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Dual-targeted controlled delivery based on folic acid modified pectin-based nanoparticles for combination therapy of liver cancer Yanxue Liu, Yuheng Zong, Zixuan Yang, Min Luo, Guiliang Li, Wang Yingsa, Yongli Cao, Meng Xiao, Tianjiao Kong, Jing He, Xingyong Liu, and Jiandu Lei ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b06586 • Publication Date (Web): 15 Jan 2019 Downloaded from http://pubs.acs.org on January 15, 2019

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ACS Sustainable Chemistry & Engineering

Dual-targeted controlled delivery based on folic acid modified pectinbased nanoparticles for combination therapy of liver cancer Yanxue Liua, b, Yuheng Zongb, Zixuan Yangb, Min Luob, Guiliang Lib, Wang Yingsab, Yongli Caob, Meng Xiaob, Tianjiao Kongb, Jing Heb, Xingyong Liua, Jiandu Leia, b* a

College of Chemistry and Environmental Engineering, Sichuan University of Science &

Engineering, Zigong 643000, PR China b Beijing Key Laboratory of Lignocellulosic Chemistry, College of Material Science and Technology,

Beijing Forestry University, Beijing, 100083, PR China *Corresponding Author’s email: [email protected]

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ABSTRACT: Dihydroartemisinin (DHA) as an effective anticancer drug is being concerned owing to the excellent efficacy, but the poor solubility and premature release confine the clinical application. Construction of a satisfied DHA-loaded delivery system with targeted recognition and specific controlled release brings opportunities and challenges, which can be triggered through an endogenous stimulus. In this paper, we designed a traceable and dual-targeted DHA-loaded nanocarrier by taking advantage of the highly expression of pectin with galactose residues to asialoglycoprotein receptors (ASGR) on the surface of liver, as well as the highly expression of folic acid (FA) to folic acid receptors (FRs). Folic acid modified pectin was coupled with DHA-loaded eight-arm polyethylene glycol conjugates to prepare folic acid-pectin-eight-arm polyethylene glycol-dihydroartemisinin prodrugs (FA-Pectin-8armPEG-DHA), and then embedded hydroxycamptothecin (HCPT) by the self-assembly of hydrophobic drugs and hydrophilic carriers to prepare folic acid-pectin-eight-arm polyethylene glycoldihydroartemisinin/ hydroxycamptothecin nanoparticles (FPPDH NPs). FPPDH NPs showed an average particle size 98 nm under maximum drug loading (7.04 wt%) and encapsulation efficiency (20.57 wt%). The enhanced cytotoxicity of FPPDH NPs were 204.5-fold (H22) and 178.4-fold (HepG2) to the free DHA, respectively. In addition, a clear synergy of drugs suggested that the dual-targeted combination therapy is a reliable therapeutic strategy. KEYWORDS: Pectin, Folic acid, Controlled delivery, Dual-targeted, Liver cancer

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INTRODUCTION Chemotherapy is the main method for cancer treatment in clinical, but it is lack of selectivity between normal cells and tumor cells.1 In the past few years, effective, specific targeted and controlled release systems have been encouraged to achieve a further development of anticancer drugs.2, 3 The advantages of drug delivery systems are to indirectly change the solubility of the drug and improve the stability of the drug, 4-11 avoid drug release in healthy tissues through modifying functional groups,12-17 and target tumor by stimulation-responsive.18-20 Pectin as a drug carrier showed a great potential for drug delivery due to the considerable biological activity of biodegradation, regulation immune response, inhibition tumor growth, and induction apoptosis.21-27 pH-modified pectin could inhibit Gal-3, which is a key target for cancer metastasis.28 Therefore, it can be speculated that pectin may play an important role in anti-tumor applications.29 In addition, asialoglycoprotein receptor (ASGPR) as a transmembrane glycoprotein mainly exists on the surface of liver cell, which can specifically recognize and bind to glycoproteins with galactose residues at the ends of the molecule, and transfer them to lysosomes of liver cells for metabolism.30-32 Each liver cell contains approximately 2 million ASGPRs binding site. An abundance of galactose residues in pectin can be selectively identified by ASGPR, and thus targeting liver cell.32, 33 In the recently reports, some novel treatment strategies of dual-targeted delivery system have attracted widely attention.34, 35 For example, transferrin targeting peptide B6 and arginine-glycineaspartic acid (RGD) were incorporated for simultaneous integrin targeting,36 folate and iron difunctionalized multiwall carbon nanotube (FA-MWCNT@Fe) was applied for delivering doxorubicin into HeLa cells,37 folate-targeting theragnostic prodrug (Doxo-S-S-Fol) had activatable fluorescence and activatable cytotoxicity when linked folate and DOX with a disulfide bond.38 In order to improve the drug loading performance of pectin and obtain perfect therapeutic effect, a novel pectin drug delivery system need to be established. In this paper, eight-arm polyethylene glycol was introduced as "stealth effect" to improve the stability of drugs and prolong blood circulation time in vivo. we designed a traceable and dual-targeted DHA-loaded nanocarrier by

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taking advantage of the highly expression of pectin with galactose residues to asialoglycoprotein receptors (ASGR) on the surface of liver, as well as the highly expression of folic acid (FA) to folic acid receptors (FRs). The dual-targeted DHA-loaded nanocarrier (FPPDH NPs) was developed by introducing targeted pectin-8arm-PEG-COOH to be modified by FA, and embedding hydroxycamptothecin (HCPT) by the self-assembly of hydrophobic drugs and hydrophilic carriers, which looked forward to achieving the combination therapy of drugs. The design of the FPPD conjugates and the self-assembly scheme of the FPPDH NPs are shown in Fig. 1.

Fig. 1 Schematic design of the FPPD conjugate synthesis and FPPDH NPs preparation. Schematic diagram of FPPDH NPs dual targeting liver cancer cells via FA receptor protein and galactose receptor protein ASGPR

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MATERIALS AND METHODS Materials Dihydroartemisinin (DHA), hydroxycamptothecin (HCPT), citrus pectin (galacturonic acid ≥65.0%), 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), folic acid (FA) and 4-dimethylaminopyridine (DMAP) were purchased from Sigma-Aldrich (Shanghai, China). Eight-arm polyethylene glycol (8arm-PEG-COOH, Mw=10 KDa) was supplied by Sigma-Aldrich, and was a FDA and EU food-grade product. RPMI-1640, DMEM and trypsin-EDTA were provided by Gibico Co., Ltd. (Shanghai, China). Fetal bovine serum (FBS) was supplied by Hyclone Co., Ltd. (Oregon, USA). CCK-8 was obtained from Dojindo Laboratories. (Dojindo, Kumamoto, Japan). Other chemicals (analytical grade) were provided by Lan Yi Chemical Products Co., Ltd. (Beijing, China). H22 and HepG2 cells were supplied by Institute of Process Engineering of Chinese Academy of Sciences (CAS). Five-week-old female mice (BALB/c, 25-30g) were provided by Beijing Hfk Biosciece Co., Ltd. The mice had free access to food and water with constantly maintaining 12-h dark & 12-h light cycle at 25 ± 2 oC. Synthesis of 8arm-PEG-DHA conjugates 8arm-PEG-DHA conjugates were synthesized following our previous work39 and the preparation scheme of the 8arm-PEG-DHA conjugates were shown in Fig. S1. Briefly, 8arm-PEG-COOH (1.0 g, 0.1 mmol), EDC (0.38 g, 2.0 mmol), DHA (1.13 g, 4.0 mmol) and DMAP (0.5 g, 4.0 mmol) were added to a round bottom flask containing pyridine (15 mL) for 48 h. Then, the mixture were precipitated with acetonitrile (Vacetonitrile: Vmixture =3:1, v/v) and centrifuged at 4000rpm for 30min. The mixture were transferred to a dialysis membrane (MWCO 1KDa) and dialyzed for 24 h. Afterwards, The dried 8arm-PEG-DHA conjugates were collected using freeze-dryer (Scientz18ND, Zhengzhou, China). Synthesis of FPPD conjugates In this study, amino group was introduced to the γ position to prepare FA-NH2.40 As shown in Fig.

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S2, simply, FA (88.3 mg, 0.2 mmol), EDC (76.7mg, 0.4 mmol) and NHS (92 mg, 0.8 mmol) were mixed in dimethyl sulfoxide (DMSO, 15 mL), and heated to 50 °C for 6 h. Then, ethylenediamine (156.26 mg, 2.6 mmol) was added overnight at room temperature. The mixture were precipitated by the addition of excess acetonitrile, separated by centrifugation, washed several times with acetonitrile, and dried under vacuum to obtain FA-NH2 powder. The preparation of folic acidpectin-eight-arm polyethylene glycol-dihydroartemisinin (FPPD) conjugates is shown in Fig. S3. 8armPEG-DHA (1.0 g, 0.1 mmol), pectin (0.09 g, 0.6 mmol), FA-NH2 (0.05 g), EDC (0.19 g, 1 mmol) and DMAP (0.12 g, 1 mmol) were added to a round bottom flask containing pyridine (25 mL) for 48 h. Afterwards, the mixture were precipitated by excess diethyl ether (Vdiethyl

ether:

Vmixture=3:1, v/v), and separated by centrifugation. The obtained precipitate was transferred to dialysis membrane (MWCO 8 KDa), and dialyzed for 24 h. The dried FPPD conjugates were collected using freeze-dryer. The structure of FA-NH2, 8armPEG-DHA, FPPD and FPPDH NPs were performed via 1H-NMR spectroscopy (DRX-600, Avance III, Bruker, Germany) using CDCl3 and D2O as the solvent, respectively, and processed by TOPSpin software for spectral analysis. Preparation and characterization of dual-targeted FPPDH NPs Dual-targeted FPPDH NPs were prepared by self-assembly method. Briefly, 15 mg of FPPD conjugates and 4 mg of HCPT were dissolved in DMSO (2 mL), and slowly dropwise added into deionized water with rapidly speed stirring for 10 min to obtain FPPDH NPs solution. The nanoparticles solution was transferred to a dialysis membrane (MWCO 3KDa) and dialyzed against PBS solution (pH 7.4) for 12 h. Afterwards, the nanoparticles solution were collected and freezedrying to obtain FPPDH NPs power. FPPD NPs was prepared the same as that of FPPDH NPs without HCPT. Transmission electron microscope (TEM) analyses of FPPDH NPs were performed on a JEM100CXa (JEOL, Japan). TEM samples were prepared by dispersing in deionized water by ultrasonic, assimilated one drop of colloidal solution and placed onto mesh copper grids with carbon film. The

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hydrodynamic size and Zeta Potential of FPPDH NPs was analyzed through dynamic light scattering (DLS) analysis on a Zeta Potential Analyzer (Brookhaven Instruments, NY, USA). Delivery measurements of FPPDH NPs The measurements of DHA and HCPT were taken at 238 nm and 254 nm with a UV-2000 spectrophotometer, respectively. Briefly, 8armPEG-DHA conjugates were dissolved in ethanol solution (75 mL, 60%) and filtered. The mixture (5mL) were pipetted and added into 23mL, 2% sodium hydroxide solution in the constant temperature (60 °C) water bath for 30 min. The concentration of DHA in 8armPEG-DHA conjugates was determined using a calibration curve: Y1=0.00737X1, R2=0.9998, Y1 represents the absorbance value of the sample, X1 represents the concentration of the sample, μg/mL. Delivery efficiency of DHA and HCPT in FPPDH NPs was also determined by a UV-Vis spectrophotometer. Suspensions of FPPDH NPs (5mg) in 25 mL of dilute hydrochloric acid (5%, v/v) were hydrolyzed and centrifuged to obtain DHA and HCPT. The precipitates were determined by DHA and HCPT calibration curves. HCPT-ethanol solution in the 4-64 μg/mL concentrations were prepared to obtain the calibration curve of HCPT: Y2=0.00737X2, R2=0.9998, Y2 represents the absorbance value of the sample, X2 represents the concentration of the sample, μg/mL. DHA loading capacity (LC) and HCPT encapsulation capacity (EC) were estimated according to the following formula. LC (%) = (total DHA in nanoparticles/ total particle weight)100%

(1)

EC (%) = (total HCPT in nanoparticles/ total particle weight) 100%

(2)

Drug release from FPPDH NPs To determine drug release behavior in vitro, 15 mg of FPPDH NPs was dispersed in 15 mL release medium (PBS, pH 5.5, 7.4 and 8.0) and incubated under gentle shaking at 37 °C. At determined time intervals, 2 mL of dialysate solution at different pH was taken out and centrifuged for 10 min, and the buffer was refreshed with 2 mL release medium. DHA and HCPT was measured by HPLC (C18 reversed phase column, HCPT: acetonitrile-water solution (30/70, v/v) at 254 nm, DHA: acetonitrile-water (45/55, v/v) at 238 nm). In addition, esterase (30 units) was added to a dialysis

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bag (MWCO 3.5 kDa) as a control. In vitro cellular uptake For the estimation of cell uptake of HepG2 cells was inoculated in RPMI-1640 supplemented with 10% FBS, 1% penicillin-streptomycin in a humidified incubator. The fluorescence distribution of free HCPT, pectin-eight-arm polyethylene glycol-dihydroartemisinin/ hydroxycamptothecin nanoparticles (PPDH NPs) and FPPDH NPs was observed by laser scanning confocal microscopy (CLSM, TCS SP5, Leica, Japan) at the absorption of 488 nm. HepG2 cells were seeded on confocal dish containing 4 cm2 slides at the density of 1.0×105 cells/mL and incubated overnight. HepG2 cells were incubated with free HCPT (IC50), PPDH NPs (IC50) and FPPDH NPs (IC50) at 37 °C for 4 h, the cells were rinsed with DPBS softly, resuspended and fixed in 4 % paraformaldehyde for 15 min. Then 20 μL 4',6-diamidino-2-phenylindole (DAPI) was added to the cells for 5 min. Afterwards, DAPI solution was removed, washed with DPBS for three times, and saved at 4 °C. Furthermore, flow cytometry was performed for quantitative analysis of capture capability of singletargeted and dual-targeted. HepG2 cells were cultivated into six-well plates at the density of 1 × 105 cells/well for 24 h. Then, free DHA (10 μg mL−1), PPDH NPs and FPPDH NPs (equivalent DHA) were added to cultivate for 2 h at 37 °C, respectively. Excess drug-mixture were removed by PBS for three times. Afterwards, the cells were digested with trypsin, precipitated by centrifugation, and resuspend in PBS. The samples were observed by a flow cytometer (BD Biosciences, San Jose, CA) at the absorption of 488 nm. In vitro cytotoxicity of DHA-loaded nanocarriers The cytotoxicity of FPPDH NPs was estimated using CCK-8 (Dojindo, Kumamoto, Japan) assay. The free DHA, free HCPT, FPPD NPs and FPPDH NPs at different concentrations (0.01-100 μg/mL DHA) in PBS were added to the H22 and HepG2 cells cultures (5×103 cells/well) in 96-well plate, followed by incubation for 24 h, 48 h and 72 h at 37 °C. Afterwards, CCK-8 solution (20 μL) was added to each cell culture and kept on incubating for 1 h at 37 °C in the dark. The cytotoxicity of samples were analyzed using a microplate reader (Infinite M200 Pro, TECAN, Zürich, Switzerland).

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IC50 method was used to calculate the concentration of drug that inhibit 50% of cell growth. The combination index (CI) of DHA and HCPT in FPPDH NPs was assessed according to the following formula. CI = DHAC/DHAS + HCPTC/HCPTS

(3)

Whereby DHAC and HCPTC represent the IC50 of DHA and HCPT in nanoparticles, while DHAS and HCPTS represent the IC50 of free DHA and free HCPT. CI 1 indicates antagonistic effect. Pharmacokinetic study Thirty healthy tumor-free BALB/c female mices (5 weeks) were randomly divided into five groups, and injected PBS (control), free DHA, free HCPT, FPPD NPs and FPPDH NPs, respectively. Blood samples were collected from the eyelids of the mice at 0, 0.1, 0.25, 0.5, 1, 2, 4, 8, 12, 16, 20, 24, 30, 36, 48 and 72h, and plasma was obtained by centrifugation at 4 °C for 10 min. 50 μL, 0.1 N NaOH was added to 100 μL of plasma and dissolved in the water bath at 37 °C for 15 min. Afterward, 50 μL, 0.1 N HCl was added to neutralize the mixture, and 100 μL of methanol was added to mix for 5 min. The supernatant was collected by centrifugation at 4000 rpm for 5 min, dried in nitrogen, and dissolved with methanol to measure by HPLC (C18 reversed-phase column, 60% acetonitrile0.05 % trifluoroacetic acid, 238nm or 254 nm). Blood levels of DHA and HCPT are plotted by percentage unit (%ID/g) after injection. In vivo antitumor efficacy The in vivo liver tumor xenograft model was developed by subcutaneously injecting H22 cells in the right auxiliary flanking region. Thirty BALB/c female mice (5 weeks) were randomly divided into five groups (n = 6), and injected 200 μL, 1×106 cells/mouse H22 cells. When the tumor volume reached 50-100 mm3, mice were administered with PBS (control group), DHA (10 mg kg-1), HCPT (10 mg kg-1) and nanoparticles (equivalent DHA) on days 0, 2, 4, 6 and 8 by intravenous injection, respectively. The tumor volume (TV) was calculated by the formula of TV = (L × W2)/2, L and W represent the longest and shortest tumor diameter (mm), respectively. The percentage of tumor

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growth inhibition (%TGI) was calculated by the formula of %TGI = [(C-T)/C] × 100%, C and T represent the mean tumor volume of control group and treatment group, respectively. Detection of Allergic Reaction Allergic reaction testing is value for chemotherapeutic drugs before entering into the body. Thirty tumor-bearing mice were randomly divided into five groups and administered every 2 days with DHA of 10 mg kg-1, HCPT of 10 mg kg-1 and nanoparticles (equality to free DHA). Blood of five groups mice were collected, and serum of samples were analyzed by IgE ELISA. Statistical analysis Dates were reported as mean values ± standard deviation (SD) by variance analysis. The statistical significance were considered as * p < 0.05, ** p < 0.01.

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RESULTS AND DISCUSSION Preparation of FPPDH NPs 1H

NMR spectra measurements of free DHA, 8armPEG-DHA, pectin, FA-NH2, pectin-FA, FPPD

conjugates and FPPDH NPs were shown in Fig. 2. The proton characteristic peak of pectin-FA (D2O) at δ 6.8, δ 7.7 and δ 8.5 was the evidence of successfully combining with FA. The methylene proton peak of 8armPEG-COOH (CDCl3) is at δ 2.5-3.91 (4nH, -(CH2CH2O)n -) and δ 4.18 (2H, CH2OC(O)O). The multiple peak of DHA around δ4.76 (1H, CH) moved to the low field of δ6.59 (1H, CH), indicating the formation of ester bond between DHA and 8armPEG-COOH. The terminal methylene proton peak of PEG moved from δ 4.15 (1H, CH) to the lower field of δ 4.17 (1H, CH), and the small peak remaining at δ 4.15 (1H, CH) indicated the presence of unreacted PEG functional groups. While proton characteristic peaks of FA in FPPD conjugates was very weak in 1H NMR, probably due to contain small amount of FA. A weak peak of FA was seen in FPPD NPs thus exposing hydrophilic FA on the surface of nanoparticles by self-assemble.

Fig. 2 1H-NMR spectra of pectin, pectin-FA, FA-NH2 and FPPD in D2O, DHA, 8armPEG-DHA and FPPDH NPs in CDCl3

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Table 1 Particle size, zeta potential and drug loading efficiency of nanoparticles Sample

DLEDHA (wt%)

DLEHCPT (wt%)

Size（nm）

Zeta potential (mV)

8armPEG-DHA FPPD NPs FPPDH NPs

18.65 ± 0.59 8.00 ± 0.31 7.04 ± 0.42

— — 20.57 ± 0.73

— 85.23 ± 4.71 98.33 ± 8.55

— 10.91 ± 0.49 12.06 ± 0.35

Physicochemical characterization of nanoparticles The drug loading and embedding rate of nanoparticles were detected by UV-Vis absorption spectroscopy. As shown in Table 1, the DHA-loading efficiency of FPPD NPs and FPPDH NPs were 8.00% and 7.04%, respectively, and the HCPT-embedding rate was as high as 20.57%. The particle size of FPPDH NPs was significantly increased after embedding HCPT, and obtained satisfactory particle size (98nm) under the maximum drug loading. Distribution of FPPDH NPs was relatively concentrated, and well dispersibility of FPPDH NPs was obtained due to the effect of HCPT-embedding as an increased hydrophobic core (Fig.3 A, B). Zeta potential plays a key for nanoparticles’ stability during the procedure of suspension, which evaluates the electrostatic repulsion of nanoparticles and the effect of nanoparticles to cell membrane.41,42 The zeta potential values were displayed in Table 1. The mean surface charge of the FPPD NPs and FPPDH NPs were 10.91 and 12.06 mV. The positive zeta potential of nanodrug may be ascribed to the presence of large numbers of carboxyl groups on FA and unreacted galacturonic acid of pectin in preparing nanoparticles by self-assemble. The exposed positive zeta potential on the surface of nanoparticles improved the stability of the nanoparticles.

Fig. 3 (A) TEM of FPPDH NPs with significant drug loading and embedding rate, (B) particle size distribution of FPPDH NPs

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In vitro drug release kinetics The capability of controllable release of the FPPDH NPs was considered due to the presence of ester bonds between free drugs and carriers. The pH-responsive FPPDH NPs were investigated at 37 °C in pH 5.5, 7.4 and 8.0 PBS, and the release kinetics of DHA and HCPT in FPPDH NPs were studied through HPLC analysis. As can be seen from Fig. 4A, FPPDH NPs of pH 5.5 were released more slowly than of pH 7.4 or 8.0 and without the phenomenon of burst release due to the swelling of the pectin at certain pH levels. Obviously, the release of DHA and HCPT in FPPDH NPs was pH dependent, showing a similar trend of increased release. Esterase was added to promote FPPDH NPs hydrolysis and HCPT release (Fig. 4B). The results indicated that at physiological pH, FPPDH NPs could protect the structure over a long period, and achieve a slow drug shedding and durable release.

Fig. 4 (A) DHA release kinetics with different pH in FPPDH NPs, (B) and the HCPT release kinetics with esterase and without esterase in PBS at pH 7.4 and 37 °C from the FPPDH NPs

In vitro cellular uptake The internalization of HCPT and HCPT-loaded NPs into HepG2 cells were detected by confocal microscopy. In Fig. 5, the intensity of fluorescence and the enrichment level of tumor surface could be detected, suggesting that free HCPT, PPDH NPs and FPPDH NPs with green fluorescence entered into HepG2 cells (blue fluorescence).The fluorescence intensity in HepG2 cells incubated with PPDH NPs and FPPDH NPs for 4 h were higher than free HCPT, indicating a higher level of cellular uptake due to the hydrophilic groups on the surface of nanoparticles, as well as specifically 13



recognitions of ASGPR on the surface of HepG2 cells to galactose residues of pectin that not involved in the reaction. In addition, the capacity of FPPDH NPs’ cellular uptake was better than PPDH NPs owing to the incorporation of FA onto the surface of the FPPDH NPs.

Fig. 5 Confocal microscopic pictures of H22 cells incubated with (A) free HCPT, (B) PPDH NPs and FPPDH NPs (C) at an equivalent concentration of HCPT (IC50) for 4 h at 37 °C

Fig. 6 Effect of FA targeted FPPDH NPs on viability of H22

The effect of FA in the cellular uptake of FPPDH NPs was also investigated in this paper. Simply, 14


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FA was chosen as a receptor competition, and HepG2 cells were used as FA receptor overexpression.43, 44 As is shown in Fig. 6, consistent with our assumptions, the efficacy of FPPDH NPs were inhibited by adding excessive FA and the cell viability changed little with the increase of FA concentration, indicating that supplementation of additional FA lead to a competitive on FAtargeted nanoparticles. The endocytosis of DHA, PPDH NPs and FPPDH NPs was further measured by flow cytometry results. Seen from Fig. 7, FPPDH NPs exhibited a 2.1-fold and 6.1-fold higher uptake than that of PPDH NPs and DHA after 48h of treatment, indicating that FPPDH NPs can enhance the endocytosis of liver cells through the specific interactions. Dual-targeted FPPDH NPs can quickly capture tumors and rapidly enrich on the surface of tumors.

Fig. 7 Flow cytometry analysis of HepG2 cells apoptotic through staining with Annexin V-FITC (AV) and propidium iodide (PI) after 48 h of treatment with free DHA, PPDH NPs and FPPDH NPs (IC50). Q1, Q2, Q3 and Q4 represent secondary necrosis cells, late apoptotic cells, normal viable cells, early apoptotic cells, respectively.

In vitro cellular cytotoxicity In vitro cytotoxicity detection is a necessary step to ensure the safety of nanoparticles before entering human’s body. The cytotoxicity of free DHA, HCPT and FPPD NPs and FPPDH NPs were measured by CCK-8 assay. As shown in Fig. 8A and C, free DHA, HCPT and FPPD NPs and FPPDH NPs all exhibited considerable cytotoxicity against H22 and HepG2 cells with the extension of incubation time (DHA: 7 μg ml-1, HCPT: 7 μg ml-1, nanocarriers equal to DHA), indicating the suitable concentrations and good biocompatibility of nanocarriers. After treating for 24h, 48h and 15



72h, only 35%, 12% and 4% H22 cells were survived after FPPDH NPs treatment. In addition, 38%, 24%, 10% and 2% HepG2 cells were survived after treating with free DHA, free HCPT, FPPD NPs and FPPDH NPs for 72 h, respectively. The cytotoxicity of free DHA, HCPT, FPPD NPs and FPPDH NPs in H22 and HepG2 cells was different because of the chemical sensitivity of different cell lines. Compared with potential drug efficacy, the corresponding half-maximal inhibitory concentration (IC50) values for samples were estimated and presented in in Fig. 8B and D. The results showed that IC50 was free DHA> free HCPT > FPPD NPs > FPPDH NPs (Table 2). In H22 and HepG2 cells, the cytotoxicity of FPPDH NPs was 204.5- and 178.4-fold of free DHA, respectively, and was 5.8- and 6.0-fold of free HCPT. IC50 of FPPDH NPs in H22 and HepG2 was 0.06 and 0.05, respectively, and the combined index (CIs) of DHA and HCPT was 0.05, indicating that FPPDH NPs could significantly achieve synergistic effect of DHA and HCPT. It is clarify that the cytotoxicity of FPPDH NPs against H22 and HepG2 cells was greatly enhanced, which was in consistent with the above intracellular uptake study and further verified the increased property of dual-targeted. Furthermore, it was demonstrated that the internalization of the polymer and the strong release in lysosomes could further enhance the efficacy of the drug.

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Fig.8 (A, C) Cellular cytotoxicity of DHA, HCPT, FPPD NPs and FPPDH NP in H22 and HepG2 cells. Cell viability of H22 and HepG2 cells treated with 10 µg mL-1 of DHA, HCPT, and nanoparticles (equivalent to native DHA) were measured by CCK-8 assay (n = 3, error bars represent standard deviation); (B, D) CCK-8 assay of DHA, HCPT and nanoparticles with different concentrations in H22 and HepG2 cells (n = 3, error bars represent standard deviation) Table 2 In vitro cytotoxicity analysis (IC50, μg mL-1) Sample DHA HCPT FPPD NPs FPPDH NPs

H22 12.27 (1.3410) 0.35 (0.07471) 0.11 (0.02933) 0.06 (0.00941)

HepG2 8.92 (1.0928) 0.30 (0.06030) 0.15 (0.03140) 0.05 (0.01003)

Pharmacokinetic in mice Pharmacokinetic of mice carrying H22 were studied by intravenous injection of free DHA, HCPT, FPPD NPs and FPPDH NPs. As shown in Fig. 9, the concentrations of DHA and HCPT in plasma slowly declined over time after administration of nanoparticles, and the presence of DHA in plasma was longer than free DHA, possibly due to the hydrolysis of nanoparticles and break of ester bond between DHA and 8arm-PEG, resulting in slowly release of DHA. In contrast, free DHA and HCPT

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in the blood circulation were disappeared rapidly, and the plasma concentration was less than 5% after 8 h of intravenous injection, while HCPT and DHA in FPPDH NPs were maintained 50 h and 80 h in plasma, respectively. The concentration of FPPDH NPs in plasma was higher than that of FPPD NPs, possibly because embedded HCPT enhanced the strength of hydrophobic core, thereby reducing the hydrolysis rate of nanoparticles. After 24 h of intravenous administration, FPPDH NPs showed prolonged clearance rates with DHA levels of 32.3 %ID/g, and HCPT levels of 16.1 %ID/g. The blood circulation half-time of DHA in FPPD NPs and FPPDH NPs was extended from 0.9 h to 9.4 h and 11.8 h, respectively, and that of HCPT in FPPDH NPs was extended from 0.6 h to 4.7 h.

Fig. 9 Blood circulation curves and half-time of PPDH NPs compared with free DHA (A), and PPDH NPs compared with free HCPT (B). Error bars were based on six mice per group at each time point

In vivo antitumor activity of nanoparticles H22-bearing model mice were built to investigate the in vivo antitumor activity of free drug and nanocarriers. The relative tumor volume (RTV) and body weight of the mice were measured for 24 days in Fig. 10A and 11, compared to the free drug group, FPPDH NPs treated group had significantly suppressed tumor growth after 24 days’ treatment. The anti-tumor capacity of free DHA, HCPT and nanocarriers were FPPDH NPs > FPPD NPs > free DHA and HCPT (Fig. 10B and Table 3). Tumor inhibition rate and survival rate of FPPDH NPs were up to 91.8 % (24 days) and 80.3 % (30 days) which were higher than free DHA (14.5%/ 24 days, 0%/ 30 days ) and HCPT (23.6%/ 24 days, 0%/ 30 days). It is indicated that the pectin and FA-modified FPPDH NPs could active target tumor cells, achieving a relatively high drug accumulation at the tumor site and further 18


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enhancing the drug delivery efficacy. Furthermore, no marked body weight change was observed for the FPPDH NPs groups (Fig. 10C), meaning that the synthesized nanocarriers had good biocompatibility in vivo. These findings were also consistent with the above evaluation results in vitro. There was no systemic toxicity, loss of appetite and abnormal body weight occurred during the experiment.

Fig. 10 In vivo antitumor activity of free DHA, free HCPT, and nanoparticles in the subcutaneous mouse model of H22. (A) Relative tumor volumes of mice during treatment with different groups; (B) Survival of mice in different treatments; (D) The animal weights were recorded once per week and expressed over the 20-day observation. (E) IgE levels of mice treated with free DHA, free HCPT, and nanoparticles for 30 min. Data as mean ± SD; n = 6. (F) White blood cell (WBC) changes during four administrations in normal mice with free DHA, free HCPT, and nanoparticles. The blood sample was collected from mice on day 2 after the last dosage treatment Table 3 H22xenograft model (q2d × 5): efficacy comparison Sample TV ± SD (mm3) a RTVa TGI (%)a Control 5820 ± 1729 47.19 ± 10.17 —— DHA 3890 ± 1588 26.15 ± 7.44 14.5 HCPT 4016 ± 1452 31.78 ± 8.93 23.6 FPPD NPs 623 ± 471 11.2 ± 1.5 73.1 FPPDH NPs 255 ± 130 7.1 ± 1.3 91.8 a mean tumor volume (TV), relative tumor volume (RTV), 24 days %TGI b survival curve % on day 30

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Cure (%)b —— —— —— 57.2 80.3


Fig. 11 Tumor photographs from each treatment group excised on day 24

Evaluation of the side effects Parameter IgE level was chosen for rapidly assessment type I hypersensitivity of DHA, HCPT, FPPD NPs and FPPDH NPs. In Fig. 10D, H22 tumor-bearing mice treated with free drugs showed a higher IgE levels than the nanoparticles group, while there were no significant various in the FPPD NPs and FPPDH NPs groups, suggesting that nanoparticles can greatly reduce the harm of allergic reactions. Mouse blood was collected to test the WBC count which was an indicator of hematological toxicity. Seen from Fig. 10E, WBC count of mice in FPPD NPs and FPPDH NPs was significantly higher than free drugs, indicating that the DHA-loaded nanocarriers can avoid serious hematological toxicity.

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CONCLUSIONS In this study, we successfully synthesized a novel dual-targeted delivery system of DHA-loaded nanocarriers (FPPDH NPs) for controlled delivery and combination therapy. FPPDH NPs showed an average particle size 98 nm under maximum drug loading (7.04 wt% DHA) and encapsulation efficiency (20.57 wt% HCPT). The in vitro drug release indicated that FPPDH NPs had the capacity of pH-dependence to achieve the passive targeting of tumors through EPR effect. The in vitro cellular uptake and flow cytometry assays demonstrated that FPPDH NPs managed to target liver cells specifically with higher efficacy than that of PPDH NPs. In addition, FPPDH NPs had a higher cytotoxicity than free DHA (204.5-fold H22, 178.4-fold HepG2) and the co-carrier Pectin8armPEG-COOH was biocompatible and essentially nontoxic. Furthermore, the DHA-loaded nanocarriers could be rapidly accumulated on the surface of tumor and simultaneously inhibit tumor growth by dual-targeted effect. We proposed the first report on the advantages of pectin-based nanocarriers in the literature, which is a promising dual-targeted strategy for cancer treatment.

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ACKNOWLEDGMENTS This study was supported by the National Natural Science Foundation of China (No. 21576029; 21406013) and the Sichuan Science and Technology Department of China (No. 2017JY0139).

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SUPPORTING INFORMATION The Supporting Information is available free of charge on the ACS Publications website at DOI: xxxxxxxxxxxx. Fig. S1 Preparation route diagram of 8armPEG-DHA. Fig. S2 Preparation route diagram of FANH2. Fig. S3 Preparation route diagram of FA-Pectin-8armPEG-Dihydroartemisinin conjugates （FPPD）

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REFERENCES (1) Shi, J.; Kantoff, P. W.; Wooster, R.; Farokhzad, O. C. Cancer Nanomedicine: Progress, Challenges and Opportunities. Nat. Rev. Cancer 2017, 17, 20–37, DOI 10.1038/nrc.2016.108. (2) Ritchie, J. W. A.; Williams, R. J. Cancer Research UK Centre for Drug Development: Translating 21st-century Science into the Cancer Medicines of Tomorrow. Drug Discovery Today 2015, 20(8), 995–1003, DOI 10.1016/j.drudis.2015.03.006. (3) Cai, P.; Wan, R. L.; Wang, X.; Wu, Y. L.; Chen, X. Programmable Nano–Bio Interfaces for Functional

Biointegrated

Devices.

Adv.

Mater.

2017,

29(26),

1605529,

DOI

10.1002/adma.201605529. (4) Dan, P.; Karp, J. M.; Hong, S.; Farokhzad, O. C.; Margalit, R.; Langer, R. Nanocarriers as an Emerging Platform for Cancer Therapy. Nat. Nanotechnol. 2007, 2(12), 751–760, DOI 10.1038/nnano.2007.387. (5) Barreto, J. A.; O’Malley, W.; Kubeil, M.; Graham, B.; Stephan, H.; Spiccia, L. Nanomaterials: Applications in Cancer Imaging and Therapy. Adv. Mater. 2011, 23(12), H18–40, DOI 10.1002/adma.201100140. (6) Song, J.; Pu, L.; Zhou, J.; Duan, B.; Duan, H. Biodegradable Theranostic Plasmonic Vesicles of Amphiphilic Gold Nanorods. Acs Nano 2013, 7(11), 9947–9960, DOI 10.1021/nn403846v. (7) Singh, S. R.; Grossniklaus, H. E.; Kang, S. J.; Edelhauser, H. F.; Ambati, B. K.; Kompella, U. B. Intravenous Transferrin, GRD Peptide and Dual-targeted Nanoparticles Enhance AntiVEGF Intraceptor Gene Delivery to Laser-induced CNV. Gene Therapy 2009, 16(5), 645–659, DOI 10.1038/gt.2008.185. (8) Huang, C.; Tang, Z.; Zhou, Y.; Zhou, X.; Jin, Y.; Li, D.; Yang, Y.; Zhou, S. Magnetic Micelles as a Potential Platform for Dual Targeted Drug Delivery in Cancer Therapy. Int. J. Pharm. 2012, 429(1-2), 113–122, DOI 10.1016/j.ijpharm.2012.03.001. (9) Pan, W.; Yang, H.; Zhang, T.; Li, Y.; Li, N.; Tang, B. Dual-targeted Nanocarrier Based on Cell Surface Receptor and Intracellular mRNA: An Effective Strategy for Cancer Cell Imaging and Therapy. Anal. Chem. 2013, 85(14), 6930–6935, DOI 10.1021/ac401405n. (10) Dicheva, B. M.; Ten Hagen, T. L.; Schipper, D.; Seynhaeve, A. L.; van Rhoon, G. C.; Eggermont, A. M.; Koning, G. A. Targeted and Heat-triggered Doxorubicin Delivery to Tumors by Dual Targeted Cationic Thermosensitive Liposomes. J. Control Release 2014, 195, 37–48, DOI 10.1016/j.jconrel.2014.07.058.

24


Page 25 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(11) Wang, Y.; Zhao, R.; Wang, S. In vivo Dual-targeted Chemotherapy of Drug Resistant Cancer by

Rationally

Designed

Nanocarrier.

Biomater.

2016,

75:

71–81,

DOI

10.1016/j.biomaterials.2015.09.030. (12) Liu, J.; Liu, J.; Chu, L.; Zhang, Y.; Xu, H.; Kong, D.; Yang, C.; Ding, D. Self-assembling Peptide of D-amino Acids Boosts Selectivity and Antitumor Efficacy of 10hydroxycamptothecin. ACS Appl. Mater. Interfaces 2014, 6(8), 5558–5565, DOI 10.1021/am406007g. (13) Wang, S.; Yang, L.; Zu, Y.; Zhao, C.; Sun, X.; Zhang, L.; Zhang, Z. H. Design and Performance Evaluation of Ionic-Liquids-Based Microwave-Assisted Environmentally Friendly Extraction Technique for Camptothecin and 10-Hydroxycamptothecin from Samara of Camptotheca acuminata. Ind. Eng. Chem. Res. 2011, 50(24), 13620–13627, DOI 10.1021/ie201370m. (14) Chen, Q.; Wang, X.; Wang, C.; Feng, L.; Li, Y.; Liu, Z. Drug-Induced Self-Assembly of Modified Albumins as Nano-theranostics for Tumor-Targeted Combination Therapy. ACS Nano 2015, 9(5), 5223–5233, DOI 10.1021/acsnano.5b00640. (15) Ernsting, M. J.; Tang, W. L.; Maccallum, N.; Li, S. D. Synthetic Modification of Carboxymethylcellulose and Use Thereof to Prepare a Nanoparticle Forming Conjugate of Docetaxel for Enhanced Cytotoxicity Against Cancer Cells. Bioconjugate Chem. 2011, 22(12), 2474–2486, DOI 10.1021/bc200284b. (16) Lee, S.; Park, Y. H.; Ki, C. S. Fabrication of PEG–carboxymethylcellulose Hydrogel by Thiolnorbornene Photo-click Chemistry. Int. J. Biol. Macromol. 2016, 83, 1–8, DOI 10.1016/j.ijbiomac.2015.11.050. (17) Dong, R.; Zhou, Y.; Huang, X.; Zhu, X.; Lu, Y.; Shen, J. Functional Supramolecular Polymers for

Biomedical

Applications.

Adv.

Mater.

2015,

27(3),

498–526,

DOI

10.1002/adma.201402975. (18) Mura, S.; Nicolas, J.; Couvreur, P. Stimuli-responsive Nanocarriers for Drug Delivery. Nature Materials 2013, 12(11), 991–1003, DOI 10.1038/nmat3776. (19) Zhang, P.; Cheng, F.; Zhou, R.; Cao, J.; Li, J.; Burda, C.; Min, Q.; Zhu, J. J. DNA-hybridgated Multifunctional Mesoporous Silica Nanocarriers for Dual-targeted and Micrornaresponsive Controlled Drug Delivery. Angewandte Chemie 2014, 126(9), 2403–2407, DOI 10.1002/ange.201308920. (20) Gupta, M.; Chashoo, G.; Sharma, P. R.; Saxena, A. K.; Gupta, P. N.; Agrawal, G. P.; Vyas, S. P. Dual Targeted Polymeric Nanoparticles Based on Tumor Endothelium and Tumor Cells for 25



Page 26 of 30

Enhanced Antitumor Drug Delivery. Molecular Pharmaceutics 2014, 11(3), 697–715, DOI 10.1021/mp400404p. (21) Morris, V. J.; Belshaw, N. J.; Waldron, K. W.; Maxwell, E. G. The Bioactivity of Modified Pectin Fragments. Bioactive Carbohydrates & Dietary Fibre 2013, 1(1), 21–37, DOI 10.1016/j.bcdf.2013.02.001. (22) Munarin, F.; Tanzi, M. C.; Petrini, P. Advances in Biomedical Applications of Pectin Gels. Int. J. Biol. Macromol. 2012, 51(4), 681–689, DOI 10.1016/j.ijbiomac.2012.07.002.

(23) Zhang, W.; Xu, P.; Zhang, H. Pectin in Cancer Therapy: A Review. Trends in Food Science & Technology 2015, 44(2), 258–271, DOI 10.1016/j.tifs.2015.04.001. (24) Conti, S.; Vexler, A.; Hagoel, L.; Kalich-Philosoph, L.; Corn, B. W.; Honig, N.; Shtraus, N.; Meir, Y.; Ron, I.; Eliza, I.; Lev-Ari, S. Modified Citrus Pectin as a Potential Sensitizer for Radiotherapy

in

Prostate

Cancer.

Integrative

Cancer

Therapies

2018,

DOI:

10.1177/1534735418790382. (25) Günter, E. A.; Popeyko, O. V. Calcium Pectinate Gel Beads Obtained from Callus Cultures Pectins as Promising Systems for Colon-targeted Drug Delivery. Carbohydr. Polym. 2016, 147, 490–499, DOI 10.1016/j.carbpol.2016.04.026. (26) Muthukumarana, C.; Kanmani, B. R.; Sharmila, G.; Manoj, K. N.; Shanmugaprakash, M. Carboxymethylation of Pectin: Optimization, Characterization and In-vitro, Drug Release Studies. Carbohydr. Polym. 2018, 194, 311–318, DOI 10.1016/j.carbpol.2018.04.042. (27) Perera, G.; Barthelmes, J.; Bernkop-Schnürch, A. Novel Pectin–4-aminothiophenole Conjugate Microparticles for Colon-specific Drug Delivery. J. Controlled Release 2010, 145(3), 240–246, DOI 10.1016/j.jconrel.2010.04.024. (28) Ndeh, D.; Rogowski, A.; Cartmell, A.; Luis, A. S.; Baslé, A.; Gray, J.; Venditto, I.; Briggs, J.; Zhang, X.; Labourel, A.; Terrapon, N.; Buffetto, F.; Nepogodiev, S.; Xiao, Y.; Field, R. A.; Zhu, Y.; O'Neil, M. A.; Urbanowicz, B. R.; York, W. S.; Davies, G. J.; Abbott, D. W.; Ralet, M. C.; Martens, E. C.; Henrissat, B.; Gilbert, H. J. Complex Pectin Metabolism by Gut Bacteria Reveals

Novel

Catalytic

Functions.

Nature

2017,

548(7648),

65–70,

DOI

10.1038/nature23659. (29) Cobs-Rosas, M.; Concha-Olmos, J.; Weinstein-Oppenheimer, C.; Zúñiga-Hansen, M. E. Assessment of Antiproliferative Activity of Pectic Substances Obtained by Different Extraction Methods from Rapeseed Cake on Cancer Cell Lines. Carbohydr. Polym. 2015, 117, 923–932, DOI 10.1016/j.carbpol.2014.10.027.

26


Page 27 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(30) Zhang, Y.; Chen, T.; Yuan, P.; Tian, R.; Hu, W.; Tang, Y.; Jia, Y.; Zhang, L. Encapsulation of Honokiol into Self-assembled Pectin Nanoparticles for Drug Delivery to HepG2 Cells. Carbohydr. Polym. 2015, 133, 31–38, DOI 10.1016/j.carbpol.2015.06.102. (31) Díaz, C.; Vargas, E.; Gätjens-Boniche, O. Cytotoxic Effect Induced by Retinoic Acid Loaded into Galactosyl-sphingosine Containing Liposomes on Human Hepatoma Cell Lines. Int. J. Pharm. 2006, 325(1-2), 108–115, DOI 10.1016/j.ijpharm.2006.06.034. (32) Nakabayashi, H.; Taketa, K.; Miyano, K.; Sato, J. Growth of Human Hepatoma Cells Lines with Differentiated Functions in Chemically Defined Medium. Cancer Research 1982, 44(9), 3858–3863, DOI 10.1016/0304-3835(82)90136-7. (33) Mu, J. Z.; Gordon, M.; Shao, J. S.; Alpers, D. H. Apical Expression of Functional Asialoglycoprotein Receptor in the Human Intestinal Cell Line HT-29. Gastroenterology 1997, 113(5): 1501-1509, DOI 10.1053/gast.1997.v113.pm9352852. (34) Cheng, L.; Yang, K.; Li, Y.; Chen, J.; Wang, C.; Shao, M.; Lee, S. T.; Liu, Z. Facile Preparation of Multifunctional Upconversion Nanoprobes for Multimodal Imaging and Dual‐targeted Photothermal

Therapy.

Angewandte

Chemie

2011,

123(32),

7523-7528,

DOI

10.1002/anie.201101447. (35) Conde, J.; Bao, C.; Tan, Y.; Cui, D.; Edelman, E. R.; Azevedo, H. S.; Byrne, H. J.; Artzi, N.; Tian, F. Dual Targeted Immunotherapy via In Vivo Delivery of Biohybrid RNAi‐Peptide Nanoparticles to Tumor‐Associated Macrophages and Cancer Cells. Advanced Functional Materials 2015, 25(27): 4183-4194, DOI 10.1002/adfm.201501283. (36) Nie, Y.; Schaffert, D.; Rödl, W.; Ogris, M.; Wagner, E.; Günther, M. Dual-targeted Polyplexes: One Step Towards a Synthetic Virus for Cancer Gene Therapy. J. Control. Release 2011, 152(1), 127-134, DOI 10.1016/j.jconrel.2011.02.028. (37) Li, R.; Wu, R.; Zhao, L.; Hu, Z.; Guo, S.; Pan, X.; Zou, H. F. Folate and Iron Difunctionalized Multiwall Carbon Nanotubes as Dual-targeted Drug Nanocarrier to Cancer Cells. Carbon 2011, 49(5), 1797-1805, DOI 10.1016/j.carbon.2011.01.003. (38) Santra, S.; Kaittanis, C.; Santiesteban, O. J.; Perez, J. M. Cell-specific, Activatable and Theranostic Prodrug for Dual Targeted Cancer Imaging and Therapy. J. Amer. Chem. Soc. 2011, 133(41), 16680-16688, DOI 10.1021/ja207463b. (39) Liu, Y. X; Qi, Q.; Li, X. M.; Liu, J.; Wang, L. Y.; He, J.; Lei, J. D. Self-Assembled PectinConjugated Eight-Arm Polyethylene Glycol–Dihydroartemisinin Nanoparticles for Anticancer Combination Therapy. ACS Sustainable Chemistry & Engineering 2017, 5(9), 8097-8107, DOI 10.1021/acssuschemeng.7b01715. 27



Page 28 of 30

(40) Dai, L.; Liu, K. F.; Si, C. L.; He, J.; Lei, J. D.; Guo, L. Q. A Novel Self-assembled Targeted Nanoparticle Platform Based on Carboxymethylcellulose Co-delivery of Anticancer Drugs. Journal of Materials Chemistry B 2015, 3(32), 6605-6617, DOI 10.1039/C5TB00900F. (41) Zeng, X.; Tao, W.; Mei, L.; Huang, L.; Tan, C.; Feng, S. S. Cholic Acid-functionalized Nanoparticles of Star-shaped PLGA-vitamin E TPGS Copolymer for Docetaxel Delivery to Cervical

Cancer.

Biomaterials

2013,

34(25),

6058-6067,

DOI

10.1016/j.biomaterials.2013.04.052. (42) Pang, J.; Zhao, L.; Zhang, L.; Li, Z.; Luan, Y. Folate-conjugated Hybrid SBA-15 Particles for Targeted Anticancer Drug Delivery. J. Colloid Interface Sci. 2013, 395: 31-39, DOI 10.1016/j.jcis.2012.12.016. (43) Gates, C. M.; Stemmer, W. P. C.; Kaptein, R.; Schatz, P. J. Affinity Selective Isolation of Ligands from Peptide Libraries Through Display on a lac Repressor "Headpiece Dimer". J. Mol. Biol. 1996, 255(3), 373–386, DOI 10.1006/jmbi.1996.0031. (44) Bharali, D. J.; Klejbor, I.; Stachowiak, E. K.; Dutta, P.; Roy, I.; Kaur, N.; Bergey, E. J.; Prasad, P. N.; Stachowiak, M. K. Organically Modified Silica Nanoparticles: A Nonviral Vector for In Vivo Gene Delivery and Expression in the Brain. Proc. Natl. Acad. Sci. U.S.A. 2005, 102(32), 11539-11544, DOI 10.1073/pnas.0504926102.

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For Table of Contents Use Only Graphic Figure

Synthesis and preparation of dual-targeted folic acid modified pectin-based nanoparticles and combination therapy of liver cancer

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Dual-Targeted Controlled Delivery Based on Folic ... - ACS Publications

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