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Brush-shaped RAFT polymer micelles as nanocarriers for a ruthenium (II) complex photodynamic anticancer drug
European Polymer Journal 113 (2019) 267–275
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European Polymer Journal journal homepage: www.elsevier.com/locate/europolj
Brush-shaped RAFT polymer micelles as nanocarriers for a ruthenium (II) complex photodynamic anticancer drug ⁎
T
⁎
Dan Wanga,b, , Jinquan Wanga, , Haien Huanga,b, Zizhuo Zhaoc, Pathiraja A. Gunatillakeb, ⁎ Xiaojuan Haob, a
Guangdong Pharmaceutical University, Guangzhou, Guangdong 510006, China Manufacturing, Commonwealth Scientific and Industrial Research Organisation (CSIRO), Clayton, Victoria 3168, Australia c Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510275, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Poly(PEGA) Ruthenium complex Anticancer drug Polymeric micelles Encapsulation RAFT polymerisation
Amphiphilic polymers containing brush-shaped poly(polyethylene glycol methyl ether acrylate) (PEGA) as a hydrophilic segment may form a stable hydrophilic shell for a polymeric micelle due to a strong interaction among the brush shaped molecules. In this paper, polymeric micelles with brush-shaped poly(PEGA) as hydrophilic shell were designed and formulated as nanocarriers for a new ruthenium (II) complex anticancer drug to address its poor water solubility. Well defined brush-shaped poly(PEGA)-containing amphiphilic block polymers were synthesised by reversible addition-fragmentation chain transfer (RAFT) polymerisation. The asprepared amphiphilic polymers formed stable polymeric micelle nanocarriers for the hydrophobic ruthenium (II) complex, resulting in greatly increased solubility of the drug by inhibiting its aggregation in aqueous environment. Cytotoxicity studies demonstrated that the use of polymeric micelle nanocarriers increased the drug’s toxicity to human liver cancer cells (SK-HEP-1) by 3 fold under dark and 12 fold under light conditions. However, both bare drug and encapsulated drug showed no toxicity to the normal cells, demonstrating the drug’s potential targeting capacity to cancer cells.
1. Introduction
anticancer drugs, ruthenium complexes are difficult to dissolve in water and tend to aggregate in aqueous environment, which limits their anticancer activity and inhibits their further study and application. Polymeric micelle nanocarriers have attracted many interests in the past years, because they can increase the solubility and colloidal stability of hydrophobic drugs in aqueous environment. More importantly, they show a number of in vivo advantages, such as a longer mean residence time in blood stream; reduced administration dose and diminished non-specific organ toxicity; as well as increased bioavailability [16–19]. This kind of nanocarriers is generally formed by amphiphilic polymers above their critical micellar concentration. One polymeric micelle nanocarrier contains an inner hydrophobic core, in which poorly water soluble drug can be entrapped, and an outer hydrophilic shell, which isolates the drug from the external medium. In some cases, the shell can be functionalised with moieties, such as folate, peptide, antibody, and so on, to induce targeting ability [20]. Poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(ε-caprolactone) (PCL), polyanhydrides, poly(amino acid) (PAA) are the common hydrophobic segments used for the amphiphilic polymers due to their biodegradable, biocompatible or pH-sensitive characteristics. Polyethylene glycol
Platinum based anticancer drugs such as cisplatin are now used in over a half of cancer treatment regimens [1]. Although these cisplatin drugs are very effective for certain types of cancers, they are significantly less effective for breast cancer, prostate cancer, and colorectal cancer. In addition, many late stage cancers are unresponsive to these drugs [2–4]. Besides this, severe side effects such as nephrotoxicity, ototoxicity, and neurotoxicity are major concerns [5]. In order to circumvent these problems, researchers have developed novel non-platinum based anticancer drugs, such as ruthenium complex, palladium complex, and gold complex [6–9]. Among them, ruthenium complex is definitely a rising star as it possesses some unique properties such as transferrin transport, anti-metastasis, slow ligand exchange kinetics, DNA binding etc [10–12]. At the same time, ruthenium complexes were intensively studied as photodynamic therapy (PDT) anticancer agents due to their high visible light absorption and long lived triplet excited state [13–15]. Up to now, two ruthenium complexes (NAMI-A and KP1339) are in phase II clinical trials and another one, RAPTA-C, is progressing towards clinical trials [13]. However, like many other ⁎
Corresponding authors. E-mail addresses: [email protected] (D. Wang), [email protected] (J. Wang), [email protected] (X. Hao).
https://doi.org/10.1016/j.eurpolymj.2019.01.074 Received 21 December 2018; Received in revised form 25 January 2019; Accepted 31 January 2019 Available online 01 February 2019 0014-3057/ © 2019 Published by Elsevier Ltd.
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(PEG) is often the first choice for the hydrophilic segment because of the observed longer circulation times and lower immunogenicities of the PEGylated biomacromolecules compared with their unmodified counterparts [21,22]. More sophisticated brush-shaped poly(polyethylene glycol methyl ether acrylate) (PEGA) polymers have also been developed with a good biocompatibility and the advantage of introducing various functional groups during the synthesis [23,24]. Also, the stability of the micelle structure may be enhanced due to the stronger interaction among the brush-shaped PEG side chains in the shell, compared with the linear PEG molecules, which has been investigated by a number of groups [25–32]. Interestingly, Obata et al. incorporated chlorin ring into poly(PEGA)-b-polystyrene block polymer and studied its photodynamic activity to human glioblastoma cell line (U251) and rat murine RGM-1 gastric carcinoma mucosal cell line (RGK-1). Lately, they used polystyrene-block-poly(PEGA) to encapsulate a zinc phthalocyanine photo sensitiser and studied its cytotoxicity to HeLa cell lines [33,34]. Amphiphilic polymer containing poly(PEGA) shows a great potential and may play an important role in many applications in the future. It is noteworthy that most of the polymers mentioned above were synthesised by the reversible additionfragmentation chain transfer (RAFT) polymerisation, a very versatile technology to precisely control the structure and the degree of polymerisation (DP) with narrow molecular weight distribution [35–37]. More importantly, it was found that poly(PEGA) polymers synthesised with trithiocarbonate RAFT agents were non-toxic, which is very attractive for biomedical use [38]. In this work, a new ruthenium (II) complex anticancer drug was synthesised and in order to improve its water solubility and bioavailability, a polymeric micelle nanocarrier with brush-shaped poly(PEGA) as a hydrophilic shell and polystyrene or –C12H25 short chain as a hydrophobic core was designed and prepared for encapsulation of the drug. It is expected that the polystyrene or –C12H25 core can assist high efficiency of drug loading by utilising the ‘like dissolves like’ principle and brush-shaped poly(PEGA) can provide a stable shell by its strong interaction among the brushes. It is expected that the stable polymeric micelle nanocarrier can increase the drug’s water solubility, colloidal stability, and anticancer efficacy.
Laboratories. Dynamic light scattering (DLS) experiments for measuring the size of the micelles were performed using a Malven Zetasizer (Nano – ZS) equipped with a 4 mW HeNe Laser operating at 633 nm. The measurements were conducted at 25 °C in standard disposable cuvettes and the scatter light was performed at an angle of 173°. UV–Vis spectra were recorded on a Perkin-Elmer Lambda 850 spectrophotometer. The fluorescence tests were performed using a FlexStation 3 multi-mode microplate reader (Molecular Devices). The excitation and emission wavelengths were 260 nm and 600 nm, respectively, which are the typical absorption and emission peaks of the as-prepared ruthenium (II) complex. Elemental analyses (C, H, and N) were performed using a Perkin Elmer 240Q elemental analyser. Electrospray ionization mass spectra (ESI-MS) were recorded on a LCQ system (Finnigan MAT, USA). 2.2. Synthesis of the ruthenium (II) complex 2.2.1. Synthesis of 4-(1-phenyl-1H-imidazo[4,5-f][1,10]phenanthrolin-2yl)benzoic acid (L1) A mixture of glacial acetic acid (10 mL), aniline (0.093 g, 1 mmol), 4-formylbenzoic acid (0.150 g, 1 mmol), ammonium acetate (1.542 g, 20 mmol), and 1,10-phenanthroline-5,6-dione (0.21 g, 1 mmol) was refluxed under argon for 24 h. Water (50 mL) was added to the cooled reaction and a yellow precipitate was formed. The precipitate was collected and washed with water. The crude product was then purified using column chromatography (DCM/ethanol) to obtain the ligand (yield = 58%). The ligand was characterised by elemental analysis, 1H NMR, and ESI-MS measurements. Anal. Calcd. for C26H16N4O2: C, 74.99; H, 3.87; N, 13.45%; Found: C, 74.58; H, 4.02; N, 13.29%. 1H NMR (400 MHz, DMSO): δ 9.14 – 8.97 (m, 3H), 7.91 (t, J = 7.5 Hz, 3H), 7.76 (dt, J = 18.6, 8.0 Hz, 7H), 7.52 (dd, J = 8.0, 4.0 Hz, 1H), 7.39 (d, J = 8.7 Hz, 1H). ESI-MS: m/z = 417.05 [M + H]+, 439.00 [M + Na]+. 2.2.2. Synthesis of the ruthenium (II) complex A mixture of L1 (0.083 mg, 0.2 mmol) and the ruthenium precursor Ru(X)2Cl2 (X = phen, 0.2 mmol) in N,N-Dimethylformamide (DMF, 10 mL) was heated to 150 °C for 8 h under nitrogen to give a clear deepred solution. The cooled reaction mixture was diluted with water (15 mL). Saturated aqueous ammonium hexafluorophosphate solution was added under vigorous stirring, and filtered. The dark red solid was collected and washed with small amounts of water and diethyl ether, then dried under vacuum, and purified by column chromatography on alumina using acetonitrile/ethanol as the eluent. The solvent was removed under reduced pressure and red microcrystals were obtained (yield = 60–80%). Anal. Calcd. for C50H32N8F12O2P2Ru: C, 51.42; H, 2.76; N, 9.59%; Found: C, 51.18; H, 2.94; N, 9.36%. ES-MS [CH3CN, m/ z]: 877.2 ([M − 2PF6 − H]+), 439.1 ([M − 2PF6]2+). 1H NMR (400 MHz, DMSO): δ9.19 (d, J = 8.1 Hz, 1H), 8.78 (dd, J = 16.3, 9.9 Hz, 4H), 8.40 (d, J = 10.7 Hz, 4H), 8.16–8.05 (m, 5H), 7.99 (d, J = 4.9 Hz, 1H), 7.88–7.70 (m, 12H), 7.54 (dd, J = 20.0, 6.7 Hz, 3H), 7.44 (d, J = 8.5 Hz, 1H).
2. Experimental 2.1. Materials and analytic techniques PEGA (Mn = 480), styrene (ST), azodiisobutyronitrile (AIBN), 1,4dioxane, dimethyl sulfoxide (DMSO) were purchased from SigmaAldrich (Australia). 4-cyano-4-(dodecylsulfanylthiocarbonyl)sulfanylpentanoic acid (RAFT agent) was purchased from Strem Chemicals (USA). Ruthenium chloride hydrate, 4-formylbenzoic acid, aniline, anhydrous N,N-dimethylformamide (DMF), N-hydroxysuccinimide (NHS), N,N′-dicyclohexylcarbodimide (DCC), 2,2′-bipyridine (bpy), 1,10-phenanthroline (phen), 4,7-dimethyl-1,10-phenanthroline (dmp), and 4,7-diphenyl-1,10-phenanthroline (dip) were purchased from Sigma-Aldrich (China) and all the chemicals were used as received. 1,10-phenanthroline-5,6-dione [39], 6-(4-formylphenoxy)hexanoic acid [40], and cis-Ru(X)2Cl2 [41] (X = phen) were prepared according to literature methods. Dialysis membrane (Mw cut-off = 3500 Da) was purchased from Spectrum Laboratories Incorporated (USA). 1 H NMR data were acquired on a Bruke Av 400 or 500 NMR spectrometer, and the spectra were processed using Topspin 3.5 NMR software. Gel permeation chromatography (GPC) measurement was conducted at 80 °C on a Shimadzu HPLC system comprising a SIL-20A HT auto-sampler, a PL 5.0 mm bead-size guard column, four linear PL (Styragel) columns, and a RID-10Arefractive index detector. N, N-dimethylacetamide (DMAc) containing lithium bromide (2.1 g/L) was used as the mobile phase (flow rate 1 mL/min). Calibration was performed using near-monodisperse poly(methyl methacrylate) (PMMA) standards from Polymer
2.3. Synthesis and characterisation of the brush-shaped amphiphilic polymers 2.3.1. Synthesis of polystyrene (PST) macroRAFT agent First, 4-cyano-4-(dodecylsulfanylthiocarbonyl)sulfanylpentanoic acid (RATF agent) (0.6 g, 0.0015 mol) and styrene (9 g, 0.086 mol) were added into a vial to form a homogenous solution under stirring. Then, the vial was sealed and deoxygenated by purging with high purity nitrogen gas for 30 min. After that, the vial was placed in an oil bath set at 110 °C, and stirred for 16 h. The mixture was then sampled and the conversion was measured by 1H NMR. The crude product was purified by precipitation in methanol three times followed by drying in a vacuum oven at 40 °C overnight. 1H NMR was used to check the purity and calculate the degree of polymerisation (DP). GPC was used to determine the molecular weight distribution and approximate molecular 268
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certain concentration and the micelle size was measured by DLS. The ruthenium (II) complex loading amount in the micelle was measured by measuring the fluorescence intensity of the solution of a certain amount product and retrieving the exact concentration value from a standard fluorescence intensity versus concentration curve.
weight. 2.3.2. Synthesis of polystyrene-block-poly(PEGA) (PST-b-PPEGA) In a typical process, a dioxane solution containing PST macroRAFT agent (0.5 g, 1.61 × 10-2M), AIBN (5 mg, 0.3 × 10-2M) and PEGA (4 g, 0.83 M) was prepared in a vial, which was then sealed and deoxygenated by purging with high purity nitrogen gas for 30 min. After that, the vial was placed in an oil bath set at 65 °C, stirred for 4 h and 10 min. The mixture was then sampled and the conversion was measured by 1H NMR. After desired conversion was achieved, the polymer was collected by precipitation in cyclohexane. The precipitate was dissolved in acetone and re-precipitated in cyclohexane for three times. Finally, the acetone solution of the product was transferred into a vial and the polymer was dried in a vacuum oven at 25 °C overnight. 1H NMR was used to check the purity and calculate the degree of polymerisation (DP). GPC was used to determine the molecular weight distribution and molecular weight equivalent to PMMA standards.
2.5. Cytotoxicity evaluation The human liver carcinoma cell line SK-HEP-1 (cancer cell lines) and human liver normal cell line L02 were obtained from the Cell Bank (Cell Institute, Sinica Academia Shanghai, Shanghai, China). Cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium, Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum and penicillin (100 units/mL)/streptomycin (50 units/mL) with 25 cm2 culture flasks, which were placed into a CO2 incubator set at 37 °C (95% relative humidity, 5% CO2). The culture was stopped when cells reached in the logarithmic growth phase. Then, cells were seeded into 96-well plates at 1 × 104 cells/well. After incubation for 24 h, the cells were treated with serially diluted solutions of the tested complex. Control wells were treated with culture medium. Then, the plates were incubated in the dark for another 24 h. After that, all the culture media were refreshed. For the detection of photo-toxicity, the cells were then exposed to an LED area light source (20 mW/cm2) for 10 min. Following that, all the cell cultures from the dark or light groups were incubated for an additional 48 h. Upon completion of the incubation period, cell viability was measured with the MTT assay [42]. Cell survival rate in the control wells of the dark group was considered to represent 100% cell survival. Each experiment was repeated at least three times to obtain the mean survival rates and standard deviation.
2.3.3. Synthesis of poly(PEGA) homopolymer (PPEGA) Typically, a dioxane solution containing PEGA (4 g, 0.83 M), 4cyano-4-(dodecylsulfanylthiocarbonyl) sulfanylpentanoic acid (RATF agent) (53 mg, 1.3 × 10-2M), and AIBN (5 mg, 3.0 × 10-3 M) was prepared in a vial, which was then sealed and deoxygenated by purging with high purity nitrogen gas for 30 min. After that, the vial was placed in an oil bath set at 65 °C, stirred for 1 h and 25 min. Then, the mixture was sampled and the conversion was measured by 1H NMR. After the desired conversion was achieved, the polymer was collected by precipitation in cyclohexane. The precipitate was dissolved in acetone and re-precipitated in cyclohexane for three times. Finally, the acetone solution of the product was transferred into a vial and the polymer was dried in a vacuum oven at 25 °C overnight. 1H NMR was used to check the purity and calculate the DP. GPC was used to determine the molecular weight distribution and molecular weight equivalent to PMMA standards.
3. Results and discussion 3.1. Synthesis and characterisation of ruthenium (II) complex According to previous reports [43–45], ruthenium (II) complexes, especially those bearing 2-phenylimidazo[4,5-f] [1,10]phenanthroline (PIP) ligands, can target mitochondria and induce mitochondria mediated apoptosis in cancer cells. They are a new type of promising anticancer drugs. Unfortunately, these complexes are poorly soluble in water, which is unfavourable for their use. So, in this work, a carboxyl group was introduced into PIP ligand in order to improve its water solubility. The new designed ligand (L1) and new ruthenium (II) complex were synthesised according to the process shown in Scheme 1. Their structures and compositions were confirmed by 1H NMR, ESI-MS, and elemental analysis. Fig. 1 shows the ultraviolet–visible absorption spectrum and fluorescence spectrum of the as-prepared ruthenium (II) complex anticancer drug. It can be seen that the as-prepared ruthenium (II) complex can absorb blue light (400–500 nm) and emit red light (600 nm). Usually, ruthenium (II) complexes exhibit photodynamic activity to cancer cells, which can be illustrated as follows. When the complex absorbs an excited light, for example a blue light, it will go to its excited triplet state. Some excited triplet state will then transfer its
2.4. Micelle encapsulation of the ruthenium (II) complex Thin-film hydration followed by dialysis was used to prepare ruthenium (II) complex encapsulated micelles. In a typical process, ruthenium (II) complex (0.05 g) and the as-prepared polymer (1 g) were dissolved in 20 mL of DMSO, then the solvent was evaporated by a rotary evaporator. When a thin film was formed on the inner surface of the round bottom flask, 20 mL of deionized water was added into the flask and the formed water solution was stirred for 2 h. Subsequently, the solution was transferred into a dialysis bag (Mw cut-off = 3500 Da) and dialysed against deionized water at room temperature until no more ruthenium (II) complex was released into the water. The colour of the solid ruthenium (II) complex is dark red. When it is slightly dissolved in water, the colour is yellow. So, the dialysis was carried out until no obvious yellow colour was observed in water outside of bag. Finally, the solution in the dialysis bag was collected and freeze dried. The obtained product was redispersed into deionized water to a
Scheme 1. Synthesis process of the ruthenium (II) complex. 269
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Fig. 1. Ultraviolet-visible absorption (a) and fluorescence (b) spectra of the as-prepared ruthenium (II) complex.
energy to the oxygen species in the surrounding environment, resulting in some reactive oxygen species (ROS) that will lead to apoptosis of the cancer cells. The synthesis of the designed ruthenium (II) complex was successful, and the water solubility of the complex was marginally improved. However, it can only dissolve in water with assistance of DMSO and tends to aggregate when water/DMSO ratio increases. DLS measurement showed that when the complex dissolved in pure DMSO the particle size was under 1 nm, which dramatically increased with addition of water. This will be discussed in details in the later section. To address this problem, a polymeric micelle nanocarrier with brushshaped poly(PEGA) as hydrophilic shell and poly(styrene) as hydrophobic core was designed and prepared as follows.
macroRAFT agent and block copolymer 7 are shown in Fig. 3(b). The GPC curve of the block copolymer 7 is monodisperse and symmetrical and no dead chains from the macroRAFT agent could be observed. In order to compare with block copolymer 7, a homopolymer of PEGA with a similar DP was prepared according to the process shown in Scheme 3. The [monomer]/RAFT agent ratio, initiator amount, and reaction time were optimised. Finally, the conversion was controlled at 48% (Fig. S3) and a homopolymer of PEGA with a DP of 30 was obtained (Fig. 4). The as-prepared poly(PEGA)30 homopolymer showed a low PDI of 1.23.
3.2. RAFT syntheses of brush-shaped PST-b-PPEGA block copolymer and PPEGA homopolymer
The synthesised ruthenium (II) complex anticancer drug is poorly soluble in water due to its hydrophobic nature. In order to use it in an aqueous environment, it was dissolved into DMSO first and then dispersed into water to form an aqueous solution. It is speculated that aggregation would occur when its DMSO solution was dispersed into water. The measurement of particle size of the drug in DMSO and aqueous solutions by DLS confirmed that the aggregation did occur. When the drug was dissolved in pure DMSO, its particle size was very small, about 0.6 nm (Fig. 5(a)). After the DMSO solution was dispersed into water, its particle size increased to 330 nm at a concentration of 0.4 mg/mL (Fig. 5(b)), which further increased to 1.1 µm at a higher concentration of 1.2 mg/mL (Fig. 5(c)). Such aggregation potentially impedes the drug’s efficiency in cancer treatment. The block copolymer (polymer 7) is an amphiphilic polymer, which is composed of a hydrophobic styrene segment and a hydrophilic PEGA segment. The homopolymer of poly(PEGA)30, although no hydrophobic segment like PST block was connected to it, was also an amphiphilic polymer due to the presence of the unremoved RAFT end group (-C12H25), which could act as a short hydrophobic segment. Both were brush-shaped amphiphilic polymers and could self-assemble into micelles in water. The micelles’ formation process is schematically presented in Scheme 4. The micelles’ size of these two polymers were measured by DLS. The results are shown in Fig. 6(a) and (d), which demonstrate that block copolymer 7 forms micelles with a diameter about 21 nm and homopolymer of poly(PEGA)30 forms micelles with a diameter about 8 nm. The drug loaded polymeric micelle nanocarriers were then prepared by block copolymer 7 and poly(PEGA)30 homopolymer using a thin film
3.3. Preparation of drug loaded polymeric micelle nanocarriers using the asprepared amphiphilic polymers
Scheme 2 shows the synthesis process of the brush-shaped PST–bPPEGA block copolymer. First, PST macroRAFT agent was synthesised according to the previously reported method [36]. Differently, the ratio of monomer to RAFT agent was adjusted in order to get a small hydrophobic segment. After 16 h reaction, the conversion was 45%, determined by 1H NMR (Fig. S1(a)) and the DP of the purified polymer calculated from 1H NMR spectrum was 26 (Fig. 2(a)), which was consistent with the DP calculated by conversion (DPcalc = 26). Polydispersity index (PDI) measured by GPC was 1.08 and the GPC curve is shown in Fig. 3(b). Repeated syntheses of PST macroRAFT agent demonstrated that PST macroRAFT agent with a DP of 26 could be synthesised with a good reproducibility. The brush-shaped PST–b-PPEGA block copolymers were synthesised using PST macroRAFT agent according to the procedure shown in Scheme 2. Initial attempts to produce high molecular weight polymers led to uncontrolled polymerisation (i.e. polymers 1–4 in Table S1 and Fig. S2 in Supporting Information for details) and it was thus necessary to reduce the targeted DP to 39–52 (polymers 5–6 in Table 1 and Fig. 3(a)). By decreasing the amount of initiator and controlling the conversion around 50%, an optimised block copolymer 7 (Table 1) was obtained with a low PDI of 1.29. The conversion of the block copolymer 7 was determined by 1H NMR at 54% (Fig. S1(b)), and the DP of PEGA calculated from the conversion was 28, which was consistent with the DP calculated by 1H NMR spectrum of the purified block copolymer (Fig. 2(b)). The molecular weight distribution curves of PST
Scheme 2. Synthesis of brush-shaped PST–b-PPEGA block copolymer. 270
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Fig. 2. 1H NMR spectra of purified polymers. (a) purified PST containing RAFT end groups, (b) purified PST–b-PPEGA containing RAFT end groups.
hydrophobicity, helping to increase drug loading. In addition, it was found that the drug loaded poly(PEGA)30 homopolymer micelles quickly dispersed in water while block copolymer 7 micelles needed a longer time to disperse in water. Based on these observations, finally, we chose drug loaded poly(PEGA)30 homopolymer micelles for the in vitro cell toxicity test. Prior the cytotoxicity study, the ultraviolet–visible absorption spectrum and fluorescence spectrum of the drug loaded poly(PEGA)30 homopolymer micelles were measured and compared with the pure drug (Fig. 7). No obvious change was observed, which demonstrated that the micelle nanocarriers did not change the optical properties of the complex. The intensity difference was due to the difference in the concentration of the drug.
hydration followed by dialysis method. The particle size of the drug loaded micelles was measured directly after dialysis (Fig. 6(b) and (e)). The drug loaded polymers were freeze dried after dialysis. In order to check whether the freeze drying process could affect particle size, the micelles’ size was measured again by redispersing the freeze dried samples in deionized water (Fig. 6(c) and (f)). It was observed that the average particle size of drug loaded micelles of polymer 7 was 21 nm after dialysis, same as the size of non-drug loaded polymer micelles (Fig. 6 (a) and (b)). It was also observed that the freeze drying process did not change the particle size (Fig. 6(b) and (c)), indicating a good colloidal stability of drug loaded polymer micelles. However, for poly (PEGA)30 homopolymer, the particle size of drug loaded micelles decreased to 5 nm compared to the non-drug loaded micelles (8 nm). No change was observed after freeze drying (Fig. 6(e) and (f)). In both cases, the particle size distribution became narrower (Fig. 6(a) vs (b) and (d) vs (e)), which might be caused by the hydrophobic interaction between the drug molecule and the hydrophobic segment. Particularly in the case of poly(PEGA)30, the interaction between the drug and –C12H25 segment of the poly(PEGA)30 made the micelles’ structure more dense compared with pure poly(PEGA)30 micelles without drug loading. The drug loading amounts in block copolymer 7 and poly(PEGA)30 homopolymer micelles were 1.49 wt% and 2.99 wt%, respectively. The lower loading capacity of polymer 7 may be due to the rigid structure of styrene segment that led to a loose micelle structure unfavourable for the loading of the drug. Another possible reason is the misplaced order of hydrophobicity and hydrophilicity in the structure as seen in Scheme 2 (HOOC-PST-PPEGA-C12H25 hydrophilic end group-hydrophobic segment-hydrophilic segmenthydrophobic segment). This can be improved by re-designing the structure of the block polymer, which will be presented in a separate work. In contrast, the short –C12H25 segment of the poly(PEGA)30 homopolymer may be interacting with the drug due to its
3.4. Cell toxicity study It has been reported that many Ru(II) polypyridyl complexes have excellent photoactive properties, showing enhanced cytotoxicity following light irradiation, and providing relatively selective and improved tumor therapy [46–48]. The main mechanism of action for most photoactive Ru(II) compounds in photodynamic therapy (PDT) involves reactive oxygen species (ROS) and reactive singlet oxygen (1O2) [13–14]. 1O2 and other cytotoxic ROS generated by the excited Ru(II) complexes, ultimately, cause cancer cell damage. Swavey et al. designed several Ru(II) polypyridyl compounds with porphyrin derivative ligands for PDT [49,50]. The results showed that after irradiation with light, the Ru(II) porphyryl complex efficiently induced apoptosis in melanoma cells, while normal cells were unaffected. And, more remarkably, the Ru(II) polypyridyl compound, TLD-1433, recently entered phase IB clinical trials as a PDT agent in patients with bladder cancer in 2015 [51]. Based on these, we investigated whether our Ru(II) complex loaded into polymeric micelle nanocarriers could show PDT effects.
Fig. 3. (a) Molecular weight distribution curves of the polymers 5–7, (b) molecular weight distribution curves of PST macroRAFT agent (dashed line) and poly (styrene)26–block-poly(PEGA)28 (polymer 7) (solid line). 271
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Table 1 Reaction conditions and results for the synthesis of PST–b-PPEGA block copolymers. Block copolymer
[M]/[macroRAFT agent]/[I]
T (°C)
Time (h)
Conv (%)
PDI (GPC)a
Mn (GPC)a
Mn (NMR)b
5 6 7
39/1/0.40 52/1/0.2 52/1/0.2
65 65 65
16 6 4.2
96 80 54
1.44 1.38 1.29
14,370 20,130 15,480
21,080 23,080 16,590
a b
PDI and number-average molecular weight (Mn) were determined by GPC using PMMA calibration curve. Mn was determined based on monomer conversion using 1H NMR.
Scheme 3. Synthesis of brush-shaped poly(PEGA) homopolymer.
Fig. 4. 1H NMR spectrum of purified poly(PEGA) containing RAFT end groups.
Fig. 5. Particle size distribution of the drug. (a) DMSO solution at a concentration of 5 mg/mL, (b) aqueous solution at a concentration of 0.4 mg/mL, (c) aqueous solution at a concentration of 1.2 mg/mL.
under light conditions (IC50 value was about 340 ± 39 μM). The increased toxicity can be attributed to the ROS created by the drug under light conditions. Furthermore, after being loaded into polymeric micelle nanocarriers, the drug’s cytotoxicity to tumour cells was increased by 3
The cytotoxicity profiles for SK-HEP-1 under dark and light conditions are showed in Fig. 8(a) and (b). The pure drug showed a relatively weak cytotoxicity to SK-HEP-1 under dark conditions (IC50 value was about 1237 ± 124 μM), while its toxicity was markedly enhanced 272
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Scheme 4. Schematic representation of the micelle’s formation and ruthenium (II) complex encapsulation.
Fig. 6. Particle size distribution curves of the micelles formed by the as-prepared brush-shaped polymers. (a) block copolymer 7, (b) drug loaded block copolymer 7 (after dialysis), (c) drug loaded block copolymer 7 (after dialysis and freeze drying), (d) poly(PEGA)30, (e) drug loaded poly(PEGA)30 (after dialysis), (f) drug loaded poly(PEGA)30 (after dialysis and freeze drying).
Fig. 7. Ultraviolet–visible absorption (a) and fluorescence (b) spectra of the as-prepared drug in pure and in its polymeric micelle nanocarriers.
fold (IC50 value was about 292 ± 21 μM in dark conditions), and the cytotoxicity was further enhanced by about 12 fold under light conditions (IC50 value was about 92 ± 21 μM) (Fig. 8(b)). The results demonstrated that the polymeric micelle nanocarrier was helpful in increasing the cytotoxicity of the drug to SK-HEP-1. The increase of cytotoxicity to cancer cells can be attributed to the prevention of aggregation of the drug in the aqueous environment, resulting in decreased particle size from micro-meter to nano-meter (Figs. 5 and 6). The control sample poly(PEGA)30 homopolymer showed no toxicity to cancer cells both under dark and light conditions (Fig. 8(a)). In order to evaluate the safety of the drug and the polymer, their
cytotoxicity to the normal cell L02 was studied (Fig. 8(c)). The results showed that polymer itself had no toxicity to normal cells. In addition, the drug’s toxicity to normal cells, whether as pure drug or as polymeric micelle nanocarriers, was significantly less than its toxicity to tumour cells both under dark and light conditions. The results demonstrated that the drug possesses targeting ability to tumour cells. According to the previous research [10–12], this targeting ability to tumour cells can be attributed to the fact that transferrin is over expressed on the tumour cells and transferrin can help ruthenium (II) complex transfer into the cell, like transferring iron. There may be other reasons for the difference in cytotoxicity, which may be attributed to the fact that ruthenium 273
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Fig. 8. Cytotoxicity of poly(PEGA)30 homopolymer, pure drug, and drug loaded micelle nanocarriers on SK-HEP-1 and L02 cell lines. (a) Human liver carcinoma cell line SK-HEP-1, (b) IC50 value to SK-HEP-1, (c) Human liver normal cell line L02. The data are presented as the mean ± the standard deviation.
complexes differ from traditional small molecule drugs such as cisplatin in their targets and mechanisms of action. These mechanisms have been well elucidated in a review article [51].
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4. Conclusions In order to address the poor water solubility of a new ruthenium (II) complex anticancer drug, polymeric micelle nanocarriers with brushshaped poly(PEGA) in the shell were designed and synthesised by RAFT polymerisation. Brush-shaped PST-b-PPEGA block copolymer and poly (PEGA) homopolymer containing a -C12H25 RAFT end group, with well controlled DP and narrow PDI, were synthesised after optimising reaction conditions. Both of them were able to form polymeric micelle nanocarriers for the as-prepared drug. The polymeric micelle nanocarriers greatly enhanced the drug’s water solubility by inhibiting the drug’s aggregation in aqueous environment. The -C12H25 RAFT end group-containing poly(PEGA) polymer micelle nanocarriers showed a higher drug loading capacity and better water solubility. Cytotoxicity studies demonstrated that the -C12H25 RAFT end group-containing poly (PEGA) polymeric micelle nanocarriers showed no toxicity to normal cells, however, they greatly increased the cytotoxicity of the ruthenium (II) complex anticancer drug to cancer cells by 3 fold under dark conditions and 12 fold under light conditions. In addition, it is attractive that whether as pure drug or as polymeric nanocarriers, the new ruthenium (II) complex showed much higher toxicity to tumour cells than normal cells, displaying a targeting ability. Acknowledgement The work is supported by China Scholarship Council (No. 201708440131), CSIRO Manufacturing, and the National Science Foundation of China (No. 21771042). Data availability The raw/processed data required to reproduce these findings cannot be shared at this time due to technical or time limitations. References [1] P.J. Dyson, G. Sava, Metal-based antitumour drugs in the post genomic era, Dalton Trans. 16 (2006) 1929–1933. [2] V. Brabec, J. Kasparkova, Modifications of DNA by platinum complexes relation to resistance of tumors to platinum antitumor drugs, Drug Resist. Updat. 8 (2005) 131–146. [3] T. Torigoe, H. Izumi, H. Ishiguchi, Y. Yoshida, M. Tanabe, T. Yoshida, T. Igarashi, I. Niina, T. Wakasugi, T. Imaizumi, Y. Momii, M. Kuwano, K. Kohno, Cisplatin resistance and transcription factors, Curr. Med. Chem.:Anti-Cancer Agents 5 (2005) 15–27. [4] D. Matei, F. Fang, C. Shen, J. Schilder, A. Arnold, Y. Zeng, W.A. Berry, T. Huang, K.P. Nephew, Epigenetic resensitization to platinum in ovarian cancer, Cancer Res. 72 (2012) 2197–2205. [5] C.A. Rabik, M.E. Dolan, Molecular mechanisms of resistance and toxicity associated with platinating agents, Cancer Treat. Rev. 33 (2007) 9–23.
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European Polymer Journal journal homepage: www.elsevier.com/locate/europolj
Brush-shaped RAFT polymer micelles as nanocarriers for a ruthenium (II) complex photodynamic anticancer drug ⁎
T
⁎
Dan Wanga,b, , Jinquan Wanga, , Haien Huanga,b, Zizhuo Zhaoc, Pathiraja A. Gunatillakeb, ⁎ Xiaojuan Haob, a
Guangdong Pharmaceutical University, Guangzhou, Guangdong 510006, China Manufacturing, Commonwealth Scientific and Industrial Research Organisation (CSIRO), Clayton, Victoria 3168, Australia c Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510275, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Poly(PEGA) Ruthenium complex Anticancer drug Polymeric micelles Encapsulation RAFT polymerisation
Amphiphilic polymers containing brush-shaped poly(polyethylene glycol methyl ether acrylate) (PEGA) as a hydrophilic segment may form a stable hydrophilic shell for a polymeric micelle due to a strong interaction among the brush shaped molecules. In this paper, polymeric micelles with brush-shaped poly(PEGA) as hydrophilic shell were designed and formulated as nanocarriers for a new ruthenium (II) complex anticancer drug to address its poor water solubility. Well defined brush-shaped poly(PEGA)-containing amphiphilic block polymers were synthesised by reversible addition-fragmentation chain transfer (RAFT) polymerisation. The asprepared amphiphilic polymers formed stable polymeric micelle nanocarriers for the hydrophobic ruthenium (II) complex, resulting in greatly increased solubility of the drug by inhibiting its aggregation in aqueous environment. Cytotoxicity studies demonstrated that the use of polymeric micelle nanocarriers increased the drug’s toxicity to human liver cancer cells (SK-HEP-1) by 3 fold under dark and 12 fold under light conditions. However, both bare drug and encapsulated drug showed no toxicity to the normal cells, demonstrating the drug’s potential targeting capacity to cancer cells.
1. Introduction
anticancer drugs, ruthenium complexes are difficult to dissolve in water and tend to aggregate in aqueous environment, which limits their anticancer activity and inhibits their further study and application. Polymeric micelle nanocarriers have attracted many interests in the past years, because they can increase the solubility and colloidal stability of hydrophobic drugs in aqueous environment. More importantly, they show a number of in vivo advantages, such as a longer mean residence time in blood stream; reduced administration dose and diminished non-specific organ toxicity; as well as increased bioavailability [16–19]. This kind of nanocarriers is generally formed by amphiphilic polymers above their critical micellar concentration. One polymeric micelle nanocarrier contains an inner hydrophobic core, in which poorly water soluble drug can be entrapped, and an outer hydrophilic shell, which isolates the drug from the external medium. In some cases, the shell can be functionalised with moieties, such as folate, peptide, antibody, and so on, to induce targeting ability [20]. Poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(ε-caprolactone) (PCL), polyanhydrides, poly(amino acid) (PAA) are the common hydrophobic segments used for the amphiphilic polymers due to their biodegradable, biocompatible or pH-sensitive characteristics. Polyethylene glycol
Platinum based anticancer drugs such as cisplatin are now used in over a half of cancer treatment regimens [1]. Although these cisplatin drugs are very effective for certain types of cancers, they are significantly less effective for breast cancer, prostate cancer, and colorectal cancer. In addition, many late stage cancers are unresponsive to these drugs [2–4]. Besides this, severe side effects such as nephrotoxicity, ototoxicity, and neurotoxicity are major concerns [5]. In order to circumvent these problems, researchers have developed novel non-platinum based anticancer drugs, such as ruthenium complex, palladium complex, and gold complex [6–9]. Among them, ruthenium complex is definitely a rising star as it possesses some unique properties such as transferrin transport, anti-metastasis, slow ligand exchange kinetics, DNA binding etc [10–12]. At the same time, ruthenium complexes were intensively studied as photodynamic therapy (PDT) anticancer agents due to their high visible light absorption and long lived triplet excited state [13–15]. Up to now, two ruthenium complexes (NAMI-A and KP1339) are in phase II clinical trials and another one, RAPTA-C, is progressing towards clinical trials [13]. However, like many other ⁎
Corresponding authors. E-mail addresses: [email protected] (D. Wang), [email protected] (J. Wang), [email protected] (X. Hao).
https://doi.org/10.1016/j.eurpolymj.2019.01.074 Received 21 December 2018; Received in revised form 25 January 2019; Accepted 31 January 2019 Available online 01 February 2019 0014-3057/ © 2019 Published by Elsevier Ltd.
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(PEG) is often the first choice for the hydrophilic segment because of the observed longer circulation times and lower immunogenicities of the PEGylated biomacromolecules compared with their unmodified counterparts [21,22]. More sophisticated brush-shaped poly(polyethylene glycol methyl ether acrylate) (PEGA) polymers have also been developed with a good biocompatibility and the advantage of introducing various functional groups during the synthesis [23,24]. Also, the stability of the micelle structure may be enhanced due to the stronger interaction among the brush-shaped PEG side chains in the shell, compared with the linear PEG molecules, which has been investigated by a number of groups [25–32]. Interestingly, Obata et al. incorporated chlorin ring into poly(PEGA)-b-polystyrene block polymer and studied its photodynamic activity to human glioblastoma cell line (U251) and rat murine RGM-1 gastric carcinoma mucosal cell line (RGK-1). Lately, they used polystyrene-block-poly(PEGA) to encapsulate a zinc phthalocyanine photo sensitiser and studied its cytotoxicity to HeLa cell lines [33,34]. Amphiphilic polymer containing poly(PEGA) shows a great potential and may play an important role in many applications in the future. It is noteworthy that most of the polymers mentioned above were synthesised by the reversible additionfragmentation chain transfer (RAFT) polymerisation, a very versatile technology to precisely control the structure and the degree of polymerisation (DP) with narrow molecular weight distribution [35–37]. More importantly, it was found that poly(PEGA) polymers synthesised with trithiocarbonate RAFT agents were non-toxic, which is very attractive for biomedical use [38]. In this work, a new ruthenium (II) complex anticancer drug was synthesised and in order to improve its water solubility and bioavailability, a polymeric micelle nanocarrier with brush-shaped poly(PEGA) as a hydrophilic shell and polystyrene or –C12H25 short chain as a hydrophobic core was designed and prepared for encapsulation of the drug. It is expected that the polystyrene or –C12H25 core can assist high efficiency of drug loading by utilising the ‘like dissolves like’ principle and brush-shaped poly(PEGA) can provide a stable shell by its strong interaction among the brushes. It is expected that the stable polymeric micelle nanocarrier can increase the drug’s water solubility, colloidal stability, and anticancer efficacy.
Laboratories. Dynamic light scattering (DLS) experiments for measuring the size of the micelles were performed using a Malven Zetasizer (Nano – ZS) equipped with a 4 mW HeNe Laser operating at 633 nm. The measurements were conducted at 25 °C in standard disposable cuvettes and the scatter light was performed at an angle of 173°. UV–Vis spectra were recorded on a Perkin-Elmer Lambda 850 spectrophotometer. The fluorescence tests were performed using a FlexStation 3 multi-mode microplate reader (Molecular Devices). The excitation and emission wavelengths were 260 nm and 600 nm, respectively, which are the typical absorption and emission peaks of the as-prepared ruthenium (II) complex. Elemental analyses (C, H, and N) were performed using a Perkin Elmer 240Q elemental analyser. Electrospray ionization mass spectra (ESI-MS) were recorded on a LCQ system (Finnigan MAT, USA). 2.2. Synthesis of the ruthenium (II) complex 2.2.1. Synthesis of 4-(1-phenyl-1H-imidazo[4,5-f][1,10]phenanthrolin-2yl)benzoic acid (L1) A mixture of glacial acetic acid (10 mL), aniline (0.093 g, 1 mmol), 4-formylbenzoic acid (0.150 g, 1 mmol), ammonium acetate (1.542 g, 20 mmol), and 1,10-phenanthroline-5,6-dione (0.21 g, 1 mmol) was refluxed under argon for 24 h. Water (50 mL) was added to the cooled reaction and a yellow precipitate was formed. The precipitate was collected and washed with water. The crude product was then purified using column chromatography (DCM/ethanol) to obtain the ligand (yield = 58%). The ligand was characterised by elemental analysis, 1H NMR, and ESI-MS measurements. Anal. Calcd. for C26H16N4O2: C, 74.99; H, 3.87; N, 13.45%; Found: C, 74.58; H, 4.02; N, 13.29%. 1H NMR (400 MHz, DMSO): δ 9.14 – 8.97 (m, 3H), 7.91 (t, J = 7.5 Hz, 3H), 7.76 (dt, J = 18.6, 8.0 Hz, 7H), 7.52 (dd, J = 8.0, 4.0 Hz, 1H), 7.39 (d, J = 8.7 Hz, 1H). ESI-MS: m/z = 417.05 [M + H]+, 439.00 [M + Na]+. 2.2.2. Synthesis of the ruthenium (II) complex A mixture of L1 (0.083 mg, 0.2 mmol) and the ruthenium precursor Ru(X)2Cl2 (X = phen, 0.2 mmol) in N,N-Dimethylformamide (DMF, 10 mL) was heated to 150 °C for 8 h under nitrogen to give a clear deepred solution. The cooled reaction mixture was diluted with water (15 mL). Saturated aqueous ammonium hexafluorophosphate solution was added under vigorous stirring, and filtered. The dark red solid was collected and washed with small amounts of water and diethyl ether, then dried under vacuum, and purified by column chromatography on alumina using acetonitrile/ethanol as the eluent. The solvent was removed under reduced pressure and red microcrystals were obtained (yield = 60–80%). Anal. Calcd. for C50H32N8F12O2P2Ru: C, 51.42; H, 2.76; N, 9.59%; Found: C, 51.18; H, 2.94; N, 9.36%. ES-MS [CH3CN, m/ z]: 877.2 ([M − 2PF6 − H]+), 439.1 ([M − 2PF6]2+). 1H NMR (400 MHz, DMSO): δ9.19 (d, J = 8.1 Hz, 1H), 8.78 (dd, J = 16.3, 9.9 Hz, 4H), 8.40 (d, J = 10.7 Hz, 4H), 8.16–8.05 (m, 5H), 7.99 (d, J = 4.9 Hz, 1H), 7.88–7.70 (m, 12H), 7.54 (dd, J = 20.0, 6.7 Hz, 3H), 7.44 (d, J = 8.5 Hz, 1H).
2. Experimental 2.1. Materials and analytic techniques PEGA (Mn = 480), styrene (ST), azodiisobutyronitrile (AIBN), 1,4dioxane, dimethyl sulfoxide (DMSO) were purchased from SigmaAldrich (Australia). 4-cyano-4-(dodecylsulfanylthiocarbonyl)sulfanylpentanoic acid (RAFT agent) was purchased from Strem Chemicals (USA). Ruthenium chloride hydrate, 4-formylbenzoic acid, aniline, anhydrous N,N-dimethylformamide (DMF), N-hydroxysuccinimide (NHS), N,N′-dicyclohexylcarbodimide (DCC), 2,2′-bipyridine (bpy), 1,10-phenanthroline (phen), 4,7-dimethyl-1,10-phenanthroline (dmp), and 4,7-diphenyl-1,10-phenanthroline (dip) were purchased from Sigma-Aldrich (China) and all the chemicals were used as received. 1,10-phenanthroline-5,6-dione [39], 6-(4-formylphenoxy)hexanoic acid [40], and cis-Ru(X)2Cl2 [41] (X = phen) were prepared according to literature methods. Dialysis membrane (Mw cut-off = 3500 Da) was purchased from Spectrum Laboratories Incorporated (USA). 1 H NMR data were acquired on a Bruke Av 400 or 500 NMR spectrometer, and the spectra were processed using Topspin 3.5 NMR software. Gel permeation chromatography (GPC) measurement was conducted at 80 °C on a Shimadzu HPLC system comprising a SIL-20A HT auto-sampler, a PL 5.0 mm bead-size guard column, four linear PL (Styragel) columns, and a RID-10Arefractive index detector. N, N-dimethylacetamide (DMAc) containing lithium bromide (2.1 g/L) was used as the mobile phase (flow rate 1 mL/min). Calibration was performed using near-monodisperse poly(methyl methacrylate) (PMMA) standards from Polymer
2.3. Synthesis and characterisation of the brush-shaped amphiphilic polymers 2.3.1. Synthesis of polystyrene (PST) macroRAFT agent First, 4-cyano-4-(dodecylsulfanylthiocarbonyl)sulfanylpentanoic acid (RATF agent) (0.6 g, 0.0015 mol) and styrene (9 g, 0.086 mol) were added into a vial to form a homogenous solution under stirring. Then, the vial was sealed and deoxygenated by purging with high purity nitrogen gas for 30 min. After that, the vial was placed in an oil bath set at 110 °C, and stirred for 16 h. The mixture was then sampled and the conversion was measured by 1H NMR. The crude product was purified by precipitation in methanol three times followed by drying in a vacuum oven at 40 °C overnight. 1H NMR was used to check the purity and calculate the degree of polymerisation (DP). GPC was used to determine the molecular weight distribution and approximate molecular 268
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certain concentration and the micelle size was measured by DLS. The ruthenium (II) complex loading amount in the micelle was measured by measuring the fluorescence intensity of the solution of a certain amount product and retrieving the exact concentration value from a standard fluorescence intensity versus concentration curve.
weight. 2.3.2. Synthesis of polystyrene-block-poly(PEGA) (PST-b-PPEGA) In a typical process, a dioxane solution containing PST macroRAFT agent (0.5 g, 1.61 × 10-2M), AIBN (5 mg, 0.3 × 10-2M) and PEGA (4 g, 0.83 M) was prepared in a vial, which was then sealed and deoxygenated by purging with high purity nitrogen gas for 30 min. After that, the vial was placed in an oil bath set at 65 °C, stirred for 4 h and 10 min. The mixture was then sampled and the conversion was measured by 1H NMR. After desired conversion was achieved, the polymer was collected by precipitation in cyclohexane. The precipitate was dissolved in acetone and re-precipitated in cyclohexane for three times. Finally, the acetone solution of the product was transferred into a vial and the polymer was dried in a vacuum oven at 25 °C overnight. 1H NMR was used to check the purity and calculate the degree of polymerisation (DP). GPC was used to determine the molecular weight distribution and molecular weight equivalent to PMMA standards.
2.5. Cytotoxicity evaluation The human liver carcinoma cell line SK-HEP-1 (cancer cell lines) and human liver normal cell line L02 were obtained from the Cell Bank (Cell Institute, Sinica Academia Shanghai, Shanghai, China). Cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium, Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum and penicillin (100 units/mL)/streptomycin (50 units/mL) with 25 cm2 culture flasks, which were placed into a CO2 incubator set at 37 °C (95% relative humidity, 5% CO2). The culture was stopped when cells reached in the logarithmic growth phase. Then, cells were seeded into 96-well plates at 1 × 104 cells/well. After incubation for 24 h, the cells were treated with serially diluted solutions of the tested complex. Control wells were treated with culture medium. Then, the plates were incubated in the dark for another 24 h. After that, all the culture media were refreshed. For the detection of photo-toxicity, the cells were then exposed to an LED area light source (20 mW/cm2) for 10 min. Following that, all the cell cultures from the dark or light groups were incubated for an additional 48 h. Upon completion of the incubation period, cell viability was measured with the MTT assay [42]. Cell survival rate in the control wells of the dark group was considered to represent 100% cell survival. Each experiment was repeated at least three times to obtain the mean survival rates and standard deviation.
2.3.3. Synthesis of poly(PEGA) homopolymer (PPEGA) Typically, a dioxane solution containing PEGA (4 g, 0.83 M), 4cyano-4-(dodecylsulfanylthiocarbonyl) sulfanylpentanoic acid (RATF agent) (53 mg, 1.3 × 10-2M), and AIBN (5 mg, 3.0 × 10-3 M) was prepared in a vial, which was then sealed and deoxygenated by purging with high purity nitrogen gas for 30 min. After that, the vial was placed in an oil bath set at 65 °C, stirred for 1 h and 25 min. Then, the mixture was sampled and the conversion was measured by 1H NMR. After the desired conversion was achieved, the polymer was collected by precipitation in cyclohexane. The precipitate was dissolved in acetone and re-precipitated in cyclohexane for three times. Finally, the acetone solution of the product was transferred into a vial and the polymer was dried in a vacuum oven at 25 °C overnight. 1H NMR was used to check the purity and calculate the DP. GPC was used to determine the molecular weight distribution and molecular weight equivalent to PMMA standards.
3. Results and discussion 3.1. Synthesis and characterisation of ruthenium (II) complex According to previous reports [43–45], ruthenium (II) complexes, especially those bearing 2-phenylimidazo[4,5-f] [1,10]phenanthroline (PIP) ligands, can target mitochondria and induce mitochondria mediated apoptosis in cancer cells. They are a new type of promising anticancer drugs. Unfortunately, these complexes are poorly soluble in water, which is unfavourable for their use. So, in this work, a carboxyl group was introduced into PIP ligand in order to improve its water solubility. The new designed ligand (L1) and new ruthenium (II) complex were synthesised according to the process shown in Scheme 1. Their structures and compositions were confirmed by 1H NMR, ESI-MS, and elemental analysis. Fig. 1 shows the ultraviolet–visible absorption spectrum and fluorescence spectrum of the as-prepared ruthenium (II) complex anticancer drug. It can be seen that the as-prepared ruthenium (II) complex can absorb blue light (400–500 nm) and emit red light (600 nm). Usually, ruthenium (II) complexes exhibit photodynamic activity to cancer cells, which can be illustrated as follows. When the complex absorbs an excited light, for example a blue light, it will go to its excited triplet state. Some excited triplet state will then transfer its
2.4. Micelle encapsulation of the ruthenium (II) complex Thin-film hydration followed by dialysis was used to prepare ruthenium (II) complex encapsulated micelles. In a typical process, ruthenium (II) complex (0.05 g) and the as-prepared polymer (1 g) were dissolved in 20 mL of DMSO, then the solvent was evaporated by a rotary evaporator. When a thin film was formed on the inner surface of the round bottom flask, 20 mL of deionized water was added into the flask and the formed water solution was stirred for 2 h. Subsequently, the solution was transferred into a dialysis bag (Mw cut-off = 3500 Da) and dialysed against deionized water at room temperature until no more ruthenium (II) complex was released into the water. The colour of the solid ruthenium (II) complex is dark red. When it is slightly dissolved in water, the colour is yellow. So, the dialysis was carried out until no obvious yellow colour was observed in water outside of bag. Finally, the solution in the dialysis bag was collected and freeze dried. The obtained product was redispersed into deionized water to a
Scheme 1. Synthesis process of the ruthenium (II) complex. 269
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Fig. 1. Ultraviolet-visible absorption (a) and fluorescence (b) spectra of the as-prepared ruthenium (II) complex.
energy to the oxygen species in the surrounding environment, resulting in some reactive oxygen species (ROS) that will lead to apoptosis of the cancer cells. The synthesis of the designed ruthenium (II) complex was successful, and the water solubility of the complex was marginally improved. However, it can only dissolve in water with assistance of DMSO and tends to aggregate when water/DMSO ratio increases. DLS measurement showed that when the complex dissolved in pure DMSO the particle size was under 1 nm, which dramatically increased with addition of water. This will be discussed in details in the later section. To address this problem, a polymeric micelle nanocarrier with brushshaped poly(PEGA) as hydrophilic shell and poly(styrene) as hydrophobic core was designed and prepared as follows.
macroRAFT agent and block copolymer 7 are shown in Fig. 3(b). The GPC curve of the block copolymer 7 is monodisperse and symmetrical and no dead chains from the macroRAFT agent could be observed. In order to compare with block copolymer 7, a homopolymer of PEGA with a similar DP was prepared according to the process shown in Scheme 3. The [monomer]/RAFT agent ratio, initiator amount, and reaction time were optimised. Finally, the conversion was controlled at 48% (Fig. S3) and a homopolymer of PEGA with a DP of 30 was obtained (Fig. 4). The as-prepared poly(PEGA)30 homopolymer showed a low PDI of 1.23.
3.2. RAFT syntheses of brush-shaped PST-b-PPEGA block copolymer and PPEGA homopolymer
The synthesised ruthenium (II) complex anticancer drug is poorly soluble in water due to its hydrophobic nature. In order to use it in an aqueous environment, it was dissolved into DMSO first and then dispersed into water to form an aqueous solution. It is speculated that aggregation would occur when its DMSO solution was dispersed into water. The measurement of particle size of the drug in DMSO and aqueous solutions by DLS confirmed that the aggregation did occur. When the drug was dissolved in pure DMSO, its particle size was very small, about 0.6 nm (Fig. 5(a)). After the DMSO solution was dispersed into water, its particle size increased to 330 nm at a concentration of 0.4 mg/mL (Fig. 5(b)), which further increased to 1.1 µm at a higher concentration of 1.2 mg/mL (Fig. 5(c)). Such aggregation potentially impedes the drug’s efficiency in cancer treatment. The block copolymer (polymer 7) is an amphiphilic polymer, which is composed of a hydrophobic styrene segment and a hydrophilic PEGA segment. The homopolymer of poly(PEGA)30, although no hydrophobic segment like PST block was connected to it, was also an amphiphilic polymer due to the presence of the unremoved RAFT end group (-C12H25), which could act as a short hydrophobic segment. Both were brush-shaped amphiphilic polymers and could self-assemble into micelles in water. The micelles’ formation process is schematically presented in Scheme 4. The micelles’ size of these two polymers were measured by DLS. The results are shown in Fig. 6(a) and (d), which demonstrate that block copolymer 7 forms micelles with a diameter about 21 nm and homopolymer of poly(PEGA)30 forms micelles with a diameter about 8 nm. The drug loaded polymeric micelle nanocarriers were then prepared by block copolymer 7 and poly(PEGA)30 homopolymer using a thin film
3.3. Preparation of drug loaded polymeric micelle nanocarriers using the asprepared amphiphilic polymers
Scheme 2 shows the synthesis process of the brush-shaped PST–bPPEGA block copolymer. First, PST macroRAFT agent was synthesised according to the previously reported method [36]. Differently, the ratio of monomer to RAFT agent was adjusted in order to get a small hydrophobic segment. After 16 h reaction, the conversion was 45%, determined by 1H NMR (Fig. S1(a)) and the DP of the purified polymer calculated from 1H NMR spectrum was 26 (Fig. 2(a)), which was consistent with the DP calculated by conversion (DPcalc = 26). Polydispersity index (PDI) measured by GPC was 1.08 and the GPC curve is shown in Fig. 3(b). Repeated syntheses of PST macroRAFT agent demonstrated that PST macroRAFT agent with a DP of 26 could be synthesised with a good reproducibility. The brush-shaped PST–b-PPEGA block copolymers were synthesised using PST macroRAFT agent according to the procedure shown in Scheme 2. Initial attempts to produce high molecular weight polymers led to uncontrolled polymerisation (i.e. polymers 1–4 in Table S1 and Fig. S2 in Supporting Information for details) and it was thus necessary to reduce the targeted DP to 39–52 (polymers 5–6 in Table 1 and Fig. 3(a)). By decreasing the amount of initiator and controlling the conversion around 50%, an optimised block copolymer 7 (Table 1) was obtained with a low PDI of 1.29. The conversion of the block copolymer 7 was determined by 1H NMR at 54% (Fig. S1(b)), and the DP of PEGA calculated from the conversion was 28, which was consistent with the DP calculated by 1H NMR spectrum of the purified block copolymer (Fig. 2(b)). The molecular weight distribution curves of PST
Scheme 2. Synthesis of brush-shaped PST–b-PPEGA block copolymer. 270
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Fig. 2. 1H NMR spectra of purified polymers. (a) purified PST containing RAFT end groups, (b) purified PST–b-PPEGA containing RAFT end groups.
hydrophobicity, helping to increase drug loading. In addition, it was found that the drug loaded poly(PEGA)30 homopolymer micelles quickly dispersed in water while block copolymer 7 micelles needed a longer time to disperse in water. Based on these observations, finally, we chose drug loaded poly(PEGA)30 homopolymer micelles for the in vitro cell toxicity test. Prior the cytotoxicity study, the ultraviolet–visible absorption spectrum and fluorescence spectrum of the drug loaded poly(PEGA)30 homopolymer micelles were measured and compared with the pure drug (Fig. 7). No obvious change was observed, which demonstrated that the micelle nanocarriers did not change the optical properties of the complex. The intensity difference was due to the difference in the concentration of the drug.
hydration followed by dialysis method. The particle size of the drug loaded micelles was measured directly after dialysis (Fig. 6(b) and (e)). The drug loaded polymers were freeze dried after dialysis. In order to check whether the freeze drying process could affect particle size, the micelles’ size was measured again by redispersing the freeze dried samples in deionized water (Fig. 6(c) and (f)). It was observed that the average particle size of drug loaded micelles of polymer 7 was 21 nm after dialysis, same as the size of non-drug loaded polymer micelles (Fig. 6 (a) and (b)). It was also observed that the freeze drying process did not change the particle size (Fig. 6(b) and (c)), indicating a good colloidal stability of drug loaded polymer micelles. However, for poly (PEGA)30 homopolymer, the particle size of drug loaded micelles decreased to 5 nm compared to the non-drug loaded micelles (8 nm). No change was observed after freeze drying (Fig. 6(e) and (f)). In both cases, the particle size distribution became narrower (Fig. 6(a) vs (b) and (d) vs (e)), which might be caused by the hydrophobic interaction between the drug molecule and the hydrophobic segment. Particularly in the case of poly(PEGA)30, the interaction between the drug and –C12H25 segment of the poly(PEGA)30 made the micelles’ structure more dense compared with pure poly(PEGA)30 micelles without drug loading. The drug loading amounts in block copolymer 7 and poly(PEGA)30 homopolymer micelles were 1.49 wt% and 2.99 wt%, respectively. The lower loading capacity of polymer 7 may be due to the rigid structure of styrene segment that led to a loose micelle structure unfavourable for the loading of the drug. Another possible reason is the misplaced order of hydrophobicity and hydrophilicity in the structure as seen in Scheme 2 (HOOC-PST-PPEGA-C12H25 hydrophilic end group-hydrophobic segment-hydrophilic segmenthydrophobic segment). This can be improved by re-designing the structure of the block polymer, which will be presented in a separate work. In contrast, the short –C12H25 segment of the poly(PEGA)30 homopolymer may be interacting with the drug due to its
3.4. Cell toxicity study It has been reported that many Ru(II) polypyridyl complexes have excellent photoactive properties, showing enhanced cytotoxicity following light irradiation, and providing relatively selective and improved tumor therapy [46–48]. The main mechanism of action for most photoactive Ru(II) compounds in photodynamic therapy (PDT) involves reactive oxygen species (ROS) and reactive singlet oxygen (1O2) [13–14]. 1O2 and other cytotoxic ROS generated by the excited Ru(II) complexes, ultimately, cause cancer cell damage. Swavey et al. designed several Ru(II) polypyridyl compounds with porphyrin derivative ligands for PDT [49,50]. The results showed that after irradiation with light, the Ru(II) porphyryl complex efficiently induced apoptosis in melanoma cells, while normal cells were unaffected. And, more remarkably, the Ru(II) polypyridyl compound, TLD-1433, recently entered phase IB clinical trials as a PDT agent in patients with bladder cancer in 2015 [51]. Based on these, we investigated whether our Ru(II) complex loaded into polymeric micelle nanocarriers could show PDT effects.
Fig. 3. (a) Molecular weight distribution curves of the polymers 5–7, (b) molecular weight distribution curves of PST macroRAFT agent (dashed line) and poly (styrene)26–block-poly(PEGA)28 (polymer 7) (solid line). 271
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Table 1 Reaction conditions and results for the synthesis of PST–b-PPEGA block copolymers. Block copolymer
[M]/[macroRAFT agent]/[I]
T (°C)
Time (h)
Conv (%)
PDI (GPC)a
Mn (GPC)a
Mn (NMR)b
5 6 7
39/1/0.40 52/1/0.2 52/1/0.2
65 65 65
16 6 4.2
96 80 54
1.44 1.38 1.29
14,370 20,130 15,480
21,080 23,080 16,590
a b
PDI and number-average molecular weight (Mn) were determined by GPC using PMMA calibration curve. Mn was determined based on monomer conversion using 1H NMR.
Scheme 3. Synthesis of brush-shaped poly(PEGA) homopolymer.
Fig. 4. 1H NMR spectrum of purified poly(PEGA) containing RAFT end groups.
Fig. 5. Particle size distribution of the drug. (a) DMSO solution at a concentration of 5 mg/mL, (b) aqueous solution at a concentration of 0.4 mg/mL, (c) aqueous solution at a concentration of 1.2 mg/mL.
under light conditions (IC50 value was about 340 ± 39 μM). The increased toxicity can be attributed to the ROS created by the drug under light conditions. Furthermore, after being loaded into polymeric micelle nanocarriers, the drug’s cytotoxicity to tumour cells was increased by 3
The cytotoxicity profiles for SK-HEP-1 under dark and light conditions are showed in Fig. 8(a) and (b). The pure drug showed a relatively weak cytotoxicity to SK-HEP-1 under dark conditions (IC50 value was about 1237 ± 124 μM), while its toxicity was markedly enhanced 272
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Scheme 4. Schematic representation of the micelle’s formation and ruthenium (II) complex encapsulation.
Fig. 6. Particle size distribution curves of the micelles formed by the as-prepared brush-shaped polymers. (a) block copolymer 7, (b) drug loaded block copolymer 7 (after dialysis), (c) drug loaded block copolymer 7 (after dialysis and freeze drying), (d) poly(PEGA)30, (e) drug loaded poly(PEGA)30 (after dialysis), (f) drug loaded poly(PEGA)30 (after dialysis and freeze drying).
Fig. 7. Ultraviolet–visible absorption (a) and fluorescence (b) spectra of the as-prepared drug in pure and in its polymeric micelle nanocarriers.
fold (IC50 value was about 292 ± 21 μM in dark conditions), and the cytotoxicity was further enhanced by about 12 fold under light conditions (IC50 value was about 92 ± 21 μM) (Fig. 8(b)). The results demonstrated that the polymeric micelle nanocarrier was helpful in increasing the cytotoxicity of the drug to SK-HEP-1. The increase of cytotoxicity to cancer cells can be attributed to the prevention of aggregation of the drug in the aqueous environment, resulting in decreased particle size from micro-meter to nano-meter (Figs. 5 and 6). The control sample poly(PEGA)30 homopolymer showed no toxicity to cancer cells both under dark and light conditions (Fig. 8(a)). In order to evaluate the safety of the drug and the polymer, their
cytotoxicity to the normal cell L02 was studied (Fig. 8(c)). The results showed that polymer itself had no toxicity to normal cells. In addition, the drug’s toxicity to normal cells, whether as pure drug or as polymeric micelle nanocarriers, was significantly less than its toxicity to tumour cells both under dark and light conditions. The results demonstrated that the drug possesses targeting ability to tumour cells. According to the previous research [10–12], this targeting ability to tumour cells can be attributed to the fact that transferrin is over expressed on the tumour cells and transferrin can help ruthenium (II) complex transfer into the cell, like transferring iron. There may be other reasons for the difference in cytotoxicity, which may be attributed to the fact that ruthenium 273
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Fig. 8. Cytotoxicity of poly(PEGA)30 homopolymer, pure drug, and drug loaded micelle nanocarriers on SK-HEP-1 and L02 cell lines. (a) Human liver carcinoma cell line SK-HEP-1, (b) IC50 value to SK-HEP-1, (c) Human liver normal cell line L02. The data are presented as the mean ± the standard deviation.
complexes differ from traditional small molecule drugs such as cisplatin in their targets and mechanisms of action. These mechanisms have been well elucidated in a review article [51].
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4. Conclusions In order to address the poor water solubility of a new ruthenium (II) complex anticancer drug, polymeric micelle nanocarriers with brushshaped poly(PEGA) in the shell were designed and synthesised by RAFT polymerisation. Brush-shaped PST-b-PPEGA block copolymer and poly (PEGA) homopolymer containing a -C12H25 RAFT end group, with well controlled DP and narrow PDI, were synthesised after optimising reaction conditions. Both of them were able to form polymeric micelle nanocarriers for the as-prepared drug. The polymeric micelle nanocarriers greatly enhanced the drug’s water solubility by inhibiting the drug’s aggregation in aqueous environment. The -C12H25 RAFT end group-containing poly(PEGA) polymer micelle nanocarriers showed a higher drug loading capacity and better water solubility. Cytotoxicity studies demonstrated that the -C12H25 RAFT end group-containing poly (PEGA) polymeric micelle nanocarriers showed no toxicity to normal cells, however, they greatly increased the cytotoxicity of the ruthenium (II) complex anticancer drug to cancer cells by 3 fold under dark conditions and 12 fold under light conditions. In addition, it is attractive that whether as pure drug or as polymeric nanocarriers, the new ruthenium (II) complex showed much higher toxicity to tumour cells than normal cells, displaying a targeting ability. Acknowledgement The work is supported by China Scholarship Council (No. 201708440131), CSIRO Manufacturing, and the National Science Foundation of China (No. 21771042). Data availability The raw/processed data required to reproduce these findings cannot be shared at this time due to technical or time limitations. References [1] P.J. Dyson, G. Sava, Metal-based antitumour drugs in the post genomic era, Dalton Trans. 16 (2006) 1929–1933. [2] V. Brabec, J. Kasparkova, Modifications of DNA by platinum complexes relation to resistance of tumors to platinum antitumor drugs, Drug Resist. Updat. 8 (2005) 131–146. [3] T. Torigoe, H. Izumi, H. Ishiguchi, Y. Yoshida, M. Tanabe, T. Yoshida, T. Igarashi, I. Niina, T. Wakasugi, T. Imaizumi, Y. Momii, M. Kuwano, K. Kohno, Cisplatin resistance and transcription factors, Curr. Med. Chem.:Anti-Cancer Agents 5 (2005) 15–27. [4] D. Matei, F. Fang, C. Shen, J. Schilder, A. Arnold, Y. Zeng, W.A. Berry, T. Huang, K.P. Nephew, Epigenetic resensitization to platinum in ovarian cancer, Cancer Res. 72 (2012) 2197–2205. [5] C.A. Rabik, M.E. Dolan, Molecular mechanisms of resistance and toxicity associated with platinating agents, Cancer Treat. Rev. 33 (2007) 9–23.
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