chitosan-based nanoparticles for in vitro synergistic combination chemotherapy of breast cancer

Journal Pre-proof Doxorubicin/cisplatin co-loaded hyaluronic acid/chitosan-based nanoparticles for in vitro synergistic combination chemotherapy of breast cancer Yaping Wang, Junmin Qian, Ming Yang, Weijun Xu, Jinlei Wang, Guanghui Hou, Lijie Ji, Aili Suo

PII:

S0144-8617(19)30873-2

DOI:

https://doi.org/10.1016/j.carbpol.2019.115206

Article Number:

115206

Reference:

CARP 115206

To appear in: Received Date:

11 July 2019

Revised Date:

13 August 2019

Accepted Date:

14 August 2019

Please cite this article as: Wang Y, Qian J, Yang M, Xu W, Wang J, Hou G, Ji L, Suo A, Doxorubicin/cisplatin co-loaded hyaluronic acid/chitosan-based nanoparticles for in vitro synergistic combination chemotherapy of breast cancer, Carbohydrate Polymers (2019), doi: https://doi.org/10.1016/j.carbpol.2019.115206

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Doxorubicin/cisplatin co-loaded hyaluronic acid/chitosan-based nanoparticles for in vitro synergistic combination chemotherapy of breast cancer

Yaping Wang a, Junmin Qian a,*, Ming Yang a, Weijun Xu a, Jinlei Wang a, Guanghui Hou a, Lijie Ji a, Aili Suo b,* a State Key Laboratory for Mechanical Behavior of Materials, Xi’an Jiaotong University, Xi’an 710049, China b Department of Oncology, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710061, China *Corresponding Authors: E-mail address: [email protected] (J.M. Qian); [email protected] (A.L. Suo); Tel: +86 29 82668614; Fax: +86 29 82663453

Highlights

DOX/CDDP-loaded polysaccharide-based nanovehicles were successfully prepared.

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CDDP crosslinking enhanced the stability of NPHER2(DOX/CDDP).

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CDDP and DOX were loaded onto AHA by chelation and Schiff’s base, respectively.

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Nanoparticles composed of drug core and polysaccharide shell were fabricated.

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Co-delivery of DOX and CDDP achieved great synergistic anticancer effect.

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Abstract Combination chemotherapy has attracted more and more attention in the field of anticancer treatment. Herein, a synergetic targeted combination chemotherapy of doxorubicin (DOX) and cisplatin in breast cancer was realized by HER2 antibody-decorated nanoparticles assembled from aldehyde hyaluronic acid (AHA) and hydroxyethyl chitosan (HECS). Cisplatin and DOX were successively conjugated onto AHA through chelation and Schiff’s base reaction, respectively, forming DOX/cisplatin-loaded AHA inner core. The core was sequentially complexed with HECS and targeting HER2 antibody-conjugated AHA. The formed near-spherical nanoplatform had an average size of ~160 nm and a zeta potential of −28 mV and displayed pH-responsive surface charge reversal and drug release behaviors. HER2 receptor-mediated active targeting significantly enhanced the cellular uptake of nanoplatform. Importantly, DOX and cisplatin exhibited a synergistic cell-killing effect in human breast cancer MCF-7 cells. These results clearly indicate that the novel nanoplatform is promising for synergistic combination chemotherapy of breast cancer. KEYWORDS: combination chemotherapy, self-assembly, pH-responsivity, targeted delivery, synergistic effect, breast cancer

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1. Introduction Breast cancer has become the primary cause of cancer incidence and mortality in women worldwide (Siegel et al., 2017). Currently, chemotherapy based on chemotherapeutic agents is still one of the most commonly used approaches in breast cancer treatment (Zhang et al., 2016). Doxorubicin (DOX), one of the most effective anticancer drugs, is an anthracycline-based topoisomerase II (TOP2) inhibitor that works by intercalating DNA and further inhibiting cell replication (Liu et al., 2016). Cisplatin (CDDP), another potent chemotherapeutic drug, acts as a DNA chelating agent by binding with DNA purine bases, which yields platinum-DNA adducts that may inhibit cellular transcription and replication (Jamieson & Lippard, 1999; Johnstone, Suntharalingam & Lippard, 2015; Wang & Lippard, 2005). However, like other small molecular chemotherapeutic agents, single DOX or CDDP chemotherapy often fails to achieve complete tumor eradication due to the rapid development of multidrug resistance in cancer cells (Bao et al., 2016; Holohan, Van Schaeybroeck, Longley & Johnston, 2013). Moreover, their intravenous administration usually causes systemic toxicity such as cardiotoxicity, myelosuppression, nausea 1

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and vomiting (Dayton et al., 2011; Kursunluoglu, Kayali & Taskiran, 2014). Some studies have suggested that the pathway through which DOX inhibits TOP2 is independent of that mediating DNA damage by CDDP, although both DOX and CDDP can intercalate DNA and seriously inhibit DNA activity (Wu et al., 2017). Moreover, DOX can inhibit the defective repair of CDDP-induced DNA damage (Guo et al., 2019), and the combination of DOX and CDDP may effectively overwhelm the DNA repair in tumor cells. It has been demonstrated that the combination chemotherapy of free DOX and CDDP exhibits a significantly enhanced therapeutic effect in endometrial cancer patients in a phase III clinical trial (Randall et al., 2006; Thigpen et al., 2004). However, this combination chemotherapy causes increased toxicity and side effects (Fuertes, Alonso & Perez, 2003). To address these problems and concurrently improve therapeutic effect, the development of efficient vehicles for the specific co-delivery of DOX and CDDP to tumor is highly urgent. In recent years, nanovehicle-based combination chemotherapy is considered as one promising strategy for overcoming the above-mentioned limitations and is being extensively explored (Anirudhan, Nair & Bino, 2017; Zhai, Hu, Hu, Wu & Xing, 2017). Combination chemotherapy based on nanotechnology has several advantages. Firstly, combination chemotherapy can effectively inhibit the occurrence of multidrug resistance (Hu, Sun, Wang & Gu, 2016). Secondly, different anticancer drugs with different action targets may improve therapeutic efficacy in a synergistic or combination fashion (Wen et al., 2019; Zong et al., 2018). Thirdly, in vitro cellular uptake and in vivo tumor accumulation of nanocarriers can be significantly enhanced through ligand-based active targeting or EPR-based passive targeting (Ou et al., 2018; Zong et al., 2018). Finally, the premature degradation of drugs loaded in nanocarriers can be avoided in the systemic circulation. Amongst various nanovehicles, the nanoparticles assembled from negatively and positively charged polyelectrolytes have attracted much interest because of their unique features, including potential targeting ability, facile loading of anticancer drugs, and pH-responsive drug release and surface charge reverse properties (Anirudhan, Vasantha & Sasidharan, 2017; Chai et al., 2017; Cui et al., 2019; Wang, Kankala, Fan, Long, Liu & Wang, 2018). Additionally, these polyelectrolyte-based nanoparticles can be decorated with targeting moieties, such as antibody (Liu, Du, Khan, Ji, Yu & Zhai, 2018), specific peptide (Fan et al., 2018), folic acid (Li et al., 2019) and other ligands (Song et al., 2017; Wang, Gu, Wang, Sun, Wu & Mao, 2017), to achieve active targeting through their specific interaction with receptors overexpressed in tumor cells. Negatively charged hyaluronic acid (HA) and positively charged chitosan (CS) are two important natural polysaccharide-based polyelectrolytes. They themselves and their derivatives are often employed to assemble with oppositely charged polymers into nanocomplexes or modify other nanoparticles due to their excellent biocompatibility and biodegradability (Lee et al., 2018; Srivastava & Purwar, 2017). Additionally, HA is widely used as active targeting moiety because it can specifically bind to CD44 receptors that are overexpressed in many cancer cells (Mattheolabakis, Milane, Singh & Amiji, 2015; Zhong et al., 2016). Some studies have indicated that nanovehicles that can perform rapid drug release under different stimuli such as temperature (Jin, Wu, Hou, Yu, Shen & Guo, 2018), pH (Chen et al., 2018; Li et al., 2018b), redox (Zhang, Shen, Zhao & Xu, 2017), photo-irradiation (Zhang, Li, Sun, Jia & Liu, 2018) or enzyme (Sharma, Kim, Shi, Lee, Chung & Kim, 2018), would improve chemotherapeutic efficacy. Especially, the nanovehicles that can respond to the acidity in tumor microenvironment have attracted the most interest since they not only can rapidly release drugs once their entrance into cancer cells but also facilitate the endosomal escape of drugs (Hu, Xu, Hu, Hu, Yang & Zhu, 2017; Zhu et al., 2017). Recently, we successfully developed multifunctional polysaccharide-based nanocomplexes from aldehyde hyaluronic acid (AHA) and hydroxyethyl chitosan (HECS) via layer-by-layer (LbL) self-assembly and accomplished combined chemotherapy and photodynamic therapy (Wang et al., 2019). Although such combination is simple and achieved good anticancer effect, this system is restricted from non-ideal stability and limited tissue penetration of excitation light in PDT. This nano-system formed via LbL self-assembly of oppositely charged polysaccharides holds great potential in the co-delivery of different anticancer drugs because of the versatility, facileness and simplexity of polyelectrolyte-based nanoparticles (Boudou, Crouzier, Ren,Blin, & 2

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Picart, 2010; Hammond, 2012). Moreover, its tumor targeting ability and cellular uptake can be enhanced by decorating targeting moieties onto its outside surface. Additionally, the pH-responsive charge-reversible and drug release properties of such nano-systems duet to the protonation of CS and the deprotonation of HA under slightly acidic tumor microenvironment can further enhance cellular uptake and cell-killing effect. It has been reported that the combination of DOX and CDDP based on nanoparticles exhibit a significant therapeutic effect in treating lung cancer in vitro (Xu et al., 2019). In the present study, we supposed that DOX and CDDP could be co-loaded into the polysaccharide-based nanovehicle via pH-responsive chemical linkages and this nano-system would show several unique benefits such as targeting ability, pH-responsive charge-reversible and drug release properties. Importantly, the introduction of CDDP crosslinking method into the nano-system would improve the stability of nanoparticles, and that the combination of CDDP and DOX could achieve synergistic antitumor ability while avoiding the application of external excitation light. Herein, to achieve synergistic DOX and CDDP combination chemotherapy, CDDP and DOX were firstly conjugated onto AHA macromolecules through the chelation between CDDP molecules and carboxyl groups and the Schiff base reaction between DOX molecules and aldehyde and carboxyl groups, respectively, forming the amphiphilic nanocores. The nanocores were sequentially coated with HECS and targeting ligand human epithelial growth factor receptor 2 (HER2) antibody-decorated AHA via Schiff base reaction. The resulting nanovehicles (NPHER2(DOX/CDDP)) were used for the DOX and CDDP combination chemotherapy in breast cancer MCF-7 cells. Moreover, the physicochemical characteristics of the nanovehicles and their in vitro chemotherapy effect were investigated systematically. We found that the nanovehicles exhibited good stability and had an average size of ~160 nm and pH-responsive drug release and charge reversal behaviors. Importantly, a highly synergistic potency of DOX and CDDP in the NPHER2(DOX/CDDP) formulation was observed. All in all, the novel DOX/CDDP-loaded polysaccharide-based nanovehicles had great potential for synergistically improving therapeutic efficacy of combination chemotherapy in breast cancer.

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Scheme 1. Schematic route for the synthesis of DOX/CDDP-loaded polysaccharide-based nanoparticles (NPHER2(DOX/CDDP)) from CDDP-crosslinked DOX/CDDP-AHA nanocore and polysaccharide shell.

2. Materials and methods 2.1. Materials. HA (MW 170 kDa) was purchased from Freda Biomedical Ltd Co., Ltd. (Shandong, China). CS (MW 150 kDa, 90% deacetylation), anti-HER2 monoclonal antibody, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 0.25% (w/v) trypsin, penicillin-streptomycin, and 4% paraformaldehyde were obtained from Sigma-Aldrich (Shanghai, China). Doxorubicin hydrochloride (DOX∙HCl) and CDDP were purchased from Dalian Meilun Biological Technology Co., Ltd. (Dalian, China) and Kunming Guiyan pharmaceutical Co., Ltd. (Kunming, China), respectively. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS) were

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purchased from Procell (Wuhan, China). An annexin V-EGFP/PI apoptosis detection kit was purchased from 7-Sea Biotech Co., Ltd. (Shanghai, China). 4',6-Diamidino-2-phenylindole (DAPI) was purchased from Dingguochangsheng Biotechnology Co. Ltd. (Beijing, China). NaIO4, 2-Chloroethanol and other chemical reagents were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Ultrapure water with a resistivity of 18.25 MΩ cm was used to prepare various aqueous solutions. 2.2. Formation of DOX/CDDP-AHA nanocores Lowly and highly oxidized HA (lAHA and hAHA) as well as HECS were synthesized according to our previously reported method (Wang et al., 2019). The degrees of oxidation of hAHA and lAHA were respectively determined to be 33.16 ± 4.06% and 6.95 ± 2.72%, and the substitution degree of hydroxyethyl groups of HECS was about 57.75%. Then, CDDP was conjugated onto hAHA via the chelation between CDDP and carboxyl groups of hAHA. Briefly, hAHA (180 mg) was dissolved in 30 mL of water, and CDDP (30 mg) was added after the pH was adjusted to 7.4. The reaction was gently shaken on the shaking table at 37 ºC for 72 h under dark conditions. CDDP-AHA was obtained by dialysis against water for 24 h and subsequent freeze-drying. After that, 60 mg of CDDP-AHA was dissolved in 10 mL of water, and then a certain amount of DOX was added. DOX was conjugated onto CDDP-AHA via Schiff's base reaction for 24 h at room temperature. Afterward, DOX/CDDP-AHA nanocores were obtained by three centrifugation-redispersion-washing cycles to remove free drugs. Similarly, DOX-loaded AHA nanoparticles were prepared by mixing AHA solution (60 mg, 10 mL) and different DOX amounts (10, 12, 15, 20, 30 or 60 mg) for 24 h at pH 7.4, followed by three centrifugation-redispersion-washing cycles. 2.3. Fabrication of NPHER2(DOX/CDDP) by LBL assembly NPHER2(DOX/CDDP) nanoparticles were obtained by successively coating DOX/CDDP-AHA nanocores with HECS and HAHER2 by LbL assembly method reported by us (Wang et al., 2019). Briefly, 2.0 mg DOX/CDDP-AHA nanocores were suspended in 1.0 mL of water, and the pH of the suspension was adjusted to 7.4. In the meanwhile, 2.0 mg of HECS was dissolved in 0.2, 0.4, 0.6, 0.8, or 1.0 mL of PBS and the pH was adjusted to 6.0. Under ultrasonic condition, HECS solution was added into DOX/CDDP-AHA suspension, followed by sequential continuous ultrasonic treatment for 5 min and stirring for 15 min. The mixture was centrifuged and water-washed to obtain HECS-coated DOX/CDDP-AHA (DOX/CDDP-AHA/HECS) nanoparticles. After that, 2.0 mg of HAHER2 was dissolved in 0.2, 0.4, 0.6, 0.8 or 1.0 mL of water and the pH was adjusted to 7.4. Meanwhile, DOX/CDDP-AHA/HECS nanoparticles were re-dispersed in 1.0 mL of water. Under ultrasonic condition, HAHER2 solution was added into DOX/CDDP-AHA/HECS suspension and the system was ultrasonically treated for 5 min and stirred for 15 min, obtaining HAHER2-coated DOX/CDDP-AHA/HECS (NPHER2(DOX/CDDP)) nanoparticles. Correspondingly, pristine AHA/HECS/AHA (NP), DOX-loaded AHA/HECS/HAHER2 (NPHER2(DOX)), and DOX-loaded AHA/HECS/AHA (NP(DOX)) nanoparticles were prepared following the similar procedure, respectively. 2.4. Structural characterization FT-IR analysis was performed on a Prestige-21 FTIR spectrophotometer (Shimadzu, Japan) in the 4000-400 cm-1 range with a resolution of 4 cm−1. Before the measurements, the sample was mixed with KBr and the mixture was compressed into a thin tablet for analysis. The zeta potential and hydrodynamic size were measured at 25 ºC using a ZetaSizer Nano ZS90 (Malvern Instruments Ltd., UK). The morphologies of DOX/CDDP-AHA nanocores, DOX/CDDP-AHA/HECS nanoparticles, and NPHER2(DOX/CDDP) nanoparticles were characterized by a JEM-200CX transmission electron microscope (TEM, Nippon Electric Co. Ltd. Japan). For TEM analysis, 5 μL of sample suspension was dropped onto a copper mesh grid and excess liquid was removed with filter paper. 2.5. Charge-reversal behavior of NPHER2(DOX/CDDP) nanoparticles To study the surface charge characteristics of NPHER2(DOX/CDDP) nanoparticles, the zeta potentials of NPHER2(DOX/CDDP) suspension (1.0 mg/mL) in PBS at different pH values were measured. 2.6. Drug loading ability Before and after DOX and CDDP loading, the amounts of DOX and CDDP were recorded, and all supernatants 4

were collected to determine drug content. The contents of DOX and CDDP in nanoparticles were determined by a UV-vis spectrophotometer (Persee TU-1810, Beijing, China) at the absorbance of 480 nm and inductively coupled plasma optical emission spectrometer (ICP-OES, ICAP 7000, Thermo Scientific, American), respectively. The drug loading capacity (DLC) and drug loading efficiency (DLE) of DOX and CDDP were calculated according to the following equations. 𝐷𝐿𝐶 (%) =

Weight of initial DOX or CDDP − Weight of DOX or CDDP in supernatant

𝐷𝐿𝐸 (%) =

Weight of DOX or CDDP in nanoparticles

Weight of nanoparticles Weight of nanoparticles

× 100

× 100

(1) (2)

Cumulative release (%) =

𝐶t 𝑉o +𝑉1 ∑𝑡−1 𝑖=1 𝐶𝑖 𝑀total

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2.7. In vitro pH-responsive drug release To determine the effect of pH on the release profiles of DOX and CDDP, the release of DOX and CDDP was evaluated at different pH values by a dialysis method. Briefly, 5.0 mg of dual-drug-loaded nanoparticles were ultrasonically dispersed in 5 mL of PBS with pH=7.4 or 5.5 and then the suspensions were transferred into dialysis bags (MWCO = 3500 Da) against 100 mL of dialysis medium (pH = 7.4 or 5.5). The release was performed in a constant temperature shaking table with a shaking speed of 100 rpm at 37 °C. At preset time intervals, 2 mL of medium was taken out and an equal volume of fresh medium was compensated. The concentrations of DOX and CDDP in samples were determined by UV-vis spectroscopy (wavelength: 480 nm) and ICP, respectively. The cumulative release (%) of DOX or CDDP at a predetermined time point (t) was calculated according to Eq. (3): × 100

(3)

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where, Mtotal indicates the loading content of DOX or CDDP within the nanoparticles. V0 and V1 indicate the initial volume of incubation medium and the volume of collected medium at preset time intervals (V0 = 100 mL and V1 =2 mL), respectively. Ct indicates the concentration of DOX or CDDP released in medium at the scheduled time points. 2.8. Cell culture Human breast cancer cell lines MCF-7 and MDA-MB-435 were provided by Medical Center of Xi’an Jiaotong University. The cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin at 37 ºC in a humidified incubator with 5% CO2 atmosphere. 2.9. In vitro cytotoxicity assay The cytotoxicity of pristine and drug-loaded nanoparticles was evaluated by the MTT assay. MCF-7 cells were seeded in a 96-well plate (2.0×104 cells/well) for 24 h. Different formulations with pristine or drug-loaded nanoparticle suspensions at different concentrations were added. Cell viability was measured by the MTT assay after incubation for another 48 or 72 h. After that, 20 μL of MTT (5 mg/mL) was added and cells were incubated for another 4 h at 37 ºC in the dark. Then, 200 μL of DMSO was added to replace the MTT solution and the optical density (OD) was measured with a microplate reader (PerkinElmer, EnSpire, America) at 492 nm. Cell viability was calculated according to Eq. (4): 𝑂𝐷sample

𝑂𝐷control

× 100

(4)

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Cell viability(%) =

Where ODsample and ODcontrol were the optical densities of sample and control wells, respectively. The average value of three measurements from different batches was adopted. The dose-effect profiles were obtained by sigmoidal logistic fitting by use of Origin 8.0 (OriginLab, Northampton, MA) and the inhibitory concentration (ICX) values were based on the fitted data. The synergistic, additive, or antagonistic effects of two different drugs were evaluated from combination index (CI) analysis based on the Chou-Talalay method (Chou, 2006), according to the following equation (5): (𝐷)DOX

𝐶𝐼𝑥 = (𝐷

x )DOX

(𝐷)CDDP

+ (𝐷

(5)

x )CDDP

Where (DX)DOX and (DX)CDDP represent the ICX values of DOX alone and CDDP alone, respectively. (D)DOX and 5

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(D)CDDP represent the concentrations of DOX and CDDP in the combination system at the ICX value. CI > 1 represents antagonism, CI = 1 represents additive and CI < 1 represents synergism. 2.10. In vitro cellular uptake The cellular uptake of NPHER2(DOX/CDDP), NPHER2(DOX), and NP(DOX) nanoparticles were evaluated. A fluorescence microscope (Olympus, AE31, Japan) was used to observe the intracellular distribution of these nanoparticles. Briefly, MCF-7 cells were seeded in 2 mL of DMEM complete culture medium in 6-well plates (5.0×104 cells/well) for 24 h, and then different formulations at an equivalent DOX concentration of 2 μg/mL were added. After incubation for another 1, 4 or 24 h, culture medium was removed, cells were washed with PBS three times, fixed in 4% paraformaldehyde, and stained with DAPI. For quantitative analysis, MCF-7 and MDA-MB-435 cells were seeded in 6-well plates (3.0×105 cells/well) for 24 h, respectively. After different formulations (2 μg DOX/mL) were added, the cells were incubated for another 1, 4 or 24 h. Untreated cells were used as control group. After incubation, the cells were digested and washed with PBS. Harvested cells were centrifuged, re-suspended in PBS, and analyzed with a flow cytometer (Becton Dickson FACS Canto II, BD Biosciences, USA). 2.11. In vitro apoptosis assay To evaluate the effects of DOX, CDDP or their combination chemotherapy, the apoptosis and necrosis ratios of MCF-7 cells were assessed using flow cytometry. Briefly, MCF-7 cells were seeded in 6-well plates (4.0×105 cells/well) for 24 h. The cells were then treated with NPHER2(DOX/CDDP), NPHER2(DOX) and NP(DOX) nanoparticles as well as free CDDP and DOX for 72 h. Untreated cells were used as a control. After incubation, the cells were washed with PBS, detached with 0.25% trypsin, collected and re-suspended in 400 μL buffer. Annexin V-EGFP and PI solutions were added and the cells were incubated for 15 min in the dark. The samples were immediately analyzed on flow cytometer.

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3. Results and discussion 3.1. Formation and characterization of DOX/CDDP-AHA and DOX-AHA nanocores A HA derivative hAHA (oxidation degree: 33.16% ± 4.06%) was employed to load DOX+CDDP or DOX alone because it may react with DOX and CDDP and has good hydrophilicity. DOX could be efficiently conjugated onto AHA via Schiff's base reaction. Additionally, since the pKa values of DOX and HA are 8.3 (Lv et al., 2013) and 3.2 (Choi et al., 2015), respectively, their electrostatic interactions could also facilitate the laoding of DOX onto AHA. Therefore, the conjugate of hydrophobic DOX and hydrophilic AHA could assemble into DOX-AHA nanocores. CDDP was conjugated onto AHA via chelation reaction. Since both CDDP and AHA are hydrophilic, the resultant CDDP-AHA chelate existed in the form of aqueous solution. Once DOX was conjugated onto CDDP-AHA chelate through Schiff's base reaction, DOX/CDDP/AHA nanocores were formed. DLS analysis indicated the size of DOX/CDDP-AHA nanocores gradually increased with the feed weight ratio of DOX and AHA (Fig. 1A), while their zeta potential was almost constant at -35 mV (Fig. 1B). The increase in size suggested the efficient loading of more DOX on CDDP-AHA chelate through chemical conjugation, while the unchanging zeta potential confirmed the stability of nanocores. Considering subsequent coating of HECS and HAHERs onto DOX/CDDP-AHA nanocores, small-sized nanocores formed at a feed weight ratio of DOX and AHA of 1/6 were chosen for the LBL process. The formation of DOX/CDDP-AHA nanocores was verified by FTIR spectrometry. As shown in Fig. 1C, the absorption peak at 1632 cm-1 was attributed to the asymmetrical stretching vibrations of carboxyl groups of AHA. However, a red shift of 10 cm-1, from 1632 cm-1 to 1642 cm-1, was observed in the FTIR spectrum of CDDP-AHA. This may be due to the formation of coordination bond between Pt in CDDP (weak Lewis acid) and oxygen in carboxylate (strong base) (Wang et al., 2014). In addition, a new peak at 1200 cm-1 was attributed to the stretching vibrations of N-H bonds in CDDP (Haririan, Alavidjeh, Khorramizadeh, Ardestani, Ghane & Namazi, 2010). The peaks at 1579, 1282, 1206, 1114 and 986 cm-1 were ascribed to the characteristic absorption peaks of DOX (Das et al., 2010). These results suggested that DOX and

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CDDP were successfully conjugated onto AHA.

Fig. 1. Characterization of DOX/CDDP-AHA nanocores. Effects of feeding weight ratio of DOX and AHA on the size (A) and zeta potential (B) of DOX/CDDP-AHA nanocores. FTIR spectra (C) of AHA, CDDP-AHA and DOX/CDDP-AHA nanocores. Error bars indicate SD (n=3).

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3.2. Preparation of NPHER2(DOX/CDDP) by LBL assembly NPHER2(DOX/CDDP) or NPHER2(DOX) nanoparticles were prepared by sequential assembly of polycationic HECS and polyanionic HAHER2 on DOX/CDDP-AHA or DOX-AHA nanocores. To accomplish LBL assembly, the inner core surface must display enough net charges to provide favorable sites for the deposition of oppositely charged polyelectrolyte. In the present study, HECS and HAHER2 were used as cationic and anionic polyelectrolytes, respectively. The mass ratio of polyelectrolyte to inner core during the LBL assembly had a decisive impact on the formation of nanoparticles. The zeta potential and average size of the nanoparticles assembled at different mass ratios of polyelectrolyte and inner core were evaluated, and the results are given in Fig. 2. Since the initial DOX/CDDP-AHA core was negatively charged, different mass ratios of HECS to DOX/CDDP-AHA (1:5, 2:5, 3:5, 4:5, 1:1) were adopted to determine a suitable HECS amount. Since the pKa value of CS is approximately equal to 6.5 (Hillberg & Tabrizian, 2006), the pH of HECS in PBS (2 mg/mL) was fixed pH=6.0 to display a high positive charge density. As shown in Fig. 2A, when the mass ratio of HECS and DOX/CDDP-AHA was less than 2:5, the zeta potential of nanoparticles was still negative. As the ratio increased up to 3:5 or was greater than this ratio, the zeta potential became from negative to positive, which indicated that enough HECS had been attached onto the nanocore surface. Although the nanoparticle size did not significantly change with increasing mass ratio, it showed a maximal value when zeta potential was approximate to zero. This may be due to nearly equal numbers of negative changes on DOX/CDDP-AHA nanocore and positive charges on HECS. Under such condition, no residual electrostatic force to prevent the nanoparticles from approaching each other (Chun, Choi, Min & Weiss, 2013), resulting a maximal particle size. After this, the particle size gradually decreased. When the mass ratio of HECS and DOX/CDDP-AHA increased to 1:1, the size and zeta potential of DOX/CDDP-AHA/HECS nanoparticles became 155 nm and +49 mV, respectively. The mass ratio was chosen for the following assemble process. In order to endow the nanoparticles with targeting ability, their surfaces were covered with HAHER2 via Schiff’s base reaction. It was found that the zeta potential of final nanoparticles obviously decreased as the mass ratio of HAHER2 and DOX/CDDP-AHA/HECS increased, while their size did not significantly change. When their mass ratio increased to 1:1, the as-formed nanoparticles had a particle size of 162 nm and a relatively low zeta potential of -28 mV, which suggested their good stability in aqueous solutions. Therefore, the mass ratio was chosen for the following studies. In addition, NP(DOX) nanoparticles were constructed according to the same procedure except replacing HAHER2 with AHA.

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Fig. 2. Effects of successive HECS (A) and HAHER2 (B) deposition on nanoparticle size and zeta potential during LBL assembly process. Error bars indicate SD (n=3). The concentration of HAHER2 solution (pH 7.4) was fixed at 2 mg/mL.

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3.3. Physicochemical characterization of assembled nanoparticles NPHER2(DOX/CDDP) was prepared by LBL assembly method using DOX/CDDP-AHA nanoparticle as nanocores, and the corresponding size distribution and morphology are shown in Fig. 3. Dynamic light scattering (DLS) measurement showed that the average sizes of DOX/CDDP-AHA, DOX/CDDP-AHA/HECS and DOX/CDDP-AHA/HECS/HAHER2 nanoparticles were 135, 155 and 162 nm, respectively. The gradual increase in average size suggested that the successful successive assembly of HECS and HAHER2 on DOX/CDDP-AHA nanocores. The TEM images in Fig. 3 indicated that the above-mentioned three kinds of nanoaprticles displayed a near-spherical morphology and their average sizes were 125, 150 and 160 nm, respectively. Obviously, the sizes of dry particles determined by TEM were slightly smaller than those in aqueous solutions measured by DLS (Gao et al., 2017). As shown in Fig. 3D, during the assembly process, the zeta potential of nanoparticle increased from initial -38 mV to +49 mV, and decreased to -28 mV. This change further confirmed the successful LBL assembly of HECS and HAHER2 on DOX/CDDP-AHA nanocores.

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Fig. 3. Hydrodynamic size distributions of DOX/CDDP-AHA nanocores (A), DOX/CDDP-AHA/HECS nanoparticles (B) and DOX/CDDP-AHA/HECS/HAHER2 nanoparticles (C). Insets are TEM images of samples. Scale bar = 200 nm. (D) Change in surface zeta potential during the assembly process. Error bars indicate SD (n=3).

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3.4. Drug loading and pH-responsive drug release and charge reversal behaviors For a potential drug delivery system, drug loading is crucial to estimate its therapeutic efficacy. In our study, considering the stability and size of NPHER2(DOX/CDDP) under physiological conditions, NPHER2(DOX/CDDP) prepared according to the optimized formulation had high DLC and DLE, 20.55% and 21.4% for DOX and 9.53% and 81.71% for CDDP, which were higher than those in the literature (Zhang et al., 2017; Zhang et al., 2018). Another key parameter for nano-vehicles is their drug release behaviors. It is well-known that tumor microenvironment is slightly acidic. Thus, the release behaviors of DOX and CDDP were investigated in PBS solutions with pH 7.4 and pH 5.5 which imitate normal physiological conditions and acidic tumor microenvironment, respectively. As shown in Fig. 4A, the release of both DOX and CDDP at pH 5.5 was greater than at pH 7.4. The reason for this is that the deionization of carboxyl groups of AHA (Pramod, Shah & Jayakannan, 2015) and the breakage of Schiff base linkage under acidic conditions disrupted the connections between AHA and DOX/CDDP. For DOX release, 51.2% of DOX could be released from NPHER2(DOX/CDDP) within 24 h at pH 5.5, while only 12.45% of DOX was released at pH 7.4. Similarly, the cumulative release rates of CDDP from NPHER2(DOX/CDDP) within 24 h were 57.68% and 25.87% at pH 5.5 and pH 7.4, respectively. It was also noted that CDDP was released faster than DOX. This may be caused by their different release 8

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mechanisms. DOX was released via the cleavage of Schiff’s base linkages, while CDDP was released through the breakage of coordination bonds between carboxyl groups and cisplatin. These results suggested that the pH-responsive drug release behaviors of NPHER2(DOX/CDDP) would enhance drug accumulation in acidic tumor tissue and achieve combined DOX + CDDP chemotherapy. The pH-responsive surface charge-reversal behavior of NPHER2(DOX/CDDP) was investigated in the pH 8-5 range. As shown in Fig. 4B, the zeta potential of NPHER2(DOX/CDDP) increased from -33.2 mV to +13.7 mV as the pH value decreased from 8.0 to 5.0, and the value of zeta potential reversed at pH 6.4-6.0. This indicated that NPHER2(DOX/CDDP) had an acid-responsive surface charge-reversal feature that may enhance cellular uptake of nanoparticles (Li et al., 2018a). In addition, the positive zeta potentials at low pH values (≤ 6.0) may be due to the protonation of HECS. Similarly, the negative surface charge at high pH values (≥ 6.4) was due to the deprotonation of HAHER2 and simultaneous HECS shrinkage. The above-mentioned pH-responsive surface charge-reversal behavior would make NPHER2(DOX/CDDP) stability during blood circulation and enhance their cellular uptake in acidic tumor microenvironment (Du, Sun, Song, Wu & Wang, 2010). Taken together, NPHER2(DOX/CDDP) displayed good physiological stability and its pH-sensitivity might achieve surface charge-reversal, enhancing its cellular uptake. These results showed that CDDP crosslinking effectively decreased the release (12.45%) of DOX in NPHER2(DOX/CDDP) at pH 7.4 within 24 h as compared to that (17.89%) of DOX/ALA-AHCS/HAHER2 and changed charge reversal point of nanoparticles (Wang et al., 2019).

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Fig. 4. (A) pH-responsive release profiles of DOX and CDDP from NPHER2(DOX/CDDP) at pH 5.5 and 7.4. (B) zeta potential of NPHER2(DOX/CDDP) at different pH values. Error bars indicate SD (n=3).

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3.5. In vitro cellular uptake The cellular uptake of NPHER2(DOX/CDDP), NPHER2(DOX), and NP(DOX) in MCF-7 cells was first investigated by using a fluorescence microscope. Cell nucleus was identified by staining DAPI (blue), while DOX emitted red fluorescence. As shown in Fig. 5A-C, these three formulations displayed a time-dependent cellular uptake behavior. After incubation for 1 h, a weak red fluorescence was observed. The red fluorescence in the nuclei became stronger when incubation time was prolonged to 4 and 24 h, confirming the successful cellular uptake of DOX (Li et al., 2014). Compared with HER2-targeted NPHER2(DOX/CDDP) and NPHER2(DOX), NP(DOX)-treated cells exhibited much weaker red fluorescence at the same incubation time. This is because anti-HER2 antibody decoration enhanced the specific binding of nanoparticles to HER2 receptors expressed on the surface of MCF-7 cells (Zhang et al., 2017). Additionally, it was noted that the cells treated with NPHER2(DOX/CDDP) showed a similar DOX fluorescence to those treated with NPHER2(DOX) at the same incubation time, indicating that the existence of CDDP did not influence the release of DOX. The cellular uptake of nanoparticles was quantitatively evaluated by flow cytometry. As shown in Fig. 5D, DOX fluorescence intensity obviously increased in three different groups when incubation time increased from 1 to 24 h, confirming the time dependency of cellular uptake of the nanoparticles. Moreover, the intensity of DOX fluorescence in HER2 antibody decoration nanoparticle groups was about 2 times higher than that of NP(DOX) group. These results were consistent with those of fluorescence microscopy analysis. To directly verify the specificity of 9

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NPHER2(DOX) toward HER2 receptors, we performed a HER2 competition experiment on MCF-7 and MDA-MB-435 cells. Cells were pretreated with free anti-HER2 antibody (2 mg/mL) and subsequently treated with NPHER2(DOX). Just as expected, the DOX fluorescence intensity in pretreatment groups was much weaker than that of the unpretreated group and was similar to that of non-targeting NP(DOX) group (Fig. 5E and F). These results demonstrated that the cellular uptake of NPHER2(DOX) in anti-HER2 antibody pretreated cells was significantly suppressed. Therefore, the decoration of LBL assembled nanoparticles with HER2 antibody could enhance their cellular uptake in breast cancer cells.

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Fig. 5. Cellular uptake of NP(DOX) (A), NPHER2(DOX) (B) and NPHER2(DOX/CDDP) (C) in MCF-7 cells after culture for 1, 4, and 24 h, as displayed in fluorescence microscopy images. The equivalent concentration of DOX was 2 μg/mL. The images in each row display DAPI-stained nuclei (blue), DOX fluorescence (red) and their overlays, respectively. Scale bars represent 25 μm. (D) Intracellular mean fluorescence intensities of MCF-7 cells incubated with NP(DOX), NPHER2(DOX), and NPHER2(DOX/CDDP) for 1, 4 and 24 h. Intracellular mean fluorescence intensities of MCF-7(E) and MDA-MB-435 (F) cells incubated for 24 h with NP(DOX), NPHER2(DOX), and NPHER2(DOX) after free anti-HER2 (2 mg/mL) pretreatment.

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3.6. In vitro cytotoxicity and synergistic profiles The in vitro cytotoxicity of different formulations (free DOX, free CDDP, mixture of free DOX and CDDP, NP(DOX), NPHER2(DOX), and NPHER2(DOX/CDDP)) was evaluated in MCF-7 cells using the MTT assay. Firstly, the cytocompatibility of pristine (blank) AHA/HECS/AHA nanoparticles was evaluated, and the results are shown in Fig. 6. It could be seen that MCF-7 cells treated with all experimental concentrations of pristine nanoparticles had a high cell viability (> 80%) and negligible toxicity during 48-h incubation, demonstrated their good safety. Then, the proliferation inhibition effect of different formulations on MCF-7 cells was measured, and the corresponding cytotoxicity profile was shown in Fig. 7 (A, B). The results indicated that all the formulations had dose- and time-dependent cell proliferation inhibition effects. Additionally, the therapeutic effect of combination chemotherapy was also time-dependent, and the effect was much greater than that of single-drug chemotherapy after 72 h of incubation. The inhibition concentrations of different formulations are displayed in Table 1, where ICx is the drug concentration with an inhibition rate of x%. For DOX, the IC50 values of NPHER2(DOX), mixture of free DOX and CDDP, and NPHER2(DOX/CDDP) were 2.89, 2.76 and 2.70 µg/mL at 48 h and 1.89, 1.63 and 1.38 µg/mL at 72 h. These results indicated that DOX+CDDP groups had a greater cell proliferation inhibition effect than NPHER2(DOX) group and that the inhibition effects were time-dependent which was consistent with the 10

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release behaviors of DOX and CDDP. The IC50 of free DOX was 1.82 µg/mL that was slightly lower than that of NPHER2(DOX). This was due to the rapid entrance of free DOX into the nuclei. It can also be seen from Fig. 7 (A, B) that the inhibition concentration of NP(DOX) was higher than that of NPHER2(DOX) at 48 and 72 h at the same ICx, suggesting that NP(DOX) had a lower inhibition effect. This is because that the HER2-antibody decoration could increase the cell proliferation inhibition effect of the nanoparticles. To explore the combination effect (antagonistic, additive or synergistic) of DOX and CDDP in the present study, combination index (CI) was introduced. CI values were determined by dose-effect profiles and could provide quantitative information about the extent of interactions between DOX and CDDP, with CI > 1, CI = 1 or CI < 1 denoting antagonism, additivity, or synergism, respectively. As shown in Fig. 7C, the CI plots for NPHER2(DOX/CDDP) clearly indicated the existence of synergistic effect in MCF-7 cells in a wide range of IC75-IC25, while the combination of free DOX and CDDP at a ratio of 2.1 yielded an antagonistic effect. The isobologram of NPHER2(DOX/CDDP) and combination of free DOX and CDDP was analyzed and the results are shown in Fig. 7D. It was found that the isoeffect level of NPHER2(DOX/CDDP) was lower than that of combination of free DOX and CDDP and the obvious synergistic effect was observed for NPHER2(DOX/CDDP), which is in accordance with the results of CI analysis. In a sharp contrast, combination of free DOX and CDDP at a ratio of 2.1 produced an incrementally increasing antagonistic effect, which was due to the resistance of partial MCF-7 cells to CDDP (Yde & Issinger, 2006). Therefore, NPHER2(DOX/CDDP) displayed a prominent synergistic effect between DOX and CDDP, which was not found in the combination of free DOX and CDDP or single drug-loaded nanoparticles. Even so, further investigation about optimal DOX/CDDP ratio and total drug content is needed to evaluate the potential of the LbL assembled AHA/HECS-based nanoplatform.

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Fig. 6. Cell viabilities of MCF-7 cells incubated with blank AHA/HECS/AHA nanoparticles for 48 h. Error bars indicate SD (n=3). Table 1 ICx values for MCF-7 cells after 48 and 72 h of incubation

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Free

48 h

72 h

IC25 IC35 IC50 IC25 IC35 IC50 IC65 IC75

Free

Free DOX/CDDP

DOX (μg/mL)

CDDP (μg/mL)

DOX (μg/mL)

CDDP (μg/mL)

1.39 2.10 3.54 0.58 0.97 1.82 3.21 4.73

2.53 4.02 8.98 0.90 1.51 2.93 5.65 9.33

0.77 1.41 2.76 0.54 0.89 1.63 2.79 4.02

0.37 0.67 1.31 0.26 0.43 0.78 1.33 1.91

NPHER2(DOX)

NPHER2(DOX/CDDP)

DOX (μg/mL)

DOX (μg/mL)

CDDP (μg/mL)

1.02 1.64 2.89 0.58 1.00 1.89 3.31 4.70

0.81 1.43 2.70 0.41 0.71 1.38 2.50 3.71

0.38 0.68 1.29 0.19 0.34 0.66 1.19 1.77

(ICx indicates drug concentration in the case of inhibition rate of x%.)

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Fig. 7. In vitro cytotoxicity profiles of MCF-7 cells incubated with free DOX, NP(DOX), NPHER2(DOX), and NPHER2(DOX/CDDP). Dose-effect profiles for MCF-7 cells after 48 h (A) and 72 h (B) of incubation. (C) CI plots for free DOX+CDDP and NPHER2(DOX/CDDP) after 72 h of incubation. (D) Dose-normalized isobologram analysis based on the isoeffective points from dose-effect curves.

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3.8. Cell apoptosis assay To explore the reason for the cell proliferation inhibition caused by the nanoparticles, annexin V-EGFP/PI double staining assay was performed by flow cytometry to analyze cell apoptosis, and the results are shown in Fig. 8. MCF-7 cells were exposed to PBS, free DOX, free CDDP, NP(DOX), NPHER2(DOX) and NPHER2(DOX/CDDP) for 72 h. The concentrations of DOX and CDDP were 2.0 μg/mL and 0.95 μg/mL, respectively. In PBS and free CDDP groups, about 1% and 23% of cell populations were at apoptotic stages, respectively. This was attributed to the resistance of partial MCF-7 cells to CDDP. In NPHER2(DOX) group, the percentage of apoptotic cells was about 39% that was higher than those (32% and 25%) in free DOX and NP(DOX) groups. The reasons for these results are that HER2 antibody decoration enhanced cellular uptake and free DOX entered cell nuclei faster than non-targeted nanoparticles. The highest apoptotic cell population (~62%) was observed in NPHER2(DOX/CDDP) group, demonstrating the therapeutic potential of NPHER2(DOX/CDDP).

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Fig. 8. Apoptotic effect of MCF-7 cells treated with PBS (a), free DOX (b), free CDDP (c), NP(DOX) (d), NPHER2(DOX) (e) and NPHER2(DOX/CDDP) (f). The lower left, lower right, upper right and upper left quadrants in each flow cytometric sorting profile present the percentages of living, early apoptotic, late apoptotic and necrotic cells, respectively.

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4. Conclusions A novel DOX/CDDP-loaded polysaccharide-based nanovehicle was successfully synthesized . Amphiphilic inner nanocore was formed by successive conjugation of CDDP and DOX onto AHA. CDDP was chelated on AHA through the coordination between CDDP and carboxyl groups of AHA, while DOX was loaded on AHA via Schiff’s base reaction between DOX molecules and aldehyde groups of AHA. The DOX/CDDP-AHA core was surrounded by oppositely charged polyelectrolytes AHA and HECS via LBL assembly process. The CDDP-crosslinked NPHER2(DOX/CDDP) had enhanced stability and displayed a near-spherical morphology and had an average size of 160 nm. Importantly, HER2 antibody decoration facilitated the internalization of NPHER2(DOX/CDDP) in MCF-7 cells, and its pH-responsive surface charge reversal and release of DOX and CDDP enhanced cell killing in breast cancer MCF-7 cells. Pristine nanoplatform exhibited good cytocompatibility. In a word, this study demonstrated the effectiveness of the new LBL assembly strategy for targeted co-delivery of DOX and CDDP to MCF-7 cells and the achievement of synergetic combination chemotherapy. These good in vitro results would encourage us to perform in vivo studies about NPHER2(DOX/CDDP) in future.

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