Poly[platinum(IV)-alt-PEI]/Akt1 shRNA complexes for enhanced anticancer therapy

Abstract

The co-delivery of platinum(IV) prodrugs and nucleic acids has emerged as a new modality for cancer therapy. However, as a result of the different molecular properties of nucleic acids and platinum(IV) prodrugs, the co-delivery of platinum(IV) prodrugs and nucleic acids encounter challenges (e.g., insufficient drug loading capacity and efficiency, less definable ratios of the two agents). To address these issues, a novel polymeric platinum(IV) prodrug, poly[platinum(IV)-alt-PEI] (DP), was synthesized by one-step Michael-addition reaction of platinum(IV) diacrylate with low molecular weight PEI (PEI 800). The loading efficiency of the platinum(IV) prodrug was nearly 100%, for cases where the platinum(IV) prodrug content in the polymer could be precisely regulated by simply changing the molar ratios of the reactants. The poly[platinum(IV)-alt-PEI]/Akt1 shRNA complex was constructed by the electrostatic condensation of negatively charged Akt1 shRNA (a RNAi sequence for serine–threonine kinase AKT pathway) with positively charged poly[platinum(IV)-alt-PEI]. Cellular assays involving A549, MCF-7, and PC-3 cancer cells indicated that poly[platinum(IV)-alt-PEI]/Akt1 shRNA complexes for platinum(IV) prodrug and Akt1 shRNA co-delivery offered combinational anticancer efficacy, inhibiting cancer cell proliferation more efficiently than the individual treatments. In conclusion, we proposed a convenient carrier assembly, which could achieve the co-delivery of small molecular chemotherapeutics and macromolecular nucleic acids.

---

1 Introduction

Cisplatin, a simple coordination compound, is now one of the most effective anticancer drugs, and cisplatin is involved in roughly half of the chemotherapeutic regimens administered in clinical treatment.¹ However, severe toxic side effects and drug resistance associated with cisplatin limit its clinical application.^2,3 A reasonable alternative to cisplatin is to develop platinum(IV) anticancer complexes, as prodrugs of cisplatin or other platinum(II) based therapeutics. Platinum(IV) complexes can be reduced to their bioactive platinum(II) counterparts in vivo primarily by ascorbic acid and glutathione (GSH).^4,5 Platinum(IV) complexes offer several potential advantages when compared with their platinum(II) congeners, which include: (i) reduced side effects and increased stability that facilitates the intravenous-to-oral switch in cancer chemotherapy, (ii) convenient structural modifications through the axial ligands to improve their pharmacological properties. For these reasons, platinum(IV) prodrugs are regarded as the next generation platinum drugs for cancer therapy.⁶

Due to the heterogeneity and complexity of cancer, combinational therapy is becoming a promising strategy for effective treatment of cancers.^7–11 Currently, RNA interference-based therapies in combination with platinum(IV) prodrugs-based chemotherapies have been studied for cancer therapy, which can overcome platinum drug resistance, and enhance the anticancer effect of platinum(IV) complexes.^12,13 The co-delivery of platinum(IV) prodrugs and nucleic acids is currently the most efficient way to maximally exert their combinational effect.¹⁴ However, due to stark differences in molecular properties of platinum(IV) prodrugs and nucleic acids, such as metabolic stability, molecular weight, and solubility, the design of co-delivery systems for platinum(IV) prodrugs and nucleic acids remains very challenging.

Polymers serve as one of the most widely used delivery vectors for the co-delivery of platinum(IV) prodrugs and nucleic acids. Wang et al. synthesized a triblock copolymer, designated as mPEG₄₅-b-PCL₈₀-b-PPEEA₁₀ for the co-delivery of a platinum(IV) prodrug and siNotch1. The platinum(IV) prodrug was covalently conjugated to the polymer by esterification catalysed with DIC/DMAP.¹⁵ Although the co-delivery system successfully exerted the combinational effect of platinum(IV) prodrug and siNotch1, the synthetic routes of the co-delivery vector were found to be very complex. These complexities include the synthesis of the polymer, chemical coupling of platinum(IV) prodrug, and purification steps. Compared with chemical conjugation, physical encapsulation provides an simple approach for loading platinum(IV) prodrugs into delivery vectors. For example, Farokhzad et al. used a versatile polymer which simultaneously delivered a hydrophobic platinum(IV) prodrug and REV1/REV3L-specific siRNAs. The platinum(IV) prodrug was physically encapsulated in the hydrophobic PLGA part. This polymeric nanoparticle revealed a synergistic cancer therapeutic effect both in vitro and in a prostate xenograft mouse model.¹⁶ However, the loading efficiency of platinum(IV) prodrug was only 10 wt%, and most of the unencapsulated drug was removed by centrifugal filtration, which led to a significant waste in production. Additionally, loading drugs by physical encapsulation has many limitations (e.g., the burst release, hardly tunable drug loading content). Therefore, the present vectors for the co-delivery of platinum(IV) prodrugs and nucleic acids need further improvement.

In this study, a novel polycation composed of PEI 800 units linked via platinum(IV) prodrug linkages was prepared for the co-delivery of platinum(IV) prodrug and short hairpin RNA (shRNA). The synthesis of polymers and drug-loading process were integrated into one step, and the polycation functioned both as a polymeric platinum(IV) prodrug and as a delivery vector. By design, the platinum(IV) prodrug linker with two axial acrylate moieties was first synthesized, and then crosslinked with PEI 800 via a simple Michael-addition reaction. The obtained polycation termed DP displayed a high drug loading efficiency (∼100%) and high drug content (35.946 wt%). Remarkably, the drug content in the polycation could be precisely adjusted by simply changing the molar ratios of the reactants. DP/Akt1 shRNA complexes were then formed by the electrostatic interaction of DP with negatively charged Akt1 shRNA. In vitro anticancer effect of DP/Akt1 shRNA was evaluated by cell viability and apoptosis assays. The ability of DP/Akt1 shRNA complexes to down-regulate the expression of Akt1 gene was also investigated by quantitative polymerase chain reaction (qPCR) and western blot analysis, respectively.

2 Materials and methods

2.1 Materials

PEI (M_w = 800 Da) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Cisplatin was purchased from Shandong Boyuan pharmaceutical co. (Jinan, China). Acrylic anhydride was brought from Shanghai Boyle Chemical Co. (Shanghai, China). RMPI 1640, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Hoechst 33342 were purchased from KeyGEN BioTECH (Nanjing, China). YOYO-1, LysoTracker® Red DND-99 and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (Waltham, USA). Trypsin-EDTA (0.25%) and Trypsin without EDTA solution (0.25%) were purchased from Gibco (Burlington, Canada). Akt1 shRNA expression plasmid (shAkt1, sequence: 5′-AUACCGGCAAAGAAGCGAUGCUGCA-3′) and scrambled shRNA expression plasmid (SCR, sequence: 5′-GTTCTCCGAACGTGTCACGT-3′) were obtained from Genechem Co., Ltd. (Shanghai, China). Plasmids were propagated in Escherichia coli, extracted by the alkali lysis technique, and purified by a E.Z.N.A.® Fastfilter Endo-free Plasmid Maxi kit (Omega, USA). All other chemicals were obtained from commercial sources with the highest purity available and used without further purification.

2.2 Cell lines

A549 (human lung cancer cells), PC-3 (human prostate cancer cells) and MCF-7 (human breast cancer cells) were purchased from the Chinese Academy of Sciences Shanghai Institute of Cell Bank (Shanghai, China) and cultured in RMPI 1640 with 10% FBS, 80 units mL⁻¹ of penicillin, and 80 μg mL⁻¹ of streptomycin at 37 °C in a humidified atmosphere of 5% CO₂.

2.3 Synthesis of cis-dichloro-cis-diammine-trans-dihydroxyplatinum(IV) [Pt(NH₃)₂Cl₂(OH)₂ (DHP)]

DHP was synthesized as previously reported.¹⁷ Briefly, 1 g cisplatin and 120 mL hydrogen peroxide (30%) were added into a 250 mL flask, and then heated to 75 °C for 5 h. The product was then concentrated in a rotary evaporator under reduced pressure, followed by recrystallization at 4 °C overnight. The crystal was collected by filtration, washed with ice-cold water, stirred in 5 mL boiling water for 10 min, and cooled to room temperature. The yellow solid was collected by filtration, washed with ice-cold water, ethyl alcohol and diethyl ether, and dried in a vacuum desiccator.

2.4 Synthesis of cis-dichloro-trans-diacylato-cis-diamineplatinum(IV) [Pt(NH₃)₂Cl₂(CO₂CHCH₂)₂ (DHPAA)]

Acrylic anhydride (12 mmol) and DHP (3 mmol) were dissolved in 10 mL anhydrous N,N-dimethylformamide (DMF). The solution was stirred at 50 °C under nitrogen-fixing condition for 24 h in the dark, and then cooled to room temperature. The resulting yellow to orange solution was filtered through celite, the solution volume was reduced under vacuum, and the product was precipitated by adding ice-cold diethyl ether.

2.5 Preparation and characterization of poly[platinum(IV)-alt-PEI] (DP)

DP was synthesized by Michael-addition polymerization of DHPAA with PEI 800. In a typical experiment, equimolar DHPAA (0.5 mmol) and PEI 800 (0.5 mmol) were first dissolved in 3 mL DMSO and 1 mL water, respectively. Then the DMSO solution of DHPAA was added dropwise to the PEI 800 solution in a reaction flask with stirring. Polymerization was carried out in the dark at 40 °C for 1 day under nitrogen atmosphere. After the completion of the reaction, DP was isolated by exhaustive dialysis against distilled water (molecular weight cut-off = 1500 Da) for 2 days, followed by lyophilization. The synthesized product was stored at −80 °C for later use.

The structure of DP was confirmed by ¹H nuclear magnetic resonance spectroscopy (¹H NMR, Bruker AV-300, USA). D₂O, DMSO-d₆ and D₂O were used as the solvents for DP, DHPAA and PEI 800, respectively.

2.6 Preparation of Rhodamine B isothiocyanate (RITC) labelled DP

All RITC labelled polymers were freshly prepared for subsequent use. Briefly, 5 mg DP was dissolved in 4 mL sodium carbonate solution (1 M, pH = 9.0), and 2 mg RITC was dissolved in 1 mL methanol. Then the methanol solution of RITC was added dropwise to the DP sodium carbonate solution in a reaction flask with stirring. The reaction was carried out in the dark at room temperature under nitrogen. After stirring for 24 h at room temperature, the solution was dialyzed against distilled water (molecular weight cut-off = 1500 Da) for 2 days. The product was obtained after lyophilization and stored at −80 °C for later use.

2.7 Preparation and characterization of DP/shAkt1 complexes

The DP/shAkt1 complexes at different weight ratios were prepared by adding shAkt1 solution to an equal volume of DP solution containing various amounts of DP with gentle vortex. The resulting mixture was incubated at room temperature for 30 min to form the complexes. All DP/shAkt1 complexes were used immediately after preparation. In the following experiments, all the ratios represented the weight ratio of DP to shAkt1.

The mean hydrodynamic particle sizes and zeta potentials of the DP/shAkt1 complexes were measured by a ZetaPlus particle size and zeta potential analyser (Brookhaven Instruments, USA).

A single drop of DP/shAkt1 complexes was placed on a copper grid and air dried. The micrographs of the shAkt1/DP complexes (weight ratio, DP : shAkt1 = 30 : 1) with 10 μg shAkt1 were observed by transmission electron microscopy (TEM, JEM-2100, JEOL, Japan).

2.8 Evaluation of retention and protection properties of shAkt1 by DP/shAkt1 complexes

shAkt1 condensation was determined by electrophoresis. Complexes prepared at different weight ratios from 0.1 to 40 with 12 μL final volume were incubated at room temperature for 30 min, and then electrophoresed in a 1% agarose gel containing GEL RED™ in Tris-acetate (TAE) running buffer at 50 V for 30 min. The image was photographed with a gel image system (Tanon 1600, China).

Protection and release properties of shAkt1 in DP/shAkt1 complexes were measured using a gel electrophoresis method. Briefly, DNase I or PBS in DNase I/Mg²⁺ digestion buffer with a volume of 1 μL was added to 4 μL complexes solution (weight ratio, DP : shAkt1 = 5 : 1) or to 0.4 μg naked shAkt1. The mixture was incubated at 37 °C with continuous stirring at 100 rpm for 30 min. Thereafter, 4 μL EDTA (250 mM) was added to all the mixed solutions, and maintained for 10 min at 65 °C for DNase I inactivation. Afterwards, the samples were mixed with 5% heparin sodium at a final volume of 18 μL, and the mixture was incubated at room temperature for 2 h, followed by electrophoresis in a 1% agarose gel containing GEL RED™ in TAE running buffer at 50 V for 30 min.

2.9 Efflux inhibition of DP/shAkt1 complexes

To elucidate the mechanisms of the cellular uptake of DP/shAkt1 complexes, A549 cells were seeded into 12-well plates at 1.2 × 10⁵ cells per well, and incubated at 37 °C overnight. Cells were pre-treated with sodium azide (1 mg mL⁻¹), methyl-β-cyclodextrin (5 mM), chlorpromazine hydrochloride (10 μg mL⁻¹) respectively for 1 h. After that, RITC-DP/shAkt1 (weight ratios, RITC-DP : shAkt1 = 30 : 1) complexes containing 1 μg shAkt1 were then added. After incubation for 1 h, the cells were washed for 3 times with cold PBS (phosphate-buffered saline), and collected for flow cytometry analysis (FACS, BD AccuriC6, USA). All experiments were performed in triplicate. Results were shown as the relative uptake percentage compared to non-treated cells.

2.10 In vitro cell uptake and endosomal escape of DP/shAkt1 complexes

The contents of RITC-labelled DP and YOYO-1 labelled shAkt1 (1 μg shAkt1 was fluorescently labelled with 5 μL 10 μM YOYO-1 iodide) in A549 cells were measured by FACS. A549 cells (1 × 10⁵/well) were seeded in 24-well plates with 1 mL of RPMI 1640 containing 10% FBS, and incubated at 37 °C for 24 h. Then the media were replaced with serum-free media containing RITC-DP/YOYO-1-shAkt1 complexes (weight ratios, RITC-DP : YOYO-1-shAkt1 = 30 : 1) loaded with 1 μg YOYO-1-shAkt1. After incubation for 4 h, a total of 1 × 10⁴ cells were immediately analysed by FACS.

A549 cells were seeded in thin glass-bottomed 35 mm petri dishes at 1.2 × 10⁴ cells per dish, and incubated for 12 h at 37 °C under 5% CO₂ atmosphere to reattach. After that, the media were removed, and the cells were washed twice with PBS, followed by incubation at 37 °C with LysoTracker red DND-99 (100 nM, for 60 min, Molecular Probes, Oregon, USA). Then, the freshly prepared DP/shAkt1 complexes (30 : 1) containing 1 μg YOYO-1 iodide labelled shAkt1 were added immediately to the petri dishes, followed by incubation at 37 °C for 1 h or 4 h. Afterwards, cells were stained with Hoechst 33342 (100 μM in PBS), fixed with 4% paraformaldehyde, and visualized under a confocal laser scanning microscope (CLSM, FV1000, Olympus).

2.11 In vitro transfection

The transfection of pGL3 plasmids mediated by DP/pGL3 complexes in A549 cells was evaluated, and PEI 25K was used as a positive control. A549 cells were seeded in 24-well plates at a density of 8 × 10⁴ per well, and cultured with 1 mL media containing 10% FBS for 24 h. Then, the media were replaced with serum-free media containing polymer/pGL3 (1 μg) complexes at different weight ratios. After incubation for 4 h, the complexes were removed. The cells were further incubated for 24 h in 1 mL fresh media containing 10% FBS. The luciferase assay was carried out according to the recommended protocol from the manufacturer. Relative light units (RLU) were recorded by a chemiluminometer (Luminoskan Ascent, ThermoFisher Scientific, USA). The total proteins were measured by a BCA protein assay kit (KeyGEN BioTECH, Nanjing, China). Luciferase activity was expressed as RLU per mg protein.

To visualize the transfection on cells, A549 cells were transfected with polymer/GFP (green fluorescent protein, 1 μg) complexes (weight ratios, DP : GFP = 30 : 1, PEI 25K : GFP = 1 : 1, respectively). After 24 h, GFP expression was observed under an inverted fluorescence microscope (Nikon ti-s, Japan).

2.12 In vitro cytotoxicity

In vitro cytotoxicity tests were evaluated as previously reported¹⁸ on A549, PC-3 and MCF-7 cancer cells. Cisplatin was dissolved in stroke-physiological saline solution as a 1 mg mL⁻¹ stock solution. A549, PC-3, and MCF-7 cells were seeded in flat-bottomed 96-well plates at 8000 cells per well. After incubation for 18 h, the media were replaced with serum-free media containing different formulations listed as follows: PEI 25K/shAkt1 complexes, PEI 25K/SCR complexes, shAkt1, cisplatin, DP/shAkt1 complexes, DP/SCR complexes and DHPAA. To make the data comparable, the final concentration of platinum was 9.54 μg mL⁻¹, and shRNA was 2 μg mL⁻¹. After 24 or 72 h, the viability of the cells was determined by MTT assay. Briefly, cells were incubated with 20 μL MTT reagent (5 mg mL⁻¹) for 4 h at 37 °C. Then, 150 μL of DMSO was added to each well to dissolve formazan crystals. The absorbance of each well was measured at 570 nm by a microplate reader (ThermoFisher Scientific Multiskan Go, USA). Data shown is representative of three independent experiments.

2.13 Apoptosis analyses

A549 cells were seeded in 6-well plates at 2 × 10⁴ per well. After incubation for 18 h, the cells were treated with 2 mL serum-free media containing different formulations as follows: shAkt1, cisplatin, DP/shAkt1 complexes, DP/SCR complexes and DHPAA. To make the data comparable, the final concentration of platinum was 9.54 μg mL⁻¹, and shRNA was 2 μg mL⁻¹. After 48 h incubation, cells were harvested, and stained with the AnnexinV-FITC Apoptosis Detection Kit (KeyGEN BioTECH, Nanjing, China) according to the recommended protocol from the manufacturer, then measured by FACS. The data were analysed with FlowJo software.

2.14 In vitro shAkt1 transfection and assay of Akt1 protein by western blot

A549 cells were seeded in 6-well plates at 2 × 10⁴ per well, and incubated at 37 °C with 5% CO₂ for 24 h. The media were then replaced with serum-free media containing different polymer/shAkt1 (4 μg), polymer/SCR (4 μg) complexes and continued to be incubated for 48 h. The cellular levels of Akt1 mRNA were measured by qPCR. Total RNAs from transfected A549 cells were extracted using TRIzol reagent (Invitrogen, Amecica), and used for cDNA synthesis using a TransScript All-in-One First-Strand cDNA Synthesis SuperMix (Transgen, Peking) according to the recommended protocol from the manufacturer. qPCR analysis was performed using the cDNAs as the template. PCR was amplified for 45 cycles. Levels of β-actin were used as the internal control. Primers for the PCR analysis were as follows: Akt1, 5′-GGAGGACAATGACTACGGC-3′ (sense) and 5′-TCATCTTGGTCAGGTGGTGT-3′ (antisense); and β-actin, 5′-CCTTCCTTCCTGGGCATGGAGTCCTG-3′ (sense) and 5′-GGAGCAATGATCTTGACTTC-3′ (antisense).

The Akt1 protein expression of A549 cells was evaluated by western blot analysis. A549 cells (2 × 10⁵ per well) seeded in 6-well plates were treated with different formulations (PEI 25K/shAkt1 complexes, PEI 25K/SCR complexes, DP/shAkt1 complexes, DP/SCR complexes and shAkt1 only) for 72 h. Akt1 protein expression was measured by Western blot analysis using Akt1 mouse mAb antibody (1:1000, Cell Signaling Technologies).

2.15 Hemolytic activity

The hemolytic activity was evaluated as previously reported.¹⁹ Briefly, 200 μL blood from a healthy rat was added to 1 mL PBS (pH 7.4) containing different amounts of polymers (0.01, 0.05, 0.1, 1 mg). 200 μL blood mixed with 800 μL PBS was used as a negative control, and PBS containing Triton X-100 to cause complete hemolysis was used as the positive controls, respectively. After incubation at 37 °C for 1 h, samples were centrifuged at 3000 rpm for 5 min. 200 μL supernatant was added in a 96-well plate, and analysed for released hemoglobin at 543 nm using Thermo Multiskan GO (ThermoFisher Scientific, USA). Percentage hemolysis was determined according to the following formula:where A_s, A_n and A_p represent the absorbance of samples, negative and positive controls, respectively.

2.16 Statistical analysis

Quantitative measurements were conducted in triplicate for each sample, and results were reported as the means ± standard deviation (SD). Statistical analysis was performed by one-way ANOVA among three or more groups or two-tailed student's t-test between two groups using SPSS 19.0. P-value < 0.05 was used to establish significant differences.

3 Results and discussion

3.1 Synthesis and characterization of cis-dichloro-cis-diammine-trans-dihydroxyplatinum(IV) [Pt(NH₃)₂Cl₂(OH)₂] (DHP) and cis-dichloro-trans-diacylato-cis-diamineplatinum(IV) [Pt(NH₃)₂Cl₂(CO₂CHCH₂)₂] (DHPAA)

The synthesis began with the oxidation of cisplatin by hydrogen peroxide. After the oxidation, DHP was obtained with two hydroxyl groups at its axial positions, as shown in Fig. 1A. The successful synthesis of DHP was confirmed by Fourier Transform Infrared Spectrometer (FTIR Spectrometer, Fig. S1†) and high resolution mass spectroscopy [HRMS, positive ion mode, m/z calcd for Pt(NH₃)₂Cl₂(OH)₂: (M + H)⁺ = 333.3367. Found = 333.3382]. The nucleophilicity of hydroxyl groups coordinated to platinum(IV) centers affords a convenient method to synthesize platinum(IV) carboxylate compounds through a condensation reaction with anhydrides.²⁰ Therefore, DHP further reacted with acrylic anhydride to synthesize DHPAA (yield, 821 mg, 62.05%) with pendant diacrylate groups (Fig. 1B). ¹H NMR spectra were used to confirm the structure of DHPAA (Fig. 1D). The peaks between δ 5.5 ppm (b) and 6.3 ppm (d) belonged to the protons of vinyl groups (–CO₂CHCH₂–) in DHPAA, while the broad peaks between δ 6.3 ppm and 6.9 ppm were attributed to the protons of ammonia molecules (NH₃) coordinated to the platinum(IV) centers. The molecular weight of DHPAA was measured by ESI-MS (negative ion mode, m/z = 441.0). All these results indicated that DHPAA was successfully synthesized.

Fig. 1 Synthesis route of (A) DHP; (B) DHPAA; (C) DP. (D) ¹H NMR spectra of DP, DHPAA and PEI 800. * and ▽ represent the solvent peaks of DMF and diethyl ether in DMSO-d₆, respectively.

3.2 Synthesis and characterization of DP

Michael-addition reaction is a versatile synthetic methodology for highly efficient conjugate addition of nucleophiles (amino groups) with electron-deficient olefins (acrylate groups). The advantages of Michael-addition reaction in terms of mild reaction conditions, high functional group tolerance, high conversions, and favorable reaction rates permit the synthesis of macromolecular structures for polymer-drug conjugates and gene delivery.^21,22

DP was synthesized by the Michael-addition reaction of platinum(IV) diacrylate with PEI 800, a convenient reaction without any byproducts.²³ PEI is a widely studied cationic material for siRNA and DNA delivery.^24,25 Here, low molecular weight PEI was chosen as the cationic parts of our polycation (Fig. 1C). The molecular weight of DP was determined by gel permeation chromatography (GPC) analysis, and the weight-average molecular weight turned out to be 2654 (Table S1†). Platinum(IV) prodrug content (35.946 wt%) in the polymer was measured by inductively coupled plasma optical emission spectrometer (ICP, iCAP6300, Thermo Fisher Scientific), and the result was very close to the theoretical determination (35.9 wt%), for cases where platinum(IV) prodrug content in the polymer could be finely tuned by simply changing the molar ratios of the reactants (Table S2†). The cisplatin-DNA adducts were detected after the reduction of DP by sodium ascorbate (Fig. S4†), indicating DP contained the platinum(IV) prodrug of cisplatin. The chemical structure of DP was characterised by means of ¹H NMR spectra (Fig. 1D). The peaks (a) between 2.7 ppm and 2.9 ppm were attributed to protons (–NCH₂–CH₂–) in the PEI 800 units of DP. The peaks at 2.45 ppm (c) and 2.89 ppm (b) belonged to the protons (–CH₂–CH₂–) which originated from the conjugate addition of the vinyl groups in DHPAA units to amino groups of PEI 800 units. The signals of vinyl groups in DHPAA units disappeared, indicating the conjugate addition of the vinyl groups in DHPAA to the amino groups of PEI 800 units was complete. Furthermore, DP was studied by means of ¹⁹⁵Pt NMR spectroscopy, which is an ideal tool for investigating the platinum-based coordination compounds.²⁶ The spectrum showed only one signal at 1219 ppm (Fig. S5†), which corresponded to the platinum(IV) prodrug of cisplatin.^27–29 All these results confirmed that DP was successfully synthesized.

3.3 Gel retardation assay and stability evaluation

Condensing DNA is one of the crucial preconditions for polycationic vectors to effectively deliver genes into target cells, and achieve gene expression. The gel retardation assay was carried out to confirm the condensation capability of DP with shAkt1 at various weight ratios. As shown in Fig. 2A, the mobility of shAkt1 was entirely retarded, when the weight ratio of DP/shAkt1 complexes was as little as 1. It indicated DP was able to efficiently bind to shAkt1.

Fig. 2 Physicochemical characterization of DP/shAkt1 complexes. (A) Agarose gel retardation of DP/shAkt1 complexes at different weight ratios. (B) Protection and release assay of shAkt1. shAkt1 was released by adding 5% heparin sodium to the DP/shAkt1 complexes at weight ratio = 5. (C) Hydrodynamic diameters and surface charges of DP/shAkt1 complexes formed at various weight rations (means ± SD, n = 3). (D) Representative TEM image of DP/shAkt1 complexes at weight ratio = 30 (scale bar: 100 nm).

Since DNA is easily degraded by nuclease, an efficient gene carrier must protect DNA from enzymatic degradation.³⁰ The protection of shAkt1 against DNase I was confirmed by gel retardation assay. The naked shAkt1 was completely degraded after DNase I treatment, as evidenced by the loss of bright band corresponding to free shAkt1 (Fig. 2B). In contrast, after the substitution of shAkt1 from DP/shAkt1 complexes by heparin sodium, no significant loss of shAkt1 integrity was observed with or without the treatment of DNase I. These results suggested that the adsorption of DP to shAkt1 could sterically hinder the access of nuclease to shAkt1, thus increasing the stability of shAkt1.

3.4 Characterization of DP/shAkt1 complexes

Particle sizes and zeta potentials measurements of DP/shAkt1 complexes at different weight ratios ranging from 10 to 80 were presented in Fig. 2C. These results showed that the particle sizes decreased slowly with an increase in weight ratios, and kept almost stable at 130 nm, indicating the compact condensation of DNA by DP.

It was reported that a particle diameter of less than 200 nm was an optimal size for cancer therapy.³¹ Therefore, DP/shAkt1 complexes at weight ratio of 30 could be a good candidate for cancer therapy. The zeta potential of DP/shAkt1 complexes were all higher than +20 mV over the weight ratios tested, and increased slowly with an increase in weight ratios.

The TEM image in Fig. 2D showed that the DP/shAkt1 complexes at weight ratio of 30 were nanometer-sized spherical particles. Moreover, the diameter measured by TEM was smaller than that obtained by dynamic light scattering measurement, which was due to the fact that the size of complexes in solid state measured by TEM images was smaller than the hydrodynamic diameter measured by dynamic light scattering.³²

Energy dispersive X-ray spectroscopy (EDX) was employed to analyse the elemental composition of DP/shAkt1 complexes. As shown in Fig. S2,† signal from platinum atom with signals for C, O, N atoms were found, suggesting the complexes were formed by DP and shAkt1.

3.5 Internalization pathway of DP/shAkt1 complexes

To optimize the nanoparticles, it is important to understand the underlying mechanisms of cellular uptake process.³³ As shown in Fig. 3, both low temperature (4 °C) and sodium azide treatment caused a significant decrease in the cellular uptake of DP/shAkt1 complexes, suggesting an energy-dependent cellular uptake was involved. Since PEI-mediated gene delivery system has been previously reported to be endocytosed via a caveolar pathway,³⁴ we intended to investigate if our complexes entered into the cells by the same way. Methyl-β-cyclodextrin (Me-β-CD), a cyclic glucose oligomer, is widely used to disrupt caveolar pathway.^35,36 As illustrated in Fig. 3, Me-β-CD pretreatment yielded a 36% decrease in cellular uptake of DP/shAkt1 complexes, implying that caveolar and cholesterol-dependent pathways were involved. Next, the clathrin-pathway inhibitor (chlorpromazine hydrochloride) and macropinocytosis inhibitor (amiloride) were used to evaluate their effects on the cellular uptake of DP/shAkt1 complexes. As demonstrated in Fig. 3, chlorpromazine hydrochloride and amiloride both negligibly inhibited the cellular internalization of DP/shAkt1 complexes, excluding the involvement of clathrin-pathways and macropinocytosis. Therefore, the cellular uptake of DP/shAkt1 complexes was demonstrated to be an active and energy-dependent process, and the DP/shAkt1 complexes were taken up by cells via caveolae-mediated endocytosis.

Fig. 3 Influence of low temperature and inhibitors on the internalization of DP/shAkt1 complexes in A549 cells. Cells without pretreatment were set as the control group (means ± SD, n = 3). Statistical analysis was performed by two-tailed student's t-test with **p < 0.01, ***p < 0.001.

3.6 In vitro analysis of the co-delivery of platinum(IV) prodrug and shAkt1

To demonstrate DP/shAkt1 complexes could simultaneously deliver platinum(IV) prodrug and shAkt1 into the same cancer cells, we analysed the cellular uptake of RITC-DP/YOYO-1-shAkt1 complexes in A549 cells. Cells were incubated with RITC-DP/YOYO-1-shAkt1 complexes for 4 h, and then analysed by FACS. Cells located at the double-positive quadrant after incubation for 4 h, indicating that DP/shAkt1 complexes simultaneously delivered two payloads into the same cancer cells (Fig. 4). Since co-delivering small molecular drugs and nucleic acid into the same cancer cells can exert their maximal therapeutic effect,¹⁴ DP/shAkt1 complexes may exert maximal therapeutic effect of platinum(IV) and shAkt1. This result indicated DP/shAkt1 complexes had the potential to be a promising co-delivery vector for cancer therapy.

Fig. 4 Cellular uptake of RITC-DP/YOYO-1-shAkt1 complexes determined by FACS.

3.7 Cell transfection in vitro

The in vitro gene transfection of DP/PGL3 complexes was quantitatively assessed using a luciferase assay in the absence of serum. In order to screen the optimal weight ratios of DP/nucleic acid complexes for gene delivery, experiments were performed at a series of weight ratios (from 10 to 80). As shown in Fig. 5A, a clear tendency between weight ratios and transfection efficiency was observed. The transfection efficiency of DP/PGL3 complexes increased gradually with an increase in weight ratios, reaching the peak value at the weight ratio of 30, and then decreasing slowly. The transfection efficiency of gene vectors based on PEI and its derivatives depends on many factors such as appropriate particle size, zeta potential, and endosomal escape capability.²⁵ The DP/shAkt1 complexes showed a relative small diameter (130.4 nm) and medium zeta potential (+22.32 mV) at weight ratio of 30 (Fig. 2C), which resulted in a fast cellular uptake of DP/shAkt1 complexes by caveolae-mediated endocytosis (Fig. 3). Moreover, the high buffer capability of DP enhanced endosomal escape of DP/DNA complexes (Fig. S3†). All these properties contributed to the high transfection efficiency of DP/DNA complexes. However, at weight ratios ranging from 40 to 80, DP/DNA complexes exhibited higher zeta potential and platinum(IV) prodrug content, which induced higher cytotoxicity, and caused decreased transfection efficiency. The green fluorescent protein expression of A549 cells mediated by DP/GFP pDNA and PEI 25K/GFP pDNA complexes is exhibited in Fig. 5B. DP could efficiently perform GPF pDNA expression in A549 cells in spite of being inferior to PEI 25K. These results were in accordance with the luciferase activity assays. Although the transfection efficiency of DP was only one fifth of PEI 25K-mediated transfection, the “golden standard” of gene vectors, we continue to believe DP can efficiently deliver the genes, and help genes play their roles in cancer combinational therapy.

Fig. 5 Transfection efficiencies and intracellular trafficking of DP/shAkt1 complexes in A549 cells. (A) Luciferase gene expression levels of DP/pGL3 complexes at different weight ratios (means ± SD, n = 3). (B) GFP expression of A549 cells treated with DP/GFP complexes at weight ratio = 30 detected by the inverted fluorescence microscope. (C) CLSM determined intracellular localization of shAkt1 in A549 cells after incubation for 1 h and 4 h. Scale bar represented 20 μm. Lysosomes were labelled with LysoTracker Red DND-99 (red), the nuclei were labelled with Hoechst 33342 (blue) and shAkt1 was stained by YOYO-1 (green).

3.8 Intracellular transportation of DP/shAkt1 complexes

To investigate the mechanism of cell transfection with DP/shAkt1 complexes, the endo/lysosomal escape of DP/shAk1 complexes was confirmed by CLSM. YOYO-1, LysoTracker Red DND-99, and Hoechst 33342 were used for staining shAkt1, endo/lysosomes, and the nuclei, respectively. Fig. 5C showed the fluorescent pattern observed for YOYO-1 stained shAkt1, and its overlay with fluorescent probes LysoTracker Red DND-99 (endo/lysosomes) and Hoechst 33342 (nuclei). The images were obtained by merging the fluorescence of LysoTracker Red DND-99 (red signal), YOYO-1 stained shAkt1 (green signal) with that of Hoechst 33342 (blue signal). As shown in 1 h group, the merged images revealed a partial overlap of YOYO-1 stained shAkt1 and LysoTracker Red DND-99 (yellow signal), indicating that most of shAkt1 did not escape from endo/lysosomes (Fig. 5C, 1 h group). However, at 4 h post-transfection, the extent of co-localization between the fluorescence of YOYO-1 and that of LysoTracker Red DND-99 significantly decreased, suggesting most of shAkt1 escaped from endo/lysosomes (Fig. 5C, 4 h group). The successful endo/lysosomal escape of shAkt1 was due to the high buffer capacity of DP (Fig. S3†), contributing to the highly efficient gene transfection of DP/shAkt1 complexes.

3.9 In vitro cytotoxicity

To investigate whether the DP/shAkt1 complexes-mediated simultaneous delivery of shAkt1 and platinum(IV) prodrug could synergistically inhibit the proliferation of cancer cells, the proliferation of three different cancer cell lines treated with different formulations was assessed by MTT assay. A549, PC-3 and MCF-7 cells were treated with different formulations containing fixed doses of shAkt1 and platinum, which were 2 μg mL⁻¹ and 9.54 μg mL⁻¹, respectively, for 24 h and 72 h. As shown in Fig. 6, all formulations showed a time-dependent cytotoxicity in the cancer cell lines tested. The comparison of cytotoxicity among free cisplatin, DP/shAkt1 complexes, DP/SCR complexes and DHPAA turned out that DP/shAkt1 complexes were more cytotoxic than DP/SCR complexes or DHPAA, indicating simultaneous delivering of shAkt1 and platinum(IV) prodrug could synergistically inhibit the survival of cancer cells and enhance the anticancer effect of platinum(IV) prodrug. However, free cisplatin showed higher cytotoxicity than DP/shAkt1 complexes, which was possibly because using platinum(IV) prodrug as a cross-linker affected the reduction and hydrolysis process of platinum(IV) prodrug inside the cells.^37,38 According to the experiments depicted in Fig. S6,† only 68.79% of platinum drug has been released in vitro after 72 h. Although DHPAA induced an obvious inhibition of cell proliferation, the extent was inferior to DP/SCR complexes. The reason for this occurrence might be that positively charged DP/SCR complexes had higher affinity with negatively charged cell membrane, thereby entering into the cells much easier than DHPAA. Both PEI 25 K/shAkt1 and PEI 25K/SCR complexes could inhibit the proliferation of the cancer cells tested due to the cytotoxicity of PEI 25K. The PEI 25K/shAkt1 complexes were more cytotoxic than PEI 25K/SCR complexes at 24 h and 72 h. No significant effect on cell death was noticed in cells incubated with shAkt1 alone, indicating the low cytotoxicity of shAkt1 itself.

Fig. 6 In vitro cytotoxicity of various formulations toward (A) A549; (B) MCF-7; (C) PC-3 cells at 24 h and 72 h. All results were presented as means ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA test).

3.10 Apoptosis test

FACS was applied to examine whether the combinational therapy of platinum(IV) prodrug and shAkt1 could induce increased apoptosis in A549 cells. As illustrated in Fig. 7, compared with DP/SCR complexes treated group, the co-delivery of platinum(IV) prodrug and shAkt1 could effectively enhance platinum(IV) prodrug-induced apoptosis. This indicated that the combinational therapy of platinum(IV) prodrug and shAkt1 was an effective method for cancer therapy. There were only 18.19% apoptotic cells induced by DHPAA alone. Conjugating DHPAA to cationic polymers further increased the degree of apoptosis to 24.1% due to the enhanced uptake of platinum(IV) prodrug. shAkt1 alone treated group induced negligible apoptosis, and free cisplatin had the most potential in inducing apoptosis, which was consistent with MTT results.

Fig. 7 In vitro apoptosis assay. (A) Flow cytometric analysis of apoptosis in A549 cells induced by various formulations. The lower-right and upper-right quadrants indicate the early apoptotic cells and the late apoptotic cells, respectively. (B) Quantitative analysis of the total population of apoptotic cells for every group (means ± SD, n = 3). **p < 0.01, ***p < 0.001.

3.11 qPCR and western blot assays

The knockdown of Akt1 gene expression in A549 cells was evaluated by qPCR and western blot analysis, respectively (Fig. 8). At mRNA level in A549 cells, compared with SCR control groups, both treatments with DP and PEI 25K loaded with shAkt1 showed efficient silencing effects on Akt1 mRNA (75.7% and 61.2% knockdown, respectively). Naked shAkt1 alone treatment represented negligible silencing efficiency in A549 cells. Consistently, at the expression level of proteins, shAkt1 delivered by PEI 25K and DP remarkably decreased the expression of Akt1 protein in A549 cells, and shAkt1 alone treatment did not affect the protein expression of Akt1 (Fig. 7B). These results suggested that DP-based shAkt1 delivery system could efficiently knockdown the expression of Akt1 gene.

Fig. 8 In vitro qPCR and western blot assays. (A) Relative Akt1 mRNA levels determined by qPCR after PEI 25K/shAkt1 complexes, PEI 25K/SCR complexes, DP/shAkt1 complexes, DP/SCR complexes and shAkt1 treatments (n = 3).***p < 0.001 (one-way ANOVA test). (B) Western blot analysis of Akt1 protein expression in A549 cells.

3.12 Hemolysis test

The hemolytic reaction is a critical issue for the in vivo application of cationic polymers.¹⁹ We next examined the hemocompatibility of DP by measuring hemolytic activity. As displayed in Fig. 9, no hemolysis happened with DP in contact with the erythrocytes up to 0.5 mg mL⁻¹, indicating negligible damage of erythrocyte membrane. In PEI 25K treatment group, hemolysis was observed at the low concentration (0.1 mg mL⁻¹), and resulted in more serious hemolysis with the increase in concentration. Compared with PEI 25K, the lower hemolytic effect was observed due to the lower molecular weight and charge density of DP. These results suggested that DP was highly hemocompatible, and could be used as an intravenous carrier for the co-delivery of platinum(IV) prodrugs and genes.

Fig. 9 Blood compatibility assay of DP and PEI 25K. (A) Comparison of DP and PEI 25K on the hemolytic activity by visual observation. (B) Relative hemolytic activity of DP and PEI 25K at various concentrations (means ± SD, n = 3).

4 Conclusions

In summary, we have synthesized a platinum(IV) prodrug conjugated polycation [poly[platinum(IV)-alt-PEI] via a one-step Michael-addition reaction. The novel polycation, which was able to form nano-sized complexes with negatively charged nucleic acid, co-delivered shAkt1 as well as platinum(IV) prodrug for cancer treatment. The formed complexes co-delivered shAkt1 and platinum(IV) prodrug into the same cancer cells through caveolae-mediated endocytosis. The silence of shAkt1 was demonstrated in vitro, which facilitates the inhibition of proliferation and restoration of cancer cells. Meanwhile, the platinum(IV) prodrug in poly[platinum(IV)-alt-PEI] was reduced into cisplatin which resulted in the apoptosis of cancer cells. The combinational anticancer effect of platinum(IV) prodrug and shAkt1 remarkably inhibited the proliferation of cancer cells, and induced cellular apoptosis in a synergistic manner (Scheme 1). This work represented a conceptually new approach in shRNA delivery systems, one in which shRNA carriers that possessed intrinsic anticancer activity allowed for integrated combinational cancer therapy in a single package.

Scheme 1 Schematic illustration of co-delivery of shAkt1 and platinum(IV) prodrug using poly[platinum(IV)-alt-PEI]/shAkt1 complexes.

Acknowledgements

We thank the National Natural Science Foundation of China (Grant No. 81573369, 21301191, 81570696 and 31270985), the Natural Science Foundation of Jiangsu Province (Grant No. BK20130661 and BK20140659). This work was also supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and National High Technology Research and Development Program of China (863 Program, Grant No. 2015AA020314).

Notes and references

1. N. J. Wheate, S. Walker, G. E. Craig and R. Oun, Dalton Trans., 2010, 39, 8113–8127.
2. H. Uchino, Y. Matsumura, T. Negishi, F. Koizumi, T. Hayashi, T. Honda, N. Nishiyama, K. Kataoka, S. Naito and T. Kakizoe, Br. J. Cancer, 2005, 93, 678–687.
3. L. Galluzzi, I. Vitale, J. Michels, C. Brenner, G. Szabadkai, A. Harel-Bellan, M. Castedo and G. Kroemer, Cell Death Dis., 2014, 5, e1257.
4. E. L. Weaver and R. N. Bose, J. Inorg. Biochem., 2003, 95, 231–239.
5. E. Volckova, L. P. Dudones and R. N. Bose, Pharm. Res., 2002, 19, 124–131.
6. T. C. Johnstone, K. Suntharalingam and S. J. Lippard, Chem. Rev., 2016, 116, 3436–3486.
7. F. Greco and M. J. Vicent, Adv. Drug Delivery Rev., 2009, 61, 1203–1213.
8. D. Lane, Nat. Biotechnol., 2006, 24, 163–164.
9. H. J. Mauceri, N. N. Hanna, M. A. Beckett, D. H. Gorski, M.-J. Staba, K. A. Stellato, K. Bigelow, R. Heimann, S. Gately, M. Dhanabal, G. A. Soff, V. P. Sukhatme, D. W. Kufe and R. R. Weichselbaum, Nature, 1998, 394, 287–291.
10. B.-F. Zhang, L. Xing, P.-F. Cui, F.-Z. Wang, R.-L. Xie, J.-L. Zhang, M. Zhang, Y.-J. He, J.-Y. Lyu, J.-B. Qiao, B.-A. Chen and H.-L. Jiang, Biomaterials, 2015, 61, 178–189.
11. F.-Z. Wang, L. Xing, Z.-h. Tang, J.-J. Lu, P.-F. Cui, J.-B. Qiao, L. Jiang, H.-L. Jiang and L. Zong, Mol. Pharmaceutics, 2016, 13, 1298–1307.
12. O. Vondálová Blanářová, I. Jelínková, Á. Szöőr, B. Skender, K. Souček, V. Horváth, A. Vaculová, L. Anděra, P. Sova, J. Szöllősi, J. Hofmanová, G. Vereb and A. Kozubík, Carcinogenesis, 2011, 32, 42–51.
13. C. He, K. Lu, D. Liu and W. Lin, J. Am. Chem. Soc., 2014, 136, 5181–5184.
14. T.-M. Sun, J.-Z. Du, Y.-D. Yao, C.-Q. Mao, S. Dou, S.-Y. Huang, P.-Z. Zhang, K. W. Leong, E.-W. Song and J. Wang, ACS Nano, 2011, 5, 1483–1494.
15. S. Shen, C.-Y. Sun, X.-J. Du, H.-J. Li, Y. Liu, J.-X. Xia, Y.-H. Zhu and J. Wang, Biomaterials, 2015, 70, 71–83.
16. X. Xu, K. Xie, X.-Q. Zhang, E. M. Pridgen, G. Y. Park, D. S. Cui, J. Shi, J. Wu, P. W. Kantoff and S. J. Lippard, Proc. Natl. Acad. Sci. U. S. A., 2013, 110, 18638–18643.
17. T. C. J. S. J. Lippard, J. Biol. Inorg. Chem., 2014, 19, 667–674.
18. Y. Zhou, M. Xu, Y. Liu, Y. Bai, Y. Deng, J. Liu and L. Chen, Colloids Surf., B, 2016, 144, 118–124.
19. J. Yang, Y. Liu, H. Wang, L. Liu, W. Wang, C. Wang, Q. Wang and W. Liu, Biomaterials, 2012, 33, 604–613.
20. T. C. Johnstone and S. J. Lippard, Inorg. Chem., 2013, 52, 9915–9920.
21. B. D. Mather, K. Viswanathan, K. M. Miller and T. E. Long, Prog. Polym. Sci., 2006, 31, 487–531.
22. P. Mukhopadhyay and P. P. Kundu, RSC Adv., 2015, 5, 93995–94007.
23. J. J. Green, R. Langer and D. G. Anderson, Acc. Chem. Res., 2008, 41, 749–759.
24. O. Boussif, F. Lezoualc'h, M. A. Zanta, M. D. Mergny, D. Scherman, B. Demeneix and J.-P. Behr, Proc. Natl. Acad. Sci. U. S. A., 1995, 92, 7297–7301.
25. Y. Shen, J. Wang, Y. Li, Y. Tian, H. Sun, O. Ammar, J. Tu, B. Wang and C. Sun, RSC Adv., 2015, 5, 46464–46479.
26. B. M. Still, P. G. A. Kumar, J. R. Aldrich-Wright and W. S. Price, Chem. Soc. Rev., 2007, 36, 665–686.
27. Y.-R. Zheng, K. Suntharalingam, T. C. Johnstone and S. J. Lippard, Chem. Sci., 2015, 6, 1189–1193.
28. M. Callari, D. S. Thomas and M. H. Stenzel, J. Mater. Chem. B, 2016, 4, 2114–2123.
29. S. Dhar, Z. Liu, J. Thomale, H. Dai and S. J. Lippard, J. Am. Chem. Soc., 2008, 130, 11467–11476.
30. K. Hagiwara, M. Nakata, Y. Koyama and T. Sato, Biomaterials, 2012, 33, 7251–7260.
31. D. K. Chatterjee, L. S. Fong and Y. Zhang, Adv. Drug Delivery Rev., 2008, 60, 1627–1637.
32. X.-h. Luo, F.-w. Huang, S.-y. Qin, H.-f. Wang, J. Feng, X.-z. Zhang and R.-x. Zhuo, Biomaterials, 2011, 32, 9925–9939.
33. P. Liu, Y. Sun, Q. Wang, Y. Sun, H. Li and Y. Duan, Biomaterials, 2014, 35, 760–770.
34. N. P. Gabrielson and D. W. Pack, J. Controlled Release, 2009, 136, 54–61.
35. P. V. Rewatkar, R. G. Parton, H. S. Parekh and M.-O. Parat, Adv. Drug Delivery Rev., 2015, 91, 92–108.
36. S. K. Rodal, G. Skretting, Ø. Garred, F. Vilhardt, B. Van Deurs and K. Sandvig, Mol. Biol. Cell, 1999, 10, 961–974.
37. H. Song, R. Wang, H. Xiao, H. Cai, W. Zhang, Z. Xie, Y. Huang, X. Jing and T. Liu, Eur. J. Pharm. Biopharm., 2013, 83, 63–75.
38. H. T. T. Duong, V. T. Huynh, P. de Souza and M. H. Stenzel, Biomacromolecules, 2010, 11, 2290–2299.

---

Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra16435h

---