Self-assembled nanoparticles covalently consisting of doxorubicin and EDB fibronectin specific peptide for solid tumour treatment

Abstract

In this study, we developed a facile modality to prepare a drug delivery system consisting of doxorubicin (DOX) and a ZD2 motif (DOX-ZD2) for potential clinical use treating solid tumours. The DOX-ZD2 nanoparticles with a diameter of ∼160 nm were prepared in a selective solvent, water. The DOX-ZD2 nanoparticles possessed great stability in phosphate buffer solution (PBS) and were fairly stable in a low concentration of thiols (20 μM). However, a high concentration of thiols (10 mM) remarkably promoted the deconjugation of DOX-ZD2. In vitro study indicated that DOX-ZD2 showed great preferential cellular uptake in PC3 cells compared with control groups, DOX-conjugated scramble peptide (sequence: CERAK) (DOX-Contr) and free DOX, and high cell suppression as well. The PC3 tumour model was used to investigate the therapeutic efficacy of DOX-ZD2, DOX-Contr, and free DOX. Encouragingly, the DOX-ZD2 nanoparticles showed strong anti-tumour ability in comparison with DOX-Contr and, in particular, free DOX. Our study certainly provides a novel modality for preparing DOX-contained drug delivery systems for solid tumour therapy in clinic.

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Introduction

With the advance of nanotechnology in the past decades, nanoparticle-based targeted drug delivery systems used for cancer therapy have received increasing attention due to great advantages they bring. First, the small size of nanoparticles enables them to escape the uptake of mononuclear phagocytic system (MPS) cells, which dramatically increases the possibility of the cargo arriving at tumour sites. Second, the targeting ligands increase the therapeutic efficacy of antitumor drugs and, simultaneously, weaken the side-effects.^1–4 Conversely, free antitumor drugs are usually uptaken by cells non-specifically, inducing serious side-effects.⁵ Furthermore, the unique size and amenability to surface modification with desired characteristics make nanoparticles well qualified for overcoming the biological barriers.⁵ However, the challenge to surmount all of the biological barriers still remains.^6,7 Fortunately, the advances in nanoparticle engineering and understanding the roles of nanoparticle characteristics including size, surface properties, shape, and targeting ligands, are bringing new opportunities for the application of nanoparticles in cancer therapy.

The major limitations of nanoparticles to be translated into the clinic for cancer treatment are their poor accumulation in the tumour sites, and, in particular, short penetration in solid tumours.^7–9 Thus, to increase the likelihood of success in cancer therapy, the selection of targeting ligands to enhance the specific accumulation of nanoparticles, through attaching molecular targeting probes to the nanoparticles, in tumours and simultaneously the delivery vehicle to improve the penetration into tumours, are of importance.^8,10,11

Prostate cancer (PCa), one of the leading cancer in men in the world, most likely grows slowly and does not cause health risks in men.^12,13 Unfortunately, some might develop relatively aggressively. In addition, the cancer cells can spread in the body from the prostate to other organs, which results in systemic metastasis.¹² The standard clinical treatments of PCa, generally, include surgery, chemotherapy, and radiotherapy.^9,12 Although these modalities have attractive advantages, they also yield side-effects, for example, hair loss, toxicity, damage to liver and kidney, and a destructive “bystander” effect to surrounding cells.^14,15

In this study, we aim to prepare an amphiphilic compound consisting of commercially available anti-cancer agent, DOX, and ZD2 peptide, used as drug delivery system via self-assembly for treatment of PCa. Herein, DOX was selected as the therapeutic drug for two major reasons. First, the fate tracking of DOX was convenient due to the self-emitting fluorescence.⁶ The other reason is that, currently, it is one of the most commonly used antitumor drugs, but showing very limited penetration in PCa.⁶ Very recently, the peptide sequence CTVRTSADC, named as ZD2, was reported to specifically target PCa,¹⁶ through binding to extradomain-B fibronectin (EDB-FN), one of the oncofetal fibronectin (onfFN) isoforms greatly expressed by the malignant tumors and a marker of angiogenesis and epithelial mesenchymal transition (EMT).^17–20 The study demonstrated that ZD2 peptide strongly bound to PC3 cancer cells in vitro and PC3-GFP prostate tumours in nude mice bearing the tumour xenografts. The DOX-ZD2 amphiphilic compound with dendritic architecture in our present study, possessing relatively small molecular size compared with normal amphiphilic copolymers, was expected to enhance the DOX loading efficacy and form self-assembled nanoparticles with small size to enhance the penetration ability in solid tumours simultaneously as illustrated in Scheme 1.

Scheme 1 Schematic illustration of the proposed self-assembled nanoparticle–tumor interaction mechanism.

Experimental section

Materials

All chemicals were purchased from Sinopharm Chemical Reagent Co., Ltd. Shanghai, China without further purification unless otherwise described. 2-Chlorotrityl chloride resin, 1-hydroxybenzotriazole hydrate (HOBt), N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uranium hexafluorophosphate (HBTU), and Fmoc protected amino acids were purchased from Chem-Impex International, Inc. Dichloromethane (DCM), N,N-dimethylformamide (DMF), trifluoroacetic acid (TFA), and anhydrous N,N-diisopropylethyl amine (DIPEA) were purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd. Doxorubicin hydrochloride was purchased from TCI (Shanghai, China). Maleimido-propionic-PFP was synthesized as described previously.²¹

Synthesis of DOX-MAL

DOX (580 mg, 1 mmol) and Maleimido-propionic-PFP (335 mg, 1 mmol) were dissolved in DMF (10 mL). DIPEA (1 mL) was added to the solution and the mixture was stirred for 45 min at room temperature. The DMF was directly removed by rotary evaporator using oil pump prior to purification with column chromatography (SiO₂, methanol : DCM = 1 : 5) to obtain DOX-MAL (625 mg, yield 90%). MALDI-TOF (m/z, [M + H]⁺): 695.5 (obsd), 694.2 (calcd).

Synthesis of ZD2

ZD2 peptide (sequence: CTVRTSAD) was synthesized using standard solid-phase chemistry on the 2-chlorotrityl chloride resin according to previous reports.¹⁶ In each reaction, HBTU, DIPEA, and amino acid were added to the reaction column for solid-phase chemistry at the amount of 3 fold of the amine group on the resin. After removal of Nα, Nε-Fmoc protecting group in the last amino acid using piperidine/DMF (1 : 4, v/v) solution, the peptide was branched to have 4 thiol groups for further conjugation with DOX-MAL using Fmoc-Lys(Fmoc)-OH and Fmoc-Cys(Trt)-OH and then cleaved from the resin using an acidic cocktail of TFA : TIS : EDT : water (95 : 1 : 2 : 2) for 5 h as shown in Fig. 1a. The mixture was precipitated in cold ether twice and dried at reduced pressure under N₂ nitrogen to give ZD2-(SH)₄ as white powder (yield 92%). MALDI-TOF (m/z, [M]⁺): 1545.2 (obsd), 1544.69 (calcd).

Fig. 1 (a) Schematic procedure for synthesis of ZD2, MALDI-TOF spectra of (b) DOX-MAL, (c) ZD2-(SH)₄, and (d) DOX-ZD2.

A scrambled peptide as control agent (sequence: CERAK) was prepared using the same procedure with a yield of 88%.

Synthesis of DOX-ZD2 and DOX-Contr

DOX-MAL (348 mg, 0.5 mmol) and ZD2 (426 mg, 0.5 mmol) were dissolved in DMF (20 mL), followed by addition of DIPEA (0.5 mL) under stirring for 30 min. The mixture was directly precipitated in cold ether twice to obtain white crude product, which was further purified with reversed-phase high performance liquid chromatography (HPLC) to have DOX-ZD2 as white powder (purity 96%, yield 84%). MALDI-TOF (m/z, [M + H]⁺): 4322.50 (obsd), 4321.50 (calcd).

The DOX-Contr was synthesized in the similar way using control peptide prepared earlier (sequence: CDSRATVT) as control group (purity 95%, yield 82%).

Preparation of self-assembled micelles

DOX-ZD2 or DOX-Contr (5 mg) was dissolved in DMF (5 mL) overnight at room temperature. Afterwards, pure water (50 mL) was added dropwise to the solution in 6 h, followed by dialysis against deionized water (MWO 100–500) overnight.

Characterizations

The mass spectra of agents were acquired from matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF, Bruker Autoflex III) using 2,5-dihydroxybenzoic acid (2,5-DHB) as a matrix. The micelle suspension was dipped onto a round glass coverslip (18 mm, diameter) and sputter-coated with a conductive layer before the scanning electronic microscopy (SEM, S-3400N, Hitachi, Japan) was used to analyze the morphology. Also, the self-assembled micelles were observed using a transmission electron micrographs (TEM, JEOL JEM-2010) at 200 kV. Prior to observation, the micelle suspension in water was dipped onto the surface of a copper grid until dried.

PC3 cellular uptake of DOX, DOX-Contr, and DOX-ZD2

PC3 cells (ATCC, USA) were cultured in RPMI medium containing 10% fetal bovine serum (FBS, Gibco, USA). The PC3 cells were incubated with TGF-β (10 ng mL⁻¹) to induce EMT before cultured with free DOX, DOX-Contr, and DOX-ZD2 in 6-well plates at 37 °C in 5% CO₂ at a density of ∼10⁵ cells per mL. After cultured for 24 h, the cells were cultured with drugs (100 μg mL⁻¹ DOX) for 45 min. Thereafter, the culture medium was removed and the cells were washed with PBS twice. Further, the cells were fixed with 4% paraformaldehyde (PFA) solution for 10 min, washed with PBS twice, and stained with rhodamine-phalloidin (RP) and diamidino-2-phenylindole (DAPI) according the manufacturers' instruction. The cellular uptake of drugs was visualized using a confocal laser scanning microscopy (CLSM, Nikon A1R, Japan).

The cellular uptakes versus drug concentration and culture time were also studied using the flow cytometry. Briefly, the induced PC3 cells were cultured as described above with drugs for different time intervals (0.5 h, 1 h, 2 h, 4 h, 6 h, and 12 h) at the DOX concentration of 100 μg mL⁻¹, and at different DOX concentrations (10 μg mL⁻¹, 25 μg mL⁻¹, 50 μg mL⁻¹, 100 μg mL⁻¹, and 200 μg mL⁻¹) for 0.5 h. The cells were analyzed by the flow cytometry using Summit software.

C2C12 cellular uptake of DOX, DOX-Contr, and DOX-ZD2

Similarly, C2C12 mouse adherent myoblasts were also used for investigating the cellular uptake and cell survival. C2C12 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS, 2 mM glutamine, 0.5% anti-myoplasm, 1% antibiotics, and 25 mM Hepes, pH 7.5. The culture condition was as described in the last section.

Cell viability

The PC3 cells were incubated with TGF-β (10 ng mL⁻¹) to induce EMT before cultured with free DOX, DOX-Contr, and DOX-ZD2 in 24-well plates at 37 °C in 5% CO₂ at a density of ∼10⁵ cells per mL. After cultured for 24 h and washed with PBS twice, the cells were cultured with drugs at the DOX concentrations of 10 μg mL⁻¹, 20 μg mL⁻¹, 50 μg mL⁻¹, and 100 μg mL⁻¹ for 24 h. After the culture medium was extracted and the cells were washed with PBS twice, the cell number was counted using a CCK-8 assay (Beyotime Institute of Biotechnology, China). 50 μL of CCK-8 solution was added to each well and incubated at 37 °C in 5% CO₂ for 3 h before 10 μL of the supernatant transferred into a 96-well plate. The optical density was read at 450 nm using a microplate reader (Synergy HT, USA). The optical density was converted to cell numbers according to a standard curve.

In vivo study

The in vivo study was performed according to a protocol approved by the First Affiliated Hospital of Jinzhou Medical University Animal Care Committees. Male BALB/c mice (18–20 g) were maintained at the Athymic Animal Core Facility at the First Affiliated Hospital of Jinzhou Medical University, according to the animal protocols. The mice were subcutaneously injected with a mixture of 50 μL PBS containing ∼10⁷ cells and 50 μL matrigel in the flanks. After 4 weeks post inoculation, the tumour grew to ∼7 mm in diameter. The mice (n = 7) were intravenously injected with free DOX, DOX-Contr, and DOX-ZD2 (PBS solutions) at a DOX dosage of 5 mg kg⁻¹ weekly over the course of 24 days, during which tumour volume was measured and calculated at each administration point. At 24 days, the tumour-bearing mice were sacrificed and tumours were collected, and weighed, followed by cryosection into slices (10 μm) for H&E staining. Stained slices were imaged under a light microscope (TE2000U, Nikon, Japan).

The biodistribution study of free DOX, DOX-Contr, and DOX-ZD2 in organs, such as tumour, kidney, lung, muscle, skin, femur, brain, liver, heart, and spleen was also performed after the tumour-bearing mice were intravenously injected with drugs for 3 h and 24 h. At time point, the mice were sacrificed and the organs were harvested. Then, the drugs in organs were extracted according to previous protocol,¹² followed by measuring the drug concentration using the HPLC. The biodistribution in organs was expressed as the weight of drugs in unit weight of organs (μg kg⁻¹, n = 6).

All experiments were performed in compliance with relevant laws or guidelines of the First Affiliated Hospital of Jinzhou Medical University, which were approved by the institutional Animal Care Committees, including S. H. Wang, T. T. He, Y. H. Ding, E. Y. Yi, and W. Sun.

Statistical analysis

Quantitative data were expressed as the mean ± standard deviation and analyzed with origin 9.0 (Origin Lab Corporation, USA). The statistical analysis were carried out using the student's t-test. With greater than 95% or 99% confidence level (p < 0.05 or 0.01, respectively), statistical significance was attained.

Results and discussion

In our present study, the ZD2 peptide with 2-generation dendritic structure was synthesized through standard solid phase chemistry as shown in Fig. 1a. The amino acid, Fmoc-Lys(Fmoc)-OH, was coupled with amine groups twice to generate dendritic structures with four amine groups, which was subsequently converted into thiols using Fmoc-Cys(Trt)-OH. To avoid formation of disulfides during cleavage from resin using TFA/TIS/H₂O mixture and drying under reduced pressure, the process was operated under nitrogen gas. The scramble peptide of CERAK as control group was prepared in the same procedure. Eventually, the DOX-Contr and DOX-ZD2 was purified with preparative HPLC (purity, ∼95%). Before the conjugation with DOX-MAL, all reactions occurred in solid phase, which enable us to synthesize DOX-ZD2 with high yield and high purity, one of our shining points in this study.

After the DOX-Contr and DOX-ZD2 were purified, the corresponding nanoparticles were prepared through addition of water to the DOX-Contr/DMF or DOX-ZD2/DMF solution dropwise. The morphologies of self-assembled nanoparticles were observed using SEM as shown in Fig. 2a. The nanoparticles exhibited spherical shape with a narrow size distribution and an average diameter of ∼160 nm, as presented by the size distribution measured by the ImageJ software in Fig. 2c. The nanoparticle was further investigated by the technique of TEM in Fig. 2b, showing that the nanoparticle, possessed not only spherical shape, but black rims, indicating heterogeneous characteristics between the outer layer and the inner sphere. In our present study, the DOX-Contr or DOX-ZD2 was composed of hydrophobic blocks, DOX, and hydrophilic block, ZD2 or scramble peptide, which enabled the self-assembly in selective solvent, H₂O here. Previously, numerous studies demonstrated that amphiphilic polymers could easily form micelles, such as spheres, fibers, and rods by controlling the molecular structures or self-assembling conditions. For example, PCL-b-PLA-PEO can form spheres with the hydrophobic component, PCL-b-PLA, as core and the hydrophilic component, PEO, as corona.²² Similarly, the DOX-ZD2 and DOX-Contr also formed spherical micelles with hydrophilic peptide as corona, surrounding the core, DOX. The self-assembly process can be depicted as Fig. 2d. Based on this discussion, it is reasonable to understand that some nanoparticles in SEM images showed cave-like features indicated by the arrows because of the hollow structure, which was also confirmed by the TEM observation.

Fig. 2 (a) The representative SEM images, (b) representative TEM images, and (c) size distribution quantified from SEM images of DOX-ZD2 and DOX-Contr. (d) Schematic illustration of the self-assembled DOX-ZD2 nanoparticles.

The in vitro stability of nanoparticles was also evaluated using PBS solution, low concentration of GSH (20 μM), and high concentration of GSH (10 mM) as shown in Fig. 3. To mimic the effects of extracellular and intracellular conditions on nanoparticles once injected in vivo,²³ 20 μM and 10 mM GSH solutions were employed to examine the stability of nanoparticles in vitro. Fig. 3a clearly showed that both nanoparticles were stable in PBS solution over the period of 15 days without size change. However, this stability was strongly influenced by the GSH solution. Both nanoparticles degraded in the first 6 days in the 20 μM GSH/PBS solution and after 6 days, the nanoparticles disappeared because of breaking down, which possibly was attributed to the concentration lower than critical aggregation concentration (CAC).²² This phenomenon was more significant when the concentration of GSH/PBS increased to 10 mM as shown in Fig. 3c. In this study, the DOX was conjugated with peptide through the formation of thiosuccinimide between thiol group in peptide and DOX-MAL. The thiosuccinimide was demonstrated to be unstable and showed deconjugation in physiological environment due to the retro-Michael reaction, promoted by the thiols.²⁴ Our purpose of this study was to develop promising drug delivery system for PCa therapy. Encouragingly, the stability of nanoparticles (DOX-ZD2 or DOX-Contr) showed environmental dependence, namely, stable before internalized into cells, an expected characteristic of a good drug delivery system.

Fig. 3 In vitro stability of the self-assembled nanoparticles in PBS solution (a), low concentration of GSH (b), and high concentration of GSH (c) at 37 °C.

The targeting efficiency of DOX, DOX-Contr, and DOX-ZD2 to PC3 cells was investigated through cellular uptake visualized by CLSM as demonstrated in Fig. 4a. Free DOX, possessing hydrophobic characteristic, had poor solubility in cell culture medium, which resulted in extremely low drug internalization into PC3 cells. Hence, the cells cultured with free DOX did not show clear green spots, the unique colour of DOX. Conversely, the cells, after cultured with DOX-Contr for 30 min, presented prominent accumulation of DOX, and it was more significant for DOX-ZD2, indicating that DOX-ZD2 supported the best therapeutic efficacy for PC3 cells. Also, the cellular uptakes of free DOX, DOX-Contr, and DOX-ZD2 against time and dosage were quantitatively examined using the flow cytometry as shown in Fig. 4b and c. Both DOX-Contr and DOX-ZD2 were covalently conjugated with DOX, as a self-emitting fluorescent marker, to determine the number of cells that internalized drugs. As the culture time increased, the cellular uptake increased for all drugs. However, clearly, the cellular uptake for DOX-ZD2 was much faster than others and reached over 80% within 30 min at the concentration of 1.0 μM, whereas the free DOX showed that poorest uptake and only ∼50% at 12 h. Similarly, when the dosage increased, the DOX-ZD2 also showed evidently better cellular uptake than DOX-Contr and the free DOX presented the slowest uptake. Besides, it can be seen that the DOX-ZD2 showed great cellular uptake burst within 30 min and 1.0 μM, and it increased slowly beyond these values. Thus, the cell culture time for CLSM images was selected as 30 min and the following cell survival study would be performed in the dose range from 0.1 μM to 1.0 μM. Extensively secreted by the PC3 cells, the network of EDB-FN facilitated the specific binding of ZD2 peptide as demonstrated earlier.¹⁶ In comparison with DOX-Contr, DOX-ZD2 easily bound to PC3 cells, advancing the further internalization of nanoparticles into PC3 cells. As discussed earlier, the thiosuccinimide tended to break down via deconjugation stimulated by thiols, especially in intracellular environment. Hence, the nanoparticles were quite stable before internalization by PC3 cells. However, once internalized, the nanoparticles quickly released DOX through the deconjugation of thiosuccinimide, inducing cellular apoptosis efficiently. Even though DOX-Contr nanoparticles did not have targeting effect to PC3 cells, the hydrophilic scramble peptide corona, similar to PEO, improved the cellular uptake greatly,²⁵ compared with hydrophobic free DOX. To further demonstrate the targeting advantage of DOX-ZD2, we also used normal cells, C2C12, as control cell line to perform the cellular uptake in terms of CLSM images and flow cytometry study under the same culture conditions as PC3 cells. The results in ESI (Fig. S1 and S2†), clearly, showed that free DOX still had poor uptake in C2C12 cells, whereas DOX-Contr and DOX-ZD2 had very similar uptake results, meaning that the C2C12 cells, not overexpressing EDB-FN, strongly weakened the targeting effect of ZD2.

Fig. 4 (a) CLSM images visualizing cellular uptakes of free DOX, DOX-Contr, and DOX-ZD2 in PC3 cells after cultured for 30 min. (b) Kinetic cellular uptake and (c) dosage efficiency of free DOX, DOX-Contr, and DOX-ZD2 in PC3 cells. Data are presented as means ± standard deviation (n = 5). (d) Cell survival normalized to TCP, measured by the CCK-8 assay at 24 h. *: p < 0.05 and **: p < 0.01, respectively. Data are presented as means ± standard deviation (n = 5). (e) Representative optical images of cells at control group (untreated) and at 1.0 μM of DOX after incubated for 24 h. Scale bar represents 100 μm.

The cell survival was evaluated using the CCK-8 assay after cells were cultured for 24 h with drugs at the dose of 0.1 μM, 0.2 μM, 0.5 μM, and 1.0 μM (DOX equivalent) as shown in Fig. 4d. Notably, DOX-ZD2 prominently suppressed cell survival in comparison with free DOX and DOX-Contr at the concentrations over 0.2 μM and this difference was more remarkable with the increase of DOX concentration. At the concentrations of 0.2 μM and 0.5 μM, DOX-Contr, although, indicated higher anti-PC3 cells ability than free DOX, they did not statistically show significant difference. However, the significant difference between free DOX and DOX-Contr was observed at the concentration of 1.0 μM. The cell proliferation was also observed by optical microscope at the concentration of 1.0 μM as revealed in Fig. 4e. Consistently, cells presented decreased density from DOX to DOX-ZD2, meaning that DOX-ZD2 had great anti-tumour effect. The C2C12 cells were also used to evaluate the cytotoxicity of free DOX, DOX-Contr, and DOX-ZD2 as shown in Fig. S3.† The data suggested that the higher concentration of DOX-Contr or DOX-ZD2 suppressed cell growth more, however, there was no significant difference between them at all concentrations.

The antitumor effect of free DOX, DOX-Contr, and DOX-ZD2 was investigated by treating the PC3 tumour model weekly at the dosage of 5 mg kg⁻¹ (DOX equivalent). Unsurprisingly, treated with free DOX, the tumour grew much faster than those treated with DOX-Contr and, particularly, DOX-ZD2, even though they grew slightly slower than those treated with PBS. On the other hand, the DOX-Contr inhibited tumour growth as compared with free DOX, however, the tumour continued growing over the course of 24 days (Fig. 5a). Encouragingly, the DOX-ZD2 substantially suppressed the tumour growth and the tumour size became smaller with treatment time, indicating DOX-ZD2 possessed strong antitumor effect. Besides, the tumour weights measured after the mice were sacrificed at day 24 in Fig. 5b, consistently, demonstrated that the tumours treated with DOX-ZD2 were significantly lighter than those treated with other drugs and the significant difference between DOX-Contr and PBS was also observed. The accumulation of free DOX, DOX-Contr, and DOX-ZD2 in organs after intravenous injection into tumour-bearing mice was investigated as shown in Fig. 5c and d. Interestingly, at both 3 h and 24 h, the DOX-Contr and especially DOX-ZD2 showed preferential accumulation in tumours, which were significantly higher than free DOX. Besides, DOX-Contr and particularly DOX-ZD2 had extremely low accumulation in heart, a good sign for their potential application in clinic due to the fact that cardiotoxicity associated with the use of DOX hampers its clinical potential.²⁶

Fig. 5 In vivo treatment of PC3 tumours with free DOX, DOX-Contr, and DOX-ZD2 over the course of 24 days. (a) Tumour size versus days. (b) Tumour weight versus days. *: p < 0.01. Data are presented as means ± standard deviation (n = 5). Biodistribution of free DOX, DOX-Contr, and DOX-ZD2 in PC3 tumour-bearing mice. Drugs were intravenously injected into mice via a tail vein and allowed to circulate for (c) 3 h and (d) 24 h. After harvesting the organs, the concentrations at time points were measured by HPLC. *: p < 0.05 and **: p < 0.01, respectively. Data are presented as means ± standard deviation (n = 5). (e) Histological evaluation (H&E staining) of PC3 tumours after treated with PBS, free DOX, DOX-Contr, and DOX-ZD2 at day 24. Green circles indicate apoptotic cells.

After the mice were treated with drugs for 24 days and sacrificed, the H&E staining for tumour slices was performed as shown in Fig. 5e. Consistent with earlier assessments, the histological results demonstrated that the tumours treated with DOX-ZD2 exhibited the reduced cell density with biggest space between cells in comparison with the tumours in other groups, and the tumour cells contracted evidently and were isolated from neighboring cells with halo-like spaces, indicating apoptosis.^27,28 Similarly, the tumour cells treated with DOX-Contr also showed apoptotic status, but grew better than those treated with DOX-ZD2. On the contrary, the tumours treated with PBS and free DOX had clearly denser cells than other groups.

The mechanism of our drug delivery system can be interpreted as shown in Scheme 1. The self-assembled nanoparticles, DOX-Contr or DOX-ZD2, circulated in blood vessel after intravenous injection. As studied earlier, the nanoparticles had great stability in PBS and low concentration of thiols, namely, extracellular environment. Hence, the nanoparticles can undergo long circulation before breakdown or uptaken into tumours. In addition, the nanoparticles containing hydrophilic corona might further enhance the circulation time and, simultaneously, reduced the recognition and clearance by the reticuloendothelial system (RES) before the cargo arrived at PCa.^29,30 Different from normal tissues, tumours consisted of vascularized area, non-uniformly, with abnormal branched loops and tortuosity, subcompartments, interstitium, and tumour cells. Nanoparticle diameter, chemistry, and hydrophilicity exert influences on the drug delivery into the interstitium. Usually, the interstitial pressure, greater than that in the periphery, hinder the drug diffusion to the tumours, resulting in extended retention time in interstitium, called enhanced permeability and retention (EPR).³⁰ As a consequence of the small size and hydrophilic corona, the nanoparticles, DOX-Contr or DOX-ZD2, might penetrate tumours easily via passive targeting because of EPR effect from blood pool. As a benefit from specific targeting of ZD2 motif to PCa expressing EDB-FN, the DOX-ZD2 nanoparticles possessed high efficacy to bind PCa and entered the tumour cells via active targeting. Afterwards, the intracellular environment, generating high concentration of thiols, stimulated the deconjugation of thiosuccinimide, triggering the fast release of DOX. Conversely, the free DOX was strongly prevented by the immune system through RES effect and the therapeutic efficacy was significantly compromised. The therapeutic efficacy was also evidenced by the biodistribution study (Fig. 5c and d), clearly showing the preferential accumulation of DOX-Contr, especially DOX-ZD2, in tumour sites. The advantages of our drug delivery system are encouraging. First, the drug loading is stoichiometrically controllable by the adjustment of dendritic arm number, which carries more DOX at higher generations. Second, the covalently conjugation of DOX with peptide motif through thiosuccinimide is more stable than other drug delivery systems using physical adsorption, which might release DOX before arriving at tumours. This, together with the active targeting effect, undoubtedly, increases the likelihood of DOX accumulation at tumour sites. As demonstrated by the biodistribution results, the reduced accumulation of DOX-ZD2 in hearts compared with others might address the big issue brought by DOX-related drug systems, namely, side effect on heart failure.³¹ More interestingly, the stability of DOX-ZD2 is environmentally changeable. Once internalized into cells, the nanoparticles can efficiently release DOX due to the deconjugation of thiosuccinimide. Last, but not least, the conventional protocols for preparing using amphiphilic polymers, normally, were more costly and complicated, preventing further application in clinic. However, this study provided a facile procedure using solid phase chemistry to prepare DOX-ZD2, which certainly improved the operability for product scale-up.

Conclusions

A facile modality to prepare a drug delivery system consisting of DOX and ZD2 motif (DOX-ZD2) for potential clinic use treating PCa was studied. The DOX-ZD2 or DOX-Contr nanoparticles with a diameter of ∼160 nm were prepared in a selective solvent. The nanoparticles possessed great stability in PBS and concentration-dependent deconjugation in thiol-containing solution. In vitro study indicated that DOX-ZD2 showed great preferential cellular uptake in PC3 cells compared with DOX-Contr and free DOX, and high cell suppression as well. The in vivo study using PC3 tumour model demonstrated that the DOX-ZD2 nanoparticles showed strong antitumor ability in comparison with DOX-Contr, in particular, free DOX. Our study certainly provide a novel modality for preparing DOX-contained drug delivery systems for solid tumour therapy in clinic.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (no. 81302681), the development plan of the outstanding young scholars of Liaoning Province (no. LJQ2015064), Aohongboze Foundation, the President Fund of Jinzhou Medical University (no. XZJJ20130106-02), and QuanminOral Graduate Sci-tech Innovation Foundation, the President Fund of Jinzhou Medical University (no. QM2014007).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra11186f

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