Design of an Anticancer Copper(II) Prodrug Based on the Lys199

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Design of an anticancer copper(II) pro-drug based on the Lys199 residue of the active targeting human serum albumin nanoparticle carrier Yi Gou, Yao Zhang, Zhenlei Zhang, Jun Wang, Zuping Zhou, Hong Liang, and Feng Yang Mol. Pharmaceutics, Just Accepted Manuscript • Publication Date (Web): 04 May 2017 Downloaded from http://pubs.acs.org on May 4, 2017

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Molecular Pharmaceutics

Design of an anticancer copper(II) pro-drug based on the Lys199 residue of the active targeting human serum albumin nanoparticle carrier Yi Gou1,3,Yao Zhang1, Zhenlei Zhang1, Jun Wang1, Zuping Zhou2, Hong Liang1*, Feng Yang1* 1

State Key Laboratory for the Chemistry and Molecular Engineering of

Medicinal Resources, Ministry of Science and Technology of China. Guangxi Normal University, Guilin, Guangxi, China. 2

Guangxi Universities Key Laboratory of Stem Cell and Pharmaceutical

Biotechnology, Guangxi Normal University, Guilin, Guangxi, China. 3

School of Pharmacy, Nantong University, Nantong, Jiangsu, China.

*

Corresponding author:

Hong Liang, [email protected] Feng Yang, [email protected]

Phone/Fax : 86-773-584-8836 Address: 15 Yucai Road, Guilin, Guangxi, China. Zip code: 541004

Keywords: human serum albumin; nanoparticles; folic acid; pro-drug; targeting.

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ABSTRACT We not only modified the types and numbers of coordinated ligands in a metal agent to enhance its anticancer activity, but we also designed a metal pro-drug based on the Ndonor residues of the human serum albumin (HSA) IIA sub-domain to improve its delivery efficiency and selectivity in vivo. However, there may be a conflict in simultaneously achieving the two goals because Lys199 and His242 in the IIA subdomain of HSA can replace its two coordinated ligands, which will decrease its anticancer activity relative to the original metal agent. Thus, to improve the delivery efficiency of the metal agent and simultaneously avoid decreasing its anticancer activity in vivo, we decided to develop an anticancer metal pro-drug by regulating its pharmacophore ligand so that it would be not displaced by the Lys199 residue of the folic acid (FA)-functionalized HSA nanoparticle (NP) carrier. To this end, we first synthesized two (E)-N'-(5-chloro-2-hydroxybenzylidene)benzohydrazide Schiff base (HL) Cu(II) compounds by designing a second ligand with a different coordinating atom with Cu2+:Cu(L)(QL)(Br) [C1, QL = quinolone] and Cu(L)(DMF)(Br) [C2, DMF = N,Ndimethylformamide]. As revealed by the structures of the two HSA complexes, the Cu compounds bind to the hydrophobic cavity in the HSA IIA subdomain. The QL ligand of C1 is replaced by Lys199, which coordinates with Cu2+, whereas the DMF ligand of C2 is kept intact and His242 is replaced with Br− of C2 and coordinates with Cu2+. The cytotoxicity of the Cu compounds was enhanced by the FA-HSA NPs in the Bel-7402 cells approximately 2−4 fold; however, they raise the cytotoxicity levels in the normal cells in vitro and the FA-HSA NPs did not. Importantly, the in vivo data showed that FAHSA-C2 NPs increased selectivity and the capacity to inhibit tumor growth and were less toxic than HSA-C2 NPs and C2. Moreover, C2/HSA-C2 NPs/FA-HSA-C2 NPs induced Bel-7402 cell death by potentially multiple mechanisms.

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1. INTRODUCTION Cisplatin and its derivatives have been used for decades in more than 50% of treatment regimens for patients suffering from cancer.1,2 They are highly effective for the treatment of many cancers; however, their use remains restricted by dose-limiting side effects and acquired or inherited resistance.3,4 Less toxic and more effective metal-based anti-cancer compounds have therefore been searched for and developed extensively.5,6 Many metal compounds have been assessed both in vitro and in vivo and some metal compounds have been included in clinical trials7,8, however, there are major challenges that remain: how to improve the tumor targeting ability, enhance the efficiency of metal drugs in killing cancer cells, and decrease their side effects in vivo.9,10 Excitingly, nanoparticles (NPs) as a targeted drug delivery system and a pro-drug strategy have been promising in overcoming the aforementioned problems.11−15 HSA NPs, among NP carriers, have shown great promise in strategies for targeted delivery of drugs because HSA is non-antigenic, non-toxic, and biocompatible. Also, its biodegradable endogenous proteins do not have immunogenicity.16 Importantly, based on the properties of cancer cells, we can design the active HSA NP carrier by covalently conjugating the active targeting moiety to HSA, such as folic acid (FA).17,18 Indeed, a large quantity of data have indicated that FA-mediated albumin NP carriers show promise in increasing the selectivity and activity of anticancer drugs.19−23 Many clinical studies have also demonstrated that HSA-based NPs are well tolerated without any serious side effects.24−27

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Interestingly, on the one hand, we can modify the types and numbers of coordinated ligands in metal agents to improve their anticancer activity,28−31 and on the other hand, metal pro-drugs can be designed to improve their delivery efficiency and selectivity in vivo based on the N‑Donor residue(s) of the HSA IIA sub-domain.32−35 There is a potential conflict, however, in achieving the two goals simultaneously because Lys199 and His242 in the IIA sub-domain of HSA can be used to replace their two coordinating ligands,32,34 which causes the metal agents released from the HSA carrier to lose two coordinated ligands. This will decrease their anti-cancer activity in comparison with the original metal agents. Therefore, to enrich the delivery efficiency of the metal agents in vivo and to avoid decreasing their anticancer activity at the same time, it is necessary to use a novel strategy that only allows a coordinated ligand (leaving group) without anti-cancer activity in a metal compound to be displaced by the Lys199 or His242 of HSA. Thus, we proposed the design of another two coordinated ligands (second and third ligands, namely one potential pharmacophore and one leaving group), with the intention to keep the first coordinated ligand unchanged and then regulate the second ligand (potential pharmacophore) so it will not be replaced by Lys199, and the third ligand (leaving group) will be replaced by His242 (Figure 1). Then, His242 of HSA is protonated in the cancer cell’s lysosomal acidic environment. Its coordination ability with metal ions is then decreased and the metal drug is allowed to be released from the HSA carrier.36−38

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Liver cancer is common worldwide and causes many cancer-related deaths.39 Cu compounds may be promising as a next-generation, metal-based anti-cancer compounds because Cu is an essential element for human physiological functions and because of its bioactivity and oxidative nature.34,40 Taking into consideration the above factors, to better develop the FA-HSA NP carrier for the metal anticancer agent to actively target treatment of liver cancer, the Cu(II) compounds derived from an aroylhydrazone Schiff base and the Bel-7402 human hepatocellular cell line were used to build a model for the following studies: (1) to design two new aroylhydrazone Cu(II)-Schiff base anticancer compounds with one leaving group and one potential pharmacophore, Cu(L)(QL)(Br) [C1, QL = quinolone] and Cu(L)(DMF)(Br) [C2, DMF = N,N-dimethylformamide] (Figure 2A), and to investigate their anticancer activity on Bel-7402 cells; (2) to furnish solid evidence for the possibility of developing a Cu pro-drug based on the Lys199 residue of the HSA carrier IIA subdomain; (3) to compare in vivo therapeutic efficacy, targeting ability and side effects of FAHSA-C2 NPs to C2/HSA-C2 NPs; (4) to investigate the possible mechanism for HSA-C2 NPs/FA-HSA-C2 NPs penetrating cancer cells; and (5) to determine the potential anticancer mechanism of the C2/HSA-C2 NPs/FAHSA-C2 NPs. 2. MATERIALS AND METHODS 2.1. Material HSA (fatty acid content < 0.05%, catalogue number A3782) was procured from Sigma-Aldrich and used without additional purification. All other chemicals and solvents were of high purity and were purchased from 5



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Sigma, Strem, Aldrich, Takara, or Alfa. They were used without further purification. Elemental analyses (C, H, and N) were performed on a PerkinElmer 2400 analyzer. Infrared spectra (IR) of the samples were obtained using KBr pellets on a Nexus 870 FT-IR spectrophotometer in the frequency range of 4,000−400 cm−1. UV−visible spectra were measured on a Cary 1E UV-visible spectrophotometer in the 200−800 nm range. 2.2. Synthesis and characterization of Cu(II) compounds (E)-N'-(5-chloro-2-hydroxybenzylidene)benzohydrazide Schiff-base (HL) was synthesized according to previously published procedures.41 2.2.1. Synthesis of [CuBr(QL)(L)](C1) C1 was synthesized according to previously published procedure.42,43 In brief, quinoline (0.26 g, 2 mmol) and HL (0.55 g, 2 mmol) were dissolved in a methanol solution (15 mL) and stirred for 1 h to produce an orange solution. A solution of CuBr2 (0.44 g, 2 mmol) in methanol solution (15 mL) was then added and the combined solution was stirred for another 30 min at room temperature to produce a celadon solution, which was then ﬁltered. The ﬁltrate was exposed to air for one week and blue block crystals were formed. The crystals were isolated, washed three times with distilled water and then dried in a vacuum desiccator that contained anhydrous CaCl2. The yield was 776 mg (71%). Anal. Calcd for C23H17BrClCuN3O2 (546.30): C, 50.56; H, 3.13 and N, 7.69. Found: C, 50.43; H, 3.18 and N, 7.80. IR (KBr, cm−1): 1616 ν(C=N); 581, 549, 487, 468, 443 ν(Cu−N/Cu−O). 2.2.2. Synthesis of [CuBr(DMF)(L)](C2) The procedure of C2 synthesis was the same as that of C1 except that quinoline was replaced by N,N-dimethylformamide (DMF). Yield: 764 mg 6


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(78%). Anal. Calcd for C17H17BrClCuN3O3 (490.24): C, 41.65; H, 3.49 and N, 8.57. Found: C, 41.45; H, 3.57 and N, 8.48. IR (KBr, cm−1): 1619 ν(C=N); 548, 505, 458, 436 422 ν(Cu−N/Cu−O). 2.3. X-ray crystallography of HSA complexes The complexes of Cu(II) compounds and HSA were prepared as previously reported.32 In brief, 960 µL palmitic acid (PA; 2.5 mM), 100 µL HSA (100 mg/mL), and 90 µL Cu compound (5 mM) were mixed and set overnight. Then a Millipore spin filter (10,000 Da cutoff) was used to concentrate the mixture to 100 mg/ml. Crystallization was carried out according to a previously published protocol.32 X-ray diffraction data were collected at the BL17U1 beamline of the Shanghai Synchrotron Radiation Facility under cryo-conditions (100 K).44 The data were then integrated and scaled with the HKL2000.45 The HSA complexes structures were solved by molecular replacement using PHASER in PHENIX suites with the initial model of the HSA-myristate structure (PDB code: 1BJ5). The ligand was stripped as in the initial searching model. All ligands were built into the model by LigandFit in PHENIX and were manually modified and adjusted in COOT.46−48 PyMOL was used to depict the structures.49 Data collection details and unit cell parameters are listed in Table 1. 2.4. Preparation and characterization of FA-HSA-C1/C2 NPs 2.4.1. Preparation of HSA-C1/C2 NPs The Cu(II) compounds were incubated with HSA (molar ratio 1:1) for 24 h at room temperature to obtain the metal-HSA pro-drugs and a constant drug-loading ratio. Then the HSA-C1/C2 NPs were prepared using an 7



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established desolvation process:50−52 the 4 mL HSA-Cu(II) compound mixture solution (1.5 mM) was adjusted to pH 8 and was then formed into HSA-C1/C2 NPs by continuous (1 mL/min) addition of 15.0 mL desolvating agent ethanol under constant stirring at room temperature. After protein desolvation, 450 µL of the 8% aqueous glutaraldehyde solution were added to achieve particle crosslinking. The mixture was then centrifuged for 10 minutes at 40,000 rpm to separate the HSA-C1/C2 NPs. NPs that were larger than about 250 nm were easily phagocytized after intravenous injection.53 The HSA-C1/C2 NPs were resuspended in water and then filtered through filter membranes (Millipore, 250 nm pore diameter). They were subsequently freeze-dried. 2.4.2. Preparation of FA-HSA-C1/C2 NPs A total of 2.5 mL of FA solution (20 mg/mL) in 0.1 Nsodium hydroxide were incubated with 20 mg of N-hydroxysuccinimide (NHS) and 50 mg of N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC) under constant shaking for 20 min in the dark. Subsequently, the HSA-C1/C2 NPs suspensions (content 15 mg/mL) were added. Shaking continued for 3 h. Next, the FA-conjugated nanoparticles were purified from unreacted FA using three cycles of differential centrifugation (40,000 rpm, 10 min) and redispersion of the pellet to the original volume in water. The resulting NPs was filtered again through filter membranes (Millipore, 250 nm pore diameter) and were then freeze-dried. 2.4.3. Hemolysis activity examinations Spectrophotometry was used to examine the hemolysis properties of FAHSA-C1/C2 NPs as reported in previous works.54 Each sample was treated 8


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to a hemolysis assay for 1 h (5 µM free Cu(II) compounds, 5 µM HSAC1/C2 NPs and 5 µM FA-HSA-C1/C2 NPs) to study erythrocyte agglutination. They were then placed on a glass slide, covered by a cover slip and analyzed using a phase contrast microscope. 2.5. In vitro anticancer activity Normal lung fibroblasts cells and the human hepatocellular cell lines Bel-7402 and WI-38 were purchased from the American Type Culture Collection and the German Collection of Microorganisms and Cell Cultures. Cells were incubated under a humidified atmosphere containing 5% CO2 at 37 °C. They were then grown in DMEM supplemented with10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. The cytotoxicity assay (MTT) of C2/HSA-C2 NPs/FA-HSA-C2 NPs to Bel-7402 and WI-38 cells according to reported methods (Shown in Supporting Information)32,55. 2.6. In vivo animal studies 2.6.1. Animal subject and tumor models Athymic nude mice and Kunming mice (aged 3～4 weeks, weighing 18～22 g, equal number of female and male subjects) were obtained from Beijing HFK Bioscience Co., Ltd. They were used at six weeks of age. All animal experiments were carried out in compliance with the guidelines for Animal Care Committee of the Institute and the Animal Management Rules of the Ministry of Health of the People’s Republic of China (document NO. 55, 2001). 2.6.2. Acute toxicity study

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The acute toxicity of C2, HSA-C2 NPs, and FA-HSA-C2 NPs were assessed in normal mice according to a method described previously.34,56 In brief, 32 healthy Kunming mice were divided into four groups (8 mice in each group). HSA-C2 NPs, free C2, and FA-HSA-C2 NPs were administrated to the different groups of mice at a dose of 15 µmol Cu/kg body weight. The control group received NaCl. Blood samples from each group of mice were drawn intravenously (i.v.) for 3 days to prepare serum samples. The serum biochemical parameters of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine kinase (CK) and blood urea nitrogen (BUN) of the blood samples were measured. 2.6.3. In vivo anti-tumor activity study The nude mice were injected subcutaneously with 200 µL of cell suspension containing 4 × 106 Bel-7402 cells in the right flank region. The Bel-7402 tumor-bearing mice (32) were randomly divided into four groups when the tumor volume was approximately 100 mm3 so they could be used in the anti-tumor activity study. The mice in different treatment groups were intravenously injected with NaCl, HSA-C2 NPs, free C2, and FA-HSA-C2 NPs at a dose of 3.5 µmol Cu/kg body weight every 3 days. All mice in all the groups were earmarked and followed individually throughout the experiments. The length and width of the tumor and the body weights of the mice were measured before every injection and at the end of the experiment. The volume was calculated using the following equation: tumor volume (V) = 1/2 × width2 × length. Mice were sacrificed after 24 days of treatment and major organs and tumor tissues were excised for histopathological analysis with hematoxylin and eosin (H&E) staining. 10


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2.6.4. In vivo targeting ability study The tumor tissues and major organs of mice were homogenized at the end of the in vivo experiment. Then 0.5 g of the sample was placed in a teflon container and mineralized in a microwave oven under pressure (system Milestone MSL 1200) in 1 mL of 30% hydrogen peroxide and in the presence of 7 mL of concentrated HNO3. The contents of Cu in the mice major organs and tumors were determined using inductively coupled plasma atomic emission spectrometry (ICP-AES). 2.7. Cu pro-drugs release from HSA complexes The Cu compounds released from the HSA complexes were analyzed by dialyzing HSA complexes at pH 4.7 and pH 7.4 buffers to simulate the cell matrix and interstitial space environments, respectively.34,37,38 In brief, 2 mL of the HSA complex suspension in the dialysis pocket were dispersed in the tube containing 40 mL of pH 4.7 and pH 7.4 buffers for 48 h, respectively. The amount of Cu compounds released from the HSA complexes was determined with a graphite furnace atomic absorption spectrometer (GFAAS). 2.8. Determining the possible mechanism of uptake of HSA-C2 NPs and FA-HSA-C2 NPs by cells Bel-7402 cells were seeded into 3.5 cm dishes and cultured at 37 ºC for 24 h. Next, HSA-C2 NPs (10 µM)/FA-HSA-C2 NPs (10 µM) were added and the mixture was incubated for 2 h at 4°C and then 37°C under 5% CO2 in a humidified atmosphere. The mechanisms of cellular internalization of FA-HSA-C2 NPs and HSA-C2 NPs in Bel-7402 cells were further determined by blocking the 11



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uptake pathway with various endocytosis inhibitors. The cells were pretreated with NaN3 (0.05% w/v), monensin (100 nmol/L), NH4Cl (10 nmol/L), brefeldin A (10 µg/mL), chlorpromazine (10 µg/mL), glucose (200 mmol/L), filipin (5 µg/mL), cytochalasin-D (50 µM), or nocodazole (20 µmol/L) for 30 min. HSA-C2 NPs (10 µM)/FA-HSA-C2 NPs (10 µM) were then added to each well and incubated for 2 h at 37 ºC. In addition, in order to investigate whether folic acid (FA) specifically mediates cellular uptake, Bel-7402cells were pre-incubated with a 500 µg/ml FA for 30 min before adding HSA-C2 NPs (10 µM)/FA-HSA-C2 NPs (10 µM) and incubated for 2 h at 37 ºC. Subsequently, the cells were harvested and lysed. The amount of copper in Bel-7402 cells was analyzed using GF-AAS. 2.9. Determine of potential anticancer mechanism of C2/HSA-C2 NPs/FA-HSA-C2 NPs To confirm the potential anticancer mechanism of C2/HSA-C2 NPs/FAHSA-C2 NPs, we design and conduct studies according to previous protocol (Shown in Supporting Information).32−34,57 2.10. Statistical analysis A Student’s t test was used to evaluate measured differences. The results were expressed as the mean ± standard deviation (SD). P< 0.05 was considered to be significant. 3. RESULTS 3.1. Design of Cu pro-drugs We focused on the Cu compounds containing the tridentate (E)-N'-(5chloro-2-hydroxybenzylidene)benzohydrazide Schiff base ligand (HL) because the hydrazone class ligands themselves are promising anticancer 12


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agents,58,59 and the hydrophobic properties of the benzyl component of HL will allow the Cu compounds to target the hydrophobic binding sites of the IIA subdomain in the HSA.60,61 Our results revealed that Lys199 of HSA acts as a nucleophile and may replace the ligand that its nitrogen atom coordinates with Cu2+.32 Therefore, we kept the HL as the primary ligand for the Cu compounds, designed different ligands (DMF and quinoline) for the second ligand, and we chose Br as the third ligand. As shown in Figure 2A, compound C1 crystallized in a monoclinic system with space group P21/c, whereas C2 crystallized in a triclinic system with space group P−1. Both C1 and C2 contain L−, one Cu atom, one DMF/quinolone, and one Br per asymmetric unit. The structural details of C1 and C2 are described in the Supporting Information. 3.2. Feasibility of developing the Cu pro-drug based on the Lys199 residue of HSA The fluorescence quenching result of HSA indicated that C1 and C2 bind close to the subdomain IIA of HSA (Figure S1A).62 The matrix-assisted laser desorption/ionization-time of the flight-mass spectrometry (MALDITOF-MS) spectrum showed an increase of 350−400 Da for the HSA complexes relative to HSA. This was equivalent to a molecular weight of ca. one C1 and C2 (Figure S1B). Furthermore, the electrospray ionization mass spectrometry (ESI-MS) of the products released from the HSA-C1 complex at pH 5 displayed an intense signal at m/z = 335.97 when the in-source energy was 0 eV; this could be identified with isotopic envelopes corresponding to [Cu(L)]+ (fit: 335.97), implying that both Br and QL ligands were lost from C1 (Figure S2A). However, the ESI-MS of the 13



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compounds released from the HSA-C2 complex had an intense signal at m/z= 409.03 for [(L)Cu(DMF)]+ at 0 eV, which suggested that the DMF ligand was inert in the HSA-C2 complex (Figure S2B). We resolved the two structures of the HSA-PA-C1/C2 complexes to provide solid evidence on the feasibility of developing a Cu(II) pro-drug by regulating the special residue of the HSA IIA subdomain to replace the ligand of the Cu compound. The electron density maps of the Cu compounds in the complex with HSA clearly demonstrated one Cu compound molecule at the IIA subdomain (Figure 2B). The overall structure of the Cu compound bound to the HSA-PA complex was heart-shaped (Figure 2C). The Cu compounds bound to a large hydrophobic pocket in the IIA subdomain of HSA, and were delimited by residues, including Arg218, Arg222, Arg257, Trp214, Leu219, Leu238, Leu260, Lys199, Phe223, His242, Ile264, Ile290, Ser287, and Ala291 (Figure 2D). The HSA-PA-C1 structure showed that Lys199 was replaced with the quinolin ligand of C1, and coordinated with Cu(II) (Figure 2D). The HSA-PA-C2 structure revealed that the DMF ligand was kinetically inert and His242 was replaced with Br− of C2 and coordinates with Cu(II) (Figure 2D). Superimposing the structures of HSAPA-C1 and HSA-PA-C2 revealed that the C2 plane was rotated by 40° compared to C1 (Figure 2D). 3.3. Characterization of FA-HSA-C1/C2 NPs The scanning electron microscope image showed that the HSA-C1/C2 NPs and FA-HSA-C1/C2 NPs were spherical with a smooth surface (Figure S3A). The size distribution and mean diameter of the prepared NPs were determined by laser light scattering. The formations of FA-HSA-C1/C2 NPs 14


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and HSA-C1/C2 NPs with diameters of about 162 nm and 220 nm, respectively, both of which had a narrow size distribution, are shown in Figure S3B. In this particle size range, the NPs would not be easily phagocytized after injection compared to larger NPs.63,64 Excellent hemocompatibility of NP vectors for intravenous administration is one of the requisites,54 therefore, the hemolysis ratio of these formulations was assessed by a hemolysis assay. As shown in Figure 3A, compared to the free Cu(II) compounds, HSA-C1/C2 NPs and FA-HSA-C1/C2 NPs showed minor hemolysis destruction in the red blood cells, even at a high concentration of 5 µM. Also, the free Cu(II) compounds induced the prominent agglutination of erythrocytes after incubation for 1 h. No agglutination, however, was observed in the erythrocytes exposed to HSAC1/C2 NPs and FA-HSA-C1/C2 NPs (Figure 3B). 3.4. In vitro anticancer activity C1/C2, HSA-C1/C2 NPs and FA-HSA-C1/C2 NPs all display micromolar toxicities against Bel-7402 cells better than that of cisplatin in vitro. Notably, FA-HSA-C1/C2 NPs exhibited increased cytotoxic activity for Bel-7402 with FA receptor overexpression compared to C1/C2 or HSAC1/C2 NPs, but a similar effect was not observed in the normal WI-38 lung fibroblast cells with low FA receptor expression (Table 2). Interestingly, HSA-C2 NPs and FA-HSA-C2 NPs exhibit higher cytotoxicity to Bel-7402 cells than HSA-C1 NPs and FA-HSA-C1 NPs, respectively (Table 2), which indicated that the coordinated ligands in the Cu compound enhance its anticancer activity because C2 has one more coordinated ligand (DMF) than C1 when they are released from the HSA NPs carrier (Figure S2). 15



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3.5. Animal studies of C2, HSA-C2 NPs, and FA-HSA-C2 NPs The xenograft Bel-7402 murine liver cancer model was established to further evaluate whether the FA-functionalized HSA NP carriers and prodrug strategy resulted in the enhancement of the therapeutic efficacy in vivo. 3.5.1. Acute toxicity of C2, HSA-C2 NPs, and FA-HSA-C2 NPs in vivo The acute toxicity of HSA-C2 NPs, C2, and FA-HSA-C2 NPs was assessed in the kidneys, heart, and liver of normal mice by measuring levels of blood urea nitrogen (BUN), creatine kinase (CK), alkaline phosphatase (ALT), and aspartate aminotransferase (AST) at 3 days after the intravenous injection of the drugs (Table S1). The levels of CK in all of the experimental groups were similar to the control (NaCl) group. This indicated low cardiotoxicity. High BUN levels meant there was higher toxicity in the kidneys. Free C2 caused significant nephrotoxicity, which was shown by a larger BUN value (18.4 ± 2.4 mmol/L) than in the control group (7.3 ± 1.1 mmol/L). In contrast, nephrotoxicity was significantly decreased in the HSA-C2 NPs- and FA-HSA-C2 NPs-treated groups, especially for the FAHSA-C2 NPs group. Serum AST and ALT levels were significantly elevated in the group treated with C2; however, the levels induced by FA-HSA-C2 NPs and HSA-C2 NPs were similar to those induced by the control group, which indicated low liver injury by HSA-C2 NPs and FA-HSA-C2 NPs. 3.5.2. Anti-tumor activity of C2, HSA-C2 NPs, and FA-HSA-C2 NPs in vivo Bel-7402 tumor-bearing mice were injected with HSA-C2 NPs, C2, and FA-HSA-C2 NPs, and the controls were injected with NaCl, to evaluate the therapeutic efficacy. Body weights and tumor volumes of the tumor-bearing 16


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mice were monitored every three days for 24 days. The tumor volume in the mice treated with FA-HSA-C2 NPs was much smaller than the tumor volume in the mice treated with saline, HSA-C2 NPs, and C2 at the end of the experiment (Figure 4A). The tumor volume after 24 days of treatment was 65.3 ± 5.9% for C2, 47.6 ± 5.1% for HSA-C2 NPs, and 29.9 ± 3.1% for FA-HSA-C2 NPs, demonstrating that FA-HSA-C2 NPs resulted in more tumor growth inhibitory efficacy than C2 and HSA-C2 NPs compared to the control group. The mice were killed and the tumors were harvested and weighed after 24 days of treatment (Figure 4B). The tumor inhibitory rate (TIR) was calculated from the tumor weight. The TIR of FA-HSA-C2 NPs was 78.2% (P< 0.001), which was significantly higher than that of C2 (34.2%, P< 0.01) and HSA-C2 NPs (54.4%, P< 0.001) compared to the NaCl group. The tumor tissues were then excised for pathology to further evaluate the antitumor effects of HSA-C2 NPs, C2, and FA-HSA-C2 NPs in the animals. The H&E-stained tissue sections showed obvious differences in tumor tissue morphology between the control groups and the treated group (Figure 5). In the NaCl group, the tumor cells had a large nucleus, spindle shape, and no visible apoptosis or necrosis, which are indicative of rapid tumor growth. However, tumor cellularity was significantly decreased, as evaluated by the average number of tumor cells in each microscopic field, and fragmentation and nuclear shrinkage were observed in the C2, HSA-C2 NPs-, and FA-HSA-C2 NPs-treated groups, especially for FA-HSA-C2 NPs-treated tumors.65 A large necrotic area was observed in the FA-HSA-C2 NPs group. These results indicated that the therapeutic efficacy of FA-HSAC2 NPs is better than that of HSA-C2 NPs and C2. 17



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3.5.3. Comparing the side effects of C2, HSA-C2 NPs, and FA-HSA-C2 NPs Figure 4C shows the body weight changes of animals treated with NaCl, free C2, HSA-C2 NPs, and FA-HSA-C2 NPs. The body weight loss in the FA-HSA-C2 NPs and HSA-C2 NPs groups was about 8.1% and 11.8% of the original weight, respectively, which was lower than that of the NaCl control group (which was about 16.5% of the original weight). In contrast, the body weight in the free C2 treated group was increased by about 18.4%. These results indicated that the HSA-C2 NPs and FA-HSA-C2 NPs reduced the side effects induced by free C2. In addition, drug-related side effects and toxicities to major organs were observed with H&E staining (Figure 5). It is important to note that there were no abnormalities observed in any of the heart sections; however, it was clearly observed that C2 induced damage to the liver (atrophy of hepatic cells and mild steatosis) and kidneys (focal abnormalities and hyaline cast of kidney tubules). In contrast, damage to the liver and kidneys was greatly decreased in the HSA-C2 NPs- and FA-HSAC2 NPs-treated groups as they only had slight inflammation and cellular atrophy. The results of the body weight changes and the pathological study demonstrated that HSA-C2 NPs and FA-HSA-C2 NPs can effectively reduce the side effects and toxicity induced by free C2, especially for folatefunctionalized HSA-C2 NPs. 3.5.4. Selectivity of FA-HSA-C2 NPs in vivo We tested the Cu content in the tumors and the main organs of the mice treated with C2, HSA-C2 NPs, and FA-HSA-C2 NPs to determine whether FA-HSA NP carriers selectively accumulate in the Bel-7402 tumors in vivo. 18


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The inductively coupled plasma atomic emission spectrometry (ICP-AES) results indicated that the increase in the copper content in the tumors of the mice treated with FA-HSA-C2 NPs and HSA-C2 NPs was approximately 2.1- and 1.5-fold higher than that in the mice treated with C2 alone (Figure 6). These data indicated that FA receptor-mediated endocytosis plays an essential role in selective accumulation in the Bel-7402 tumor. Furthermore, our data revealed that HSA NPs and FA-HSA NPs were helpful for decreasing C2 accumulation in the liver and kidneys. 3.6. Release behavior of Cu pro-drug from HSA We simulated the inside environment of a cancer cell and measured C1 and C2 release from the HSA carrier in buffers of pH 4.7 and pH 7.4, respectively. Approximately 5% of the loaded Cu compounds were released from the HSA complex in the pH 7.4 buffer within 48 h. Up to 80% of the Cu compounds were released from HSA in the pH 4.7 buffer (Figure S4). Furthermore, the binding affinity of the Cu compounds to HSA in the pH 7.4 buffer (K = 7.54 (± 0.06) × 106 M−1 for C1 and 6.17 (± 0.03) × 106 M−1 for C2) was significantly stronger than that of HSA for Cu compounds in the pH 4.7 buffer (K = 4.62 (± 0.03) × 104 M−1 for C1 and 4.28 (± 0.03) × 104 M−1 for C2). The results indicated that the Cu compounds are more easily released from HSA when bound weakly in an acidic environment. These results suggested that the HSA-C1/C2 complexes would be stable in the blood during in vivo circulation. C1 and C2 are released after accumulating in the acidic lysosome of the cancer cells. 3.7. Possible mechanism of HSA-C2 NPs and FA-HSA-C2 NPs absorbed by cancer cells 19



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Our results showed that the Bel-7402 cells that were incubated at 4°C internalized less Cu than those incubated at 37°C (Figure 7). This indicated that the uptake of HSA NPs by the Bel-7402 cells was energy-dependent. NaN3 can prevent cells from absorbing HSA-C2 NPs and FA-HSA-C2 NPs, which suggested that the uptake of HSA NPs by the Bel-7402 cells should rely on metabolic energy.66,67 Pre-added brefeldin A and NH4Cl (disrupting the function and structure of the Golgi apparatus) inhibited the uptake of HSA NPs (Figure 7). This revealed that the Golgi apparatus is involved in the uptake process.68 The pre-added substances blocked the transfer from an endosomal to lysosomal fraction. Monensin markedly inhibited the uptake, which suggested that the acidic endosomes are involved in the uptake process.69 The cellular uptake of FA-HSA-C2 NPs and HSA-C2 NPs was significantly inhibited in the presence of chlorpromazine. Measurement of sucrose indicated the involvement of clathrin-mediated endocytosis in the internalization of nanoparticles by the Bel-7402 cells.70 However, the cellular uptake of HSA NPs was marginally influenced by nocodazole (microtubule inhibitor), filipin (disrupting internalization via caveolae), and cytochalasin D (actin filament disrupting),suggesting that the internalization of HSA-C2 NPs and FA-HSA-C2 NPs rarely relies on micropinocytosis and caveolae-mediated endocytosis.71,72 Together, our data indicated that the Bel7402 cellsabsorbedHSA-C2 NPs and FA-HSA-C2 NPs by potentially multiple mechanisms. It is interesting to note that the Bel-7402 cells can absorb more FA-HSAC2 NPs than HSA-C2 NPs; however, the uptake of FA-HSA-C2 NPs by the Bel-7402 cells pre-incubated with folic acid was less than that of HSA-C2 20


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NPs by the Bel-7402 cells (Figure 7), suggesting that the uptake of FA-HSA NPs by the Bel-7402 cells was specifically regulated through the interactions between folic acid conjugated with HSA NPs and the folic acid receptor of Bel-7402 cells. 3.8. Potential anticancer mechanism of C2, HSA-C2 NPs, and FA-HSAC2 NPs DNA and DNA topoisomerases are the primary intracellular targets of many metallodrugs,7 thus metallodrug–DNA interactions and the activity of topoisomerases are of paramount importance in understanding their mechanism. C2 exhibited a hypochromism of about 17.3% and 16.6% with a hyperchromic shift of 1 nm and 2 nm at 323 nm and 385 nm, as shown in Figure S5A. This suggested that the binding of the compound to CT-DNA may be assigned to groove binding. The ethidium bromide (EB) displacement experiment showed that C2 could bind to DNA through the groove binding mode, which released EB molecules from the DNA-EB system (Figure S5B). Concentration-dependent DNA cleavage by C2 was also performed. As shown in Figure S6A, C2 is capable of converting supercoiled pBR322 plasmid DNA (Form I) into an open circular form (Form II) in a concentration-dependent manner. Topoisomerase I (Topo I) is an essential nuclear enzyme that induces DNA modification, which is required during transcription, replication, chromatin remodeling, and repair.73 Disruption of Topo I leads to irreversible DNA strand breaks, activation of apoptosis, and cell cycle arrest.74 It was observed that C2 could inhibit Topo I and that the inhibition was concentration dependent (Figure

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S6B). These results suggested that C2 can induce DNA damage through its interaction with DNA and Topo I. Next, immunoblot analyses were conducted to examine changes in biomarker expression related to the DNA damage pathway in Bel-7402 cells. The data indicated that protein expression of the phosphorylated forms of p53 (Ser15), H2AX (γH2AX), CHK1, and CHK2 was slightly upregulated with C2 and HSA-C2 NPs treatment compared to the untreated controls. The expression of these phosphorylated proteins was markedly enhanced by treatment with FA-HSA-C2 NPs (Figure S6C). These data further indicated that FA-HSA-C2 NPs kill cells, possibly by damaging DNA.75,76 DNA damage is considered to affect apoptosis and the cell cycle.77 Therefore, we monitored these processes after treatment with C2, FA-HSAC2 NPs and HSA-C2 NPs. Figure S6D shows that these compounds caused an accumulation of cells in the G2/M phase of the cell cycle by inhibiting or delaying cell cycle progression. Progression through the cell cycle is tightly controlled by cyclin complexes and cyclin-dependent kinases (CDKs) at different phases.78 Therefore, cyclin B1 and CDK1 expression was examined to further elucidate the mechanisms involved in anticancer actions of FAHSA-C2 NPs. C2/HSA-C2 NPs/FA-HSA-C2 NPs downregulated cyclindependent kinase 1 (CDK1) and cyclin B1 expression, compared with vehicle-treated controls, whereas it upregulated intracellular p21 waf1/Cip1 (Figure S6E). We explored the rate of apoptosis inBel-7402 cells treated with FAHSA-C2 NPs with a dual Annexin V staining/PI flow cytometry assay. The flow cytometry analysis indicated that the rate of apoptosis in cells was 28.5% 22


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and 30.3% after treatment with C2 and HSA-C2 NPs, respectively. The rate increased to 47.8% after incubation with FA-HSA-C2 NPs (Figure S7A). FA-HSA-C2 NPs promoted a much higher apoptotic rate in the Bel-7402 cells with the same dose in comparison to other formulations. This result was in agreement with the Hoechst 33258 staining results (Figure S7B). Hence, both the flow cytometry assay and morphological features provided confirmation that these drugs work through the apoptosis pathway. Mitochondrial dysfunction plays a key role in triggering apoptosis.79 The mitochondrial transmembrane potential (∆ψm) was evaluated with a JC-1 mitochondrial potential assay kit and flow cytometry to establish whether apoptosis induced by C2, HSA-C2 NPs, and FA-HSA-C2 NPs was related to mitochondrial dysfunction. Figure S7C shows treating Bel-7402 cells with C2, HSA-C2 NPs, or FA-HSA-C2 NPs caused a decrease in ∆ψm to varying degrees. This confirmed activation of mitochondria-mediated apoptosis. FAHSA-C2 NPs exerted the most profound effects on mitochondrial integrity, with 39.7% of the cells showing depolarized mitochondria upon treatment with 1.2 µM for 12 h. Western blot analysis (Figure S7D) showed that C2, FA-HSA-C2 NPs and HSA-C2 NPs upregulated the expression of Bad (proapoptosis Bcl-2 family of proteins) and suppressed the expression of Bcl-2 and Bcl-xl (prosurvival Bcl-2 family proteins). The ratios of Bcl2/Bax and Bcl-xl/Bad were decreased. This indicated that the Bcl-2 family of proteins regulated the loss of ∆ψm. Furthermore, Western blotting showed that cytochrome c, poly ADP ribose polymerase (PARP), and the cleaved caspase family of proteins; i.e., cleaved caspase -3, -7, and -9 were upregulated. These results showed that the FA-HSA-C2 NPs can effectively 23



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trigger mitochondria-mediated apoptosis through regulation of the expression of the Bcl-2 family of proteins. Taken together, our results revealed that C2/HSA-C2 NPs/FA-HSA-C2 NPs kill cancer cells potential by multiply anticancer mechanism at the same time (Figure 8A). 4. DISCUSSION Our results revealed that Lys199 of HSA can displace the ligand coordinated to Cu2+ with a nitrogen atom.32 To protect the ligand from being replaced by Lys199, we designed the ligand so that its coordination atom with Cu2+ was not nitrogen, but an oxygen atom. Thus, we chose QL (nitrogen atom) and DMF (ketonic oxygen atom) coordination with Cu2+ to confirm our hypothesis. The HSA-PA-C1 structure revealed that QL was replaced by Lys199, which again confirmed that the heterocyclic nitrogen ligand in the metal compound can be replaced by Lys199. The HSA-PA-C2 structure revealed that only Br of C2 was replaced by His242, keeping the DMF intact. Clearly, it is feasible to modify the coordination group with a metal ion to avoid the ligand being displaced by Lys199 of HSA. What is the release behavior of C1 and C2 from HSA in vivo and inside cancer cells due to the different coordination modes to HSA? Both HSA-PAC1 and HSA-PA-C2 structures have shown that C1 and C2 bind in the hydrophobic cavity of the HSA IIA subdomain. This occurs by His242/Lys199 of HSA coordinating to Cu2+ and by forming stable HSA complexes. In pH 7.4, a few C1 and C2 (ca. 5%) were released from HSA, whereas in acidic environments, the binding affinity of C1 and C2 to HSA was dramatically decreased and approximately 80% of C1 and C2 were 24


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released from HSA. Obviously, the number of coordination bonds of residue of HSA to Cu2+ has little influence on the release behavior of the Cu compound from HSA in vivo and inside cancer cells. These profiles indicated that the HSA-C1/C2 complexes would be stable in the blood during in vivo circulation. However, C1 and C2 are released after accumulation in the acidic lysosomes of the cancer cells. The therapeutic efficiency of C2/HSA-C2 NPs/FA-HSA-C2 NPs in vivo was evaluated by the tumor inhibition rate (TIR), side effects, and selectivity. The TIR of the FA-HSA-C2 NPs group almost reached 78% and was about 1.4 and 2.3 times higher than those of the HSA-C2 NP and C2 groups. Mice tolerate FA-HSA-C2 NPs well and they cause less weight loss relative to C2 alone and HSA-C2 NPs. HSA-C2 NPs and FA-HSA-C2 NPs can effectively reduce liver and kidney damage induced by free C2, according to H&E staining, especially for folate-functionalized HSA-C2 NPs. Furthermore, the results from ICP-AES showed that FA-HSA-C2 NPs assist with accumulation of the Cu compound in tumors in vivo. In mice, liver cancer xenograft experiments further indicated that FA-HSA NPs helped with decreasing side effects and improving anti-tumor activity. The selectivity of the Cu compound, the latter of which is attributable to FA-HSA NP carriers, accumulates in the tumor cells by the enhanced permeability, the retention (EPR) effect and folate receptor-mediated endocytosis in tumors (Figure 8B). 5. CONCLUSIONS Due to rationally regulating the coordinated ligand in the Cu(II) pro-drug and replacing it with the special residue in the HSA IIA subdomain, we not 25



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only improved the delivery efficiency of the Cu compound, but we also avoided the decrease of its anticancer activity. Compared with C2 alone and HSA-C2 NPs, FA-HSA-C2 NPs had superior in vivo anti-tumor efficacy, were well tolerated in mice, and had lower organ toxicity and weight loss. Collectively, our results demonstrated that active FA-HSA NP carriers and a pro-drug strategy may be promising for the intravenous use of novel, active benzohydrazide HL containing a Cu(II) compound for targeted cancer therapy. ACKNOWLEDGMENTS The authors are grateful to staff of BL17U1 beamline of Shanghai Synchrotron

Radiation

Facility

for

their

technical

assistance

in

crystallographic data collection. This work received financial support from the Natural Science Foundation of China (21431001, 31460232), the Natural Science Foundation of Guangxi (2014GXNSFDA118016), Ministry of Education of China (CMEMR2015-A01), Technology division of Guilin (20150102-13), Innovation Project of Guangxi Graduate Education (2017), Guangxi ‘Bagui’ scholar program to ZP Zhou, High-level Innovation team and distinguished scholar program of Guangxi universities to F Yang.

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Figure Legends Figure 1 The hypothesis of developing metal pro-drugs by regulating the ligand in metal pro-drug to be replaced by Lys199 or His242 of HSA. 37



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Figure 2 (A) Chemical structure of two Cu(II) pro-drugs (C1 and C2). (B) The experimental σA weighted 2Fo − Fc electron density map of Cu compounds in HSA. (C) The overall structure of HSA complex. (D) The structural binding environment of Cu compounds in HSA. Figure 3 (A) Hemolysis induced by free compounds, HSA-C1/C2 NPs and FA-HSA-C1/C2 NPs after 1 h. (B) Agglutination of red blood cells observed by phase microscopy after 1 h of incubation with different formulations at the same concentration (5 µM). Each value represents means ± SD (n = 3). Figure 4 (A) Variations of tumor volume after treatment with different formulations. (B) Mean weight of tumors separated from mice after different treatments. (C) Body weight changes of different formulations. Statistical significance: *P < 0.05; **P < 0.01; ***P < 0.001. Figure 5 H&E staining analysis of Bel-7402 tumor and organs sections treated with various treatments (magnification ×400). Figure 6 Tissue copper of mice after treatment with saline, C2, HSA-C2 NPs and FA-HSA-C2 NPs. Results are the mean ± SD (n = 3): *P < 0.05; **P < 0.01. Figure 7 Uptake mechanism of HSA-C2 NPs and FA-HSA-C2 NPs. Statistical significance: *P < 0.05; **P < 0.01. Figure 8 (A) Potential anticancer mechanism of C2/HSA-C2 NPs/FA-HSAC2 NPs. (B) The potential mechanism of FA-HSA NPs deliver Cu pro-drug into cancer cells.

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Table 1 Data collection statistics and crystallographic analysis of HSA complexes. HSA complex

HSA-PA-C1

HSA-PA-C2

P1

P1

95.03, 95.60, 38.63

38.71, 95.90, 95.48

Data collection Space group Cell parameters, a, b, c (Å) Cell parameters,

105.08, 90.12,101.94 74.98, 89.96, 78.59

Resolution range (Å)

45-2.3

46-2.3

4.2

4.4

97% (98.1%)

97% (97.8%)

14.3 (4.5)

14.2 (4.4)

7.1% (23.6%)

7.4% (26.6%)

Rmodel (%)c

24.58%

24.69%

Rfree (%)d

29.79%

29.88%

R.m.s. deviation from ideal bond lengths

0.009 Å

0.009 Å

1.198

1.201

Data redundancy Completeness (%)a I/σ Rmerge (%)b Model refinement

R.m.s. deviation from ideal angles (°) a

Values for the outermost resolution shell are given in parentheses.

b

Rmerge=100×ΣhΣj| Ihj-Ih|/ΣhΣj Ihj where Ih is the weighted mean intensity of the symmetry-

related refractions Ihj. c

Rmodel=100×Σhkl|Fobs -Fcalc|/ΣhklFobs where Fobs and Fcalc are the observed and calculated

structure factors, respectively. d

Rfree is the Rmodel calculated using a randomly selected 5% sample of reflection data

omitted from the refinement.

39



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Page 40 of 49

Table 2 IC50a (µM) values of Cu(II) compounds, HSA-C1/C2 NPs and FA-HSA-C1/C2 NPs toward a panel of human cell lines for 48 h. compound

Bel-7402

WI-38

HL

87.63 ± 7.91

79.86 ± 6.52

C1

2.09 ± 0.26

3.65 ± 0.27

HSA-C1 NPs

2.21 ± 0.21

4.06 ± 0.34

FA-HSA-C1 NPs

1.17 ± 0.09

4.11 ± 0.29

C2

1.98 ± 0.14

3.74 ± 0.37

HSA-C2NPs

1.91 ± 0.21

3.52 ± 0.33

FA-HSA-C2 NPs

0.58 ± 0.08

3.69 ± 0.26

>100

>100

14.23 ± 1.22

15.17 ± 1.35

FA-HSA NPs Cisplatin a

Antitumor activity IC50 (µM)

IC50 values are presented as the mean ± SD from three separated experiments.

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Figure 1 The hypothesis of developing metal pro-drugs by regulating the ligand in metal pro-drug to be replaced by Lys199 or His242 of HSA. 98x54mm (600 x 600 DPI)



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Figure 2 (A) Chemical structure of two Cu(II) pro-drugs (C1 and C2). (B) The experimental σA weighted 2Fo − Fc electron density map of Cu compounds in HSA. (C) The overall structure of HSA complex. (D) The structural binding environment of Cu compounds in HSA. 134x159mm (600 x 600 DPI)


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Figure 3 (A) Hemolysis induced by free compounds, HSA-C1/C2 NPs and FA-HSA-C1/C2 NPs after 1 h. (B) Agglutination of red blood cells observed by phase microscopy after 1 h of incubation with different formulations at the same concentration (5 µM). Each value represents means ± SD (n = 3). 96x82mm (300 x 300 DPI)



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Figure 4 (A) Variations of tumor volume after treatment with different formulations. (B) Mean weight of tumors separated from mice after different treatments. (C) Body weight changes of different formulations. Statistical significance: *P < 0.05; **P < 0.01; ***P < 0.001. 56x18mm (600 x 600 DPI)


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Figure 5 H&E staining analysis of Bel-7402 tumor and organs sections treated with various treatments (magnification ×400). 86x65mm (300 x 300 DPI)



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Figure 6 Tissue copper of mice after treatment with saline, C2, HSA-C2 NPs and FA-HSA-C2 NPs. Results are the mean ± SD (n = 3): *P < 0.05; **P < 0.01. 59x45mm (600 x 600 DPI)


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Figure 7 Uptake mechanism of HSA-C2 NPs and FA-HSA-C2 NPs. Statistical significance: *P < 0.05; **P < 0.01. 76x33mm (300 x 300 DPI)



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Figure 8 (A) Potential anticancer mechanism of C2/HSA-C2 NPs/FA-HSA-C2 NPs. (B) The potential mechanism of FA-HSA NPs deliver Cu pro-drug into cancer cells. 94x54mm (300 x 300 DPI)


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Graphical Abstract 127x106mm (300 x 300 DPI)


Design of an Anticancer Copper(II) Prodrug Based on the Lys199

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