectin release. The accumulation of SPD micelles in the tumor microenvironment was largely on account of the overexpression of E-selectin on tumors VECs. Previous reports have also shown an up-regulation of selectins on cancer cells24-26. In agreement with this, a higher expression of E-selectin was observed on the Bel-7402 cells compared to normal human hepatic cells (Supporting S6). Therefore, presence of SA not only enhanced the influx of SPD micelles in the tumor microenvironment but also promoted their internalization into the Bel-7402 cells.

Figure 4. Evaluation on targeting and pH-triggering functions of SPD micelles in


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vivo. (A) Fluorescence images of tumor-bearing nude mice treated with SPD/ICG, PD/ICG, PP/ICG micelles and free ICG at different point-in-times. (B) Fluorescence images of tumor and tissues in tumor-bearing mice injected intravenously with SPD/ICG, PD/ICG, PP/ICG micelles and free ICG at 48 h. (C) The semi-quantitative analysis of fluorescence intensity of tumor and tissues in Figure 4B. (D) Fluorescence images of tumor and major organs in tumor-bearing mice injected intravenously with SPDD micelles at different time points. (E) The semi-quantitative analysis of fluorescence intensity of tumor and tissues in Figure 4D. (F) Fluorescence images of tumor stained with E-selectin antibody (green) and DAPI (blue). (G) Fluorescence images of tumor in mice injected with SPDD and PDD (red). The blue staining showed nuclei and the green channel showed E-selectin receptor. (H) Fluorescence images of tumor-bearing mice treated with SPDD, PDD and PPD micelles by intratumor injection. To validate the hypothesis that the improved internalization of the SPD micelles into tumor cells was mediated via E-selectin, the in vivo co-localization of E-selectin and DOX-loaded micelles was analyzed. The fluorescence signal of DOX was significantly stronger in the SPD micelles treated group compared to the PD and PPD treated groups (Figure 4G). Furthermore, E-selectin and SPD micelles were co-localized on the Bel-7402 tumor cells, thereby proving that the tumor-cell selective uptake of SPD micelles was predominantly mediated by E-selectin receptors. Nile Red-loaded micelles were injected directly into the tumors to verify the pH responsiveness of the micelles in vivo. As shown in Figure 4H, the entire tumor



tissues in the SPD/NR and PD/NR group were fluorescently stained, while only weak signals were observed in tumors injected with the PP/NR micelles. It is the likely result of the cleavage of acid-labile hydrazone that released the Nile Red dye into the tissues.

In vivo anti-tumor activity As shown in Figure 5A, anti-tumor activity of PPD was no better than the positive control groups (the tumor inhibition: 57.42 % vs 54.00 %), which can be ascribed to the excessively slow drug release, although tumor accumulation of the PPD micelles was improved due to EPR effect. In addition, the anti-tumor effect in the PDD-group were significantly boosted compared to the PPD-group (the tumor inhibition: 73.80 % vs 57.42 %) despite similar bio-distribution of these two micelles, this suggested that the improved effects were due to the faster drug release. Furthermore, tumor-bearing mice treated with SPDD micelles showed a markedly improved tumor recession compared to the PDD treated mice from day 18 to day 25 ((tumor inhibition was 86.47 % vs 73.80 %, P < 0.01). This can be explained by the improved tumor accumulation of the SPDD micelles and the fast pH-responsive drug release.


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Figure 5. The antitumor efficiency of DOX loaded SPD micelles in ectopic hepatocarcinoma model. (A) tumor volume changes. (B) H&E staining of tumor sections from ectopic hepatoma mice. (C) Fluorescence images of TUNEL positive cells. Histology in tumor was identified using immunohistochemical staining for CD31 (D), Ki67 (E). (F) Western blot results of Bax, Bcl-2 in the tumor sections from tumor-bearing mice injected with saline, free DOX, PDD and SPDD micelles. β-actin was used as internal control. (G) Densitometric measurement of Bax and Bcl-2 bands in Figure 5F. H&E staining was used to further confirm the enhanced antitumor effect of SPDD. In Figure 5B, tumor cells in the SPDD-group showed more karorrhexis



(fragmentation) and karyolysis (dissolution) compared to that in PDD-group. In addition, more apoptotic cells observed by TUNEL kits (Figure 5C) and lower proliferation rates detected by Ki67 staining (Figure 5D) in the SPDD-group further confirmed the superior anti-tumor effect of SPDD micelles. CD31 (Figure 5E) staining indicated less microvessel density in the Bel-7402 tumor-bearing mice treated with SPDD, consistent with all the findings above. The ratio of Bax and Bcl-2 determines the susceptibility of cells to apoptosis35. Treatment with SPDD micelles shifted the Bax/Bcl-2 balance towards Bax by 2-fold compared to PDD micelles (Figure 5F, 5G), which triggered apoptosis in the Bel-7402 cells. In addition, this ratio was higher in the PDD-group compared to the PPD-group, indicating that the acid-triggered linker was likely to accelerate apoptosis in the target cells. The liver receives approximately 75% of the blood supply at the flow rate of 1500-2000 mL/min in an adult human. Despite the different microenvironments of ectopic and in situ tumors, the lysosomal environment responsible for the fast DOX release in tumor cells is the same in both models. We established an orthotropic hepatoma model to monitor the anti-tumor effect of SPDD and PDD micelles. Tumor-bearing mice treated with HCl·DOX were exploited as the positive control. MRI showed tumor shrinkage in the mice treated with SPDD micelles (Figure 6A, from 77.57 mm3 to 62.13 mm3), while the other two groups showed fast rates of tumor growth (Figure 6A, PDD: from 4.63 mm3 to 54.27 mm3, HCl·DOX: from 276.70 mm3 to 366.9 mm3). Therefore, the specific tumor targeting and induced


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release clearly translated to a higher anti-tumor effect.

Staining for H&E, Ki67 and

CD31 confirmed the excellent inhibition effect of SPDD micelles including more nuclear fragmentation (Figure 6B), less expression of the nuclear protein associated with cellular proliferation (Figure 6C) and invisible microvessels angiogenesis (Figure 6D).

Figure 6. The antitumor activity of DOX loaded SPD micelles in orthotopic hepatocarcinoma model. (A) Tumor volume examined by MRI at Day 0 and Day 25. (B) H&E staining of tumor sections from orthotropic hepatoma mice. Histology in tumor was identified using immunohistochemical staining for CD31 (C), Ki67 (D).

Bio-safety of SPDD A commonly and potentially fatal side-effect of DOX is a decreased hematopoiesis, manifested as thrombocytopenia and aleukocytosis. Free DOX led to a



significant decrease in the levels of white blood cells (WBC, 440.0 ± 90.0 vs 976.7 ± 58.9×107 /L, **p < 0.01) and platelets (PLT, 487.3 ± 224.9 vs 1512.3 ± 89.5×109 /L, **

p < 0.01) compared to the saline treated group, whereas no significant differences

were seen between the saline and SPDD-groups in terms of WBC (976.7 ± 58.9 vs 850 ± 164.8 ×107 /L) and PLT (1512.3 ± 89.5 vs 1323.3± 113.0 ×109 /L) levels (Figure 7A and 7B).

Figure 7. Adverse effects of treatment with free DOX or Micelles-DOX. (A) White blood cells counts of tumor-bearing mice treated with SPDD, PDD, PPD, HCl·DOX and saline. (B) Platelet counts of tumor-bearing mice injected with different preparations. (C) Serum levels of ALT/AST on tumor-bearing mice treated with different preparations. (D) Histopathology of livers was examined by hematoxylin– eosin staining. (E) Histopathology of hearts was examined by hematoxylin–eosin staining. (F) TEM images of hearts harvested from tumor-bearing mice treated with different preparations.


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Due to the considerable accumulation of both free and micellar DOX in the liver tissues, we also analyzed liver function. Compared to saline group, AST/ALT ratio in the HCl·DOX group was significantly higher (6.0 ± 0.35 vs 3.2 ± 0.7,

**

p < 0.01,

Figure 7C), indicating adverse effects of DOX treatment. However, no significant increase was seen in AST/ALT ratio after treatment with SPDD micelles (3.8 ± 0.2 vs 3.2 ± 0.7). In addition, histopathological analysis revealed massive oxidative stress in the liver tissues of the HCl·DOX treated mice, as indicated by the presence of numerous physalides (Figure 7D). In contrast, the SPDD-group showed no visible changes, whereas PDD and PPD micelles led to adverse effects of varying degrees. The minor side effects in the SPDD-group were mainly ascribed to enhanced targeting efficiency, which is consistent with previous results. The most dangerous adverse effect of DOX is cardiomyopathy. As shown in Figure 7E and 7F, mice treated with HCl·DOX suffered from serious myocardial damage, including disordered myocardial fiber arrangement, myocardial fiber rupture and mitochondria damage. Surprisingly, the SPDD-group exhibited no apparent signs of heart failure. In the orthotopic tumor model, similar adverse cardiac effects were observed in mice treated with HCl·DOX, including broken and necrotic muscle fibers (Supporting S7). In addition, metastasis was also observed in the liver tissues of the HCl·DOX and PDD-groups (Supporting S8), while the SPDD group maintained normal liver morphology. This suggested that the anti-tumor potential of SPDD



micelles was not limited to reducing adverse effects, but also in inhibiting tumor metastasis.

Conclusions In summary, DOX loaded SPD micelles were fabricated that showed enhanced tumor tissue and tumor cell targeting in both ectopic and orthotropic hepatocarcinoma models. This novel DDS accumulated in the tumors due to dual targeting function: while the E-selectin overexpressed on the surface of tumor VECs guided the SPD micelles to the tumor tissue via SA docking, high levels of E-selectin on the plasma of tumor cells enabled their quick internalization. After cellular uptake, the hydrazone linker was cleaved in the acidic environment of the lysosomes, resulting in rapid drug release and superior anti-tumor outcome in vivo. The SA modified DOX loaded micelles expand the range of targeted cancer therapy by enhanced tumor tissue accumulation as well as rapid tumor cell internalization. Its therapeutic potential extends beyond cancers to other inflammatory diseases where E-selectin overexpression is seen on the blood vessels and require a high drug exposure for a short time.

EXPERIMENTAL SECTION Material and animals. Sialic acid and HCl·DOX were obtained from Aladdin Bio-chem Technology Co. Limited (Shanghai, China). Indocyanine green (ICG) and 3- (4,5-dimethylthiazol-2-yl) - 2,5 - diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St. Louis, MO, USA). H2N-PEG-NH2 (Mw=2.0 kDa)


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was obtained from Shanghai Seebio Biotech Co., Ltd. Primary antibodies, including Anti-Bax, Anti-Bcl-2, Anti-CD62E (E-selectin) antibodies were obtained from Abcam (UK). All other solvents were of analytical or chromatographic grade. Female BALB/c nude mice (16-20 g) were obtained from the Shanghai Silaike Laboratory Animal Limited Liability Company. All the mice were free of pathogen and fed with enough food and water. All experiments were performed in compliance with guidelines set by Zhejiang University Institutional Animal Care and Use Committee. All animal procedures were conducted in accordance with national regulations and approved by the local animal experiments ethical committee. Cell culture. The human hepatic cell LO2, human hepatic tumor cell Bel-7402 and human vascular endothelial cells (HUVEC) were purchased from Cell Resource Center of China Science Academe. These cells were cultured in Dulbecco's Modified Eagle Medium (Gibco BRL, USA) containing 10 % fetal bovine serum (FBS, Sijiqing Biologic, China), 100 U/mL penicillin and 100 U/mL streptomycin at 37 °C and a humidified atmosphere of 5 % CO2. Cells were digested by trypsin/EDTA every other day. Synthesis and characterization of SPD conjugates. Synthesis of sialic acid-PEG-hydrazone linker-DOX (SPD) conjugate was performed as mentioned above (Figure 1A). Firstly, precisely weighed SA and DMAP were placed in a 50 mL dry round bottom flask, anhydrous dimethyl sulfoxide (DMSO) was then added to make the initial dissolution. Afterwards, TSC (n

SA :

n

DMAP :

n

TSC

= 1: 5: 1.2) was

added dropwisely with stirring, and the reaction continued for another 4 h under



Page 26 of 41

nitrogen protection. After that, the mixture of H2N-PEG-NH2 and acyl chlorided SA-OTs (n

SA :

n H2N-PEG-NH2 = 1: 1) dissolved in anhydrous DMSO was kept reacting

for another 24 h at 100 ℃ with protection of nitrogen. SA-PEG-NH2 was obtained after dialysis (MWCO 2.0 kDa, Spectrum Laboratories, Laguna Hills, CA) and lyophilization. The pH of fresh water used for dialysis was kept at 8.0 to avoid formation of electrostatic compound of free SA and H2N-PEG-NH2 or SA-PEG-NH2. Secondly, the resulting SA-PEG-NH2, NPCF and DMAP (n

SA-PEG：n NPCF:

n

DMAP =

1:1.2:3) were weighed and reacted in 10 mL of anhydrous DMSO for 48 h. The reaction mixture was then dialyzed against fresh water and SA-PEG-NPCF was obtained after lyophilization. Thirdly, NH2NH2·H2O （n

SA-PEG-NBCF :

n

NH2NH2·H2O

=

1:1.5 ） was put into the SA-PEG-NPCF solution and the mixture was stirred continuously for 24 h to get SA-PEG-hyd. At last, under the prevention from light condition, DOX（n SA-PEG-hyd : n DOX= 1:1.5）was transferred into the existing solution with stirring for another 48 h. Dialysis by frequent exchange of deionized water was utilized to remove unwanted agents (MWCO 3.5 kDa, Spectrum Laboratories, Laguna Hills, CA). Ultimately, the precipitated SPD was received after lyophilization. 1H NMR and Fourier Transform infrared spectroscopy were adopted to verify the structures of H2N-PEG-NH2, DOX, SA and SPD. The molecular weights and distributions of the conjugates were measured by Size-Exclusion Chromatography with DMSO as an eluent. Calibrated with narrow dextran monodisperse standards, PLgel MIXED-C columns (particle size: 5 µm; dimensions: 7.5 mm × 300 mm; flow rate: 0.6 mL/min) were employed with a differential refractive index detector. As a


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fluorescence probe36, pyrene was adopted to measure the critical micelle concentration (CMC) of SPD and PD. The detailed settings of fluorescence spectrophotometer (F-2500, Hitachi Co., Japan) were as below: Excitation wavelength: 337 nm, Excitation slit: 2.5 nm, Emission slit:10 nm. SPD and PD solution were diluted to suitable concentrations varying from 3.9 to 500 µg/mL comprising 5.94×10-7 mol/L of pyrene. The emission intensity ratio of peak at 374 nm and 385 nm was recorded to calculate CMC. Preparation and characterization of SPDD micelles. SPDD micelles were dissolved in deionized water to form blank micelles with the treatment of probe-type ultrasonicator (400 W, 30 times, active every 2 s for a 3 s duration). Then DOX dissolved in DMSO was quickly added into micelle solutions with continuously stirring for 2 h. Excessive DMSO was removed by dialysis method (MWCO 3.5 kDa) with frequent exchange of fresh water. Ultimately, the suspension was centrifuged at 4000 rpm for 10 min to remove unencapsulated DOX. The supernatant was lyophilized to obtain SPDD micelles. PDD micelles were prepared by the same method. The size distribution of micelles were detected by dynamic light scattering (DLS, Zetasizer, Malvern Co., UK). The morphology of blank micelles, DOX-loaded micelles and blank micelles treated at pH 5.0 were examined by transmission electron microscopy (TEM, JEOL JEM-1230, Japan). A drop of the micelle solution was added onto a copper grid coated by a carbon film and stayed overnight to make dry. Afterwards, the samples were further stained by phosphotungstic acid solution (2%, w/v). Then TEM images of micelles was obtained. The encapsulated DOX amount in



micelles was measured by fluorescence spectrophotometry37. The detailed settings of fluorescence spectrophotometer were as below: Excitation wavelength: 505 nm, Emission wavelength: 565 nm, Excitation slit: 5.0 nm, Emission slit: 5.0 nm. Encapsulation efficiency (EE) and drug loading (DL) were calculated according to the following formulas: DL (%) = (mass of encapsulated DOX in micelles/mass of DOX-loaded micelles) ×100%, EE (%) = (mass of encapsulated DOX in micelles/mass of DOX added) ×100%. pH-triggered release of DOX from micelles. The dialysis membrane diffusion technique was utilized to evaluate drug release behavior. A known amount of SPDD and PDD solution were transferred into a dialysis membrane bag (MWCO 3.5 kDa) with 30 mL of phosphate buffer solution (PBS) at pH 7.4, pH 6.5 and pH 5.0, respectively. An incubator shaker was used to maintain 37 ℃ and shake horizontally at 60 rpm. The release medium was withdrawn and replaced with fresh PBS at predefined time points. Cytotoxicity assays. MTT assay was used to evaluate the in vitro cytotoxicity. Briefly, Bel-7402 cells were seeded into 96-well plates with 0.5×104 cells/well and then incubated overnight. Various concentrations of PDD micelles, SPDD micelles and DOX-loaded commercial PP micelles were added. 48 h later, 20 µL of MTT solution (5 mg/mL) was added to each well for further incubation. After 4 h, the medium was withdrawn, and 100 µL of DMSO were added to each well to dissolve purple formazan crystals. The absorbance at 570 nm of each well was detected by a


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microplate reader (Bio-Rad, Model 680, USA). Untreated cells served as control group. Cell viability was calculated according to the following formulas: Cell viability (%) = Asample/Acontrol ×100% To visualize the anti-tumor effects of the different treatments, cells were seeded into 24-well plates with 5 × 104 cells/well and allowed to attach for 24 h. Then different preparations were added into the medium with a DOX concentration of 1 µg/mL and incubated for 48 h. Afterwards, cells were exposed to Calcein-AM for 30 min and observed under Fluorescence Inverse Microscope (DMIL, Leica Microsystems Ltd, Germany). Cellular uptake. Bel-7402 cells and LO2 cells were seeded into 24-well plates with 5 × 104 cells/well and cultured overnight. SPD micelles and PD micelles were then added and cells were incubated for another 0.5 h, 1 h, 2 h and 4 h. After that, cells were washed three times by PBS, and ﬁxed in 4 % paraformaldehyde solution at room temperature for 30 min. The cellular uptake was observed by Fluorescence Inverse Microscope. Flow cytometry (FC 500 MCL; Beckman Coulter, USA) was used to quantify the fluorescence intensity of cellular uptake. Briefly, cells were seeded and treated in the same way as the qualitative experiments, and after incubation with different preparations at predefined time points, cells were digested with trypsin/EDTA and resuspended in PBS to be detected. For the competitive analysis, free SA with concentration of 2 mg/mL and 6 mg/mL were added firstly to compete with micelles for binding E-selectins. After 0.5



h, cells were exposed to different preparations for 1 h. Cells were then collected and resuspended in PBS to be detected. Immunofluorescence of E-selectin. Bel-7402 were seeded into 6-well plates with 1 × 105 cells/well on sterile round coverslips and cultured overnight. Subsequently, SPD micelles and PD micelles were added and cells were incubated for another 0.5 h. After being washed by PBS for three times, cells were fixed with 4 % paraformaldehyde. Later, cells were treated with 0.1% Triton X-100 at first to enhance the permeability of cell membrane. 5% FBS in PBS was used to block the nonspecific binding for 1 h at 25℃. Then cells were exposed to anti-CD62E antibody (10µg/mL) for 24 h at 4 ℃. After being rinsed with PBS for five times, cells were exposed with FITC-conjugated secondary antibody (1:200 dilutions) at 25℃ for 2 h. Next, DAPI was used to stain the nuclei at 25℃ for 10 min. Confocal laser scanning microscope (CLSM) was applied to capture the cellular fluorescence. Western blot. Proteins from activated HUVEC, non-activated HUVEC, Bel-7402 and LO2 cells were extracted and measured by bicinchoninic acid assay. Equal amounts of proteins were separated by 12% SDS-PAGE and transferred onto PVDF membranes. Membranes were then infiltrated in 5% bovine serum albumin for 1 h at 25℃ to block the nonspecific sites. After overnight incubation with anti-CD62E antibody (4 µg/mL) at 4℃, membranes were then washed and incubated with HRP-conjugated secondary antibodies for 2 h at 25℃. Proteins were developed by an enhanced chemiluminescence kit and were quantified by densitometry using Image Lab 3.0 software. β-actin was exploited as control.


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Intracellular drug release.

Bel-7402 cells were seeded into 96-well plates

with 0.5×104 cells/well and incubated overnight. Cells were exposed to Nile Red loaded SPD, PD and PEG-PLGA (PP) micelles (50 µg/mL) for 0.5 h, 1 h, 2 h, 4 h, 8 h and 12 h. Intracellular drug release was observed by IVIS® Spectrum system (Caliper, Hopkington, MA, USA). Lysosome tracking assay. Bel-7402 cells were seeded into 6-well plates with 1 × 105 cells/well on sterile round coverslips and cultured overnight. Later, cells were exposed to SPD and PD micelles for 0.5 h. After incubation with Lysotracker Green DND-26 for lysosome tracking, Bel-7402 cells were fixed and washed by PBS at 25℃ for three times. DAPI was performed to stain the nuclei for 30 min. At last, CLSM was applied to capture the cellular fluorescence. Biodistribution of micelles in tumors and the accumulative strategy. The tumor-bearing mouse models were built by subcutaneous injection of Bel-7402 cells into the flanks of female BALB/c nude mice. The cells injected were resuspended in DMEM without FBS. Xenogafted Bel-7402 tumor-bearing mice were injected intravenously with 1.0 mg/kg ICG equivalent of ICG-tetrabutylammonium iodide complex-loaded SPD micelles, PD micelles, PP micelles or free ICG. Biodistribution of micelles in the Bel-7402 tumor bearing mice were visualized by the IVIS® Spectrum system at predefined time points (2 h, 6 h, 12 h, 24 h and 48 h). At 48 h, major tissues of sacrificed mice were collected and imaged to observe the fluorescence signals. After imaged and transferred into liquid nitrogen, tumors were stained with anti-CD62E antibody. CLSM was applied to observe the introtumoral



fluorescence. To determine the DOX concentration in tumors, mice were injected intravenously with DOX-loaded SPD micelles at a dose of 5.0 mg/kg. At predefined time points (2 h, 6 h, 12 h, 24 h and 48 h), major tissues of sacrificed mice were collected and imaged by the IVIS® Spectrum system. Intratumoral drug release. Tumor-bearing mice were treated with Nile red-loaded micelles by intratumoral injections for 8 h. After sacrifice, the tumors were harvested and immediately immersed in liquid nitrogen. The frozen sections were stained with DAPI. CLSM was applied to observe the intratumoral fluorescence.

In vivo anti-tumor activity in extopic tumor models. Tumor-bearing mice (6/group) were injected intravenously with saline, and 5.0 mg/kg DOX equivalent of SPDD micelles, PDD micelles, PPD micelles and HCl·DOX on days 0, day 3 and day 6 after the tumor volume reached approximately 100 mm3. The smallest diameters (a) and largest diameters (b) of the corresponding tumors were determined using a digital caliper. Tumor volume was defined as the formula a2×b/2. The tumor volume was recorded every three days from day 1 to day 25. Isolated tissues (livers, hearts and tumors) were collected and fixed with 4 % paraformaldehyde. Once completed, specimens were embedded in paraffin, cut into 5 µm thick paraffin sections and stained with hematoxylin and eosin (H&E). Deparaffinized sections were treated with anti-CD31 and anti-Ki67 antibodies, respectively, followed by peroxidase/DAB secondary antibodies. At the same time, TUNEL apoptosis detection kit were utilized to test the apoptotic cells in tumors


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according to the specification. Hearts from sacrificed mice were removed and cut into 1 mm3 slabs. After fixation, dehydration, embedding and sectioning, ultrathin sections were obtained and stained with uranyl acetate and lead citrate. The pathological changes of hearts were examined by TEM.

In vivo anti-tumor activity in orthotopic tumor models. Orthotopic hepatoma models were established by transplanting with Bel-7402 tumors into livers of BALB/c nude mice. The normal liver tissues were exposed by cutting longitudinally to abdominal cavity, and then 1 mm3 tumor tissue was implanted into the liver parenchyma. Biodegradable stitches were applied to suture the wound between the abdominal wall and skin. After transplantation for two weeks, the tumor-bearing mice were classified into three groups and imaged by 3.0 T MRI. The detailed settings of MRI were as below: FOV, 60 × 60 mm2; TR, 3000 ms; TE, 78.6 ms; slice thickness, 2.0 mm. Mice were received intravenous treatments of different preparations with an equivalent dosage of DOX (5.0 mg/kg) every three days. Monitored for another 21 days, the tumor volumes were determined once again by MRI. Tumors and other representative tissues (hearts and livers) were collected and stained

with

H&E.

Histopathology

of

tumors

were

identified

using

immunohistochemical staining for CD31 and Ki67. Adverse effects in tumor-bearing mice. On day 25, venous blood of all the mice were collected. The whole blood was used to test cell counts including white



blood cells (WBC) and platelets (PLT). While serum separated was used to test alanine aminotransferase (ALT) levels and aspartate transaminase (AST) levels. Whole blood and serum samples were measured in triplicate by an automated Beckman Analyzer. Statistical analysis. Comparative analysis of differences between groups was calculated by one-way analysis of variance (ANOVA) or repeated ANOVA with post hoc Tukey tests with SPSS 18.0 (95 % confidence interval). A significant difference was set at **p

Sialic Acid-functionalized pH-triggered Micelles for Enhanced Tumor

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