pH-sensitive nanomedicine based on PEGylated nanodiamond for enhanced tumor therapy

Abstract

Enhancing chemotherapeutic efficiency through enriched drug load and controlled drug release is urgent for alleviating the suffering of cancer patients. Here, a novel pH-sensitive nanomedicine was constructed to acquire high drug loading capacity and better therapeutic efficiency. Interestingly, with the assistance of a pH 8.0 sodium borate buffer solution, PEGylated nanodiamond vehicles loaded with doxorubicin (DOX) achieved nearly 50% loading efficiency, low premature drug release in physiological conditions and effective stimuli-response release under a tumor microenvironment. In addition, assessment by flow cytometry and cell migration assay illustrated that NP/D could induce cell apoptosis, cycle abnormality and inhibit cell migration. The results from the confocal fluorescence microscopy study showed that NP/D could be internalized into cells and distributed into the cytoplasm, subsequently DOX detaches from NP/D and could migrate and enter the nucleus to inhibit cell proliferation. NP/D can open the window for new nanodrugs for a broad spectrum of anticancer agents.

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1. Introduction

In order to overcome the severe side effects induced by chemotherapy agents during cancer therapy,^1,2 a wide range of nanocarriers, such as iron oxide magnetic nanoparticles,³ gold nanoparticles⁴ and polymer nanoparticles⁵ were explored to deliver anticancer drugs so as to enhance efficiency of chemotherapy. However, despite many advantages of nanocarriers promoting their availability, the instability and toxicity of nanocarriers limited their further clinical application.^6–9

Nanodiamond (ND), a new family member of carbon nanoparticles, has been unearthed for various biomedical applications.^10–13 ND has been a nouveau nanocarrier for drug delivery^14–16 and cellular imaging,¹⁷ which is due to not only possessing the least toxicity compared to other carbon-based materials, and having excellent biocompatibility, chemical stability and optical properties,^18–20 but also it could be modified to make it have more virtues.^21–23 Choi et al. demonstrated that ND could improve the cellular uptake of DOX and be employed as drug carriers for efficient cancer therapy.²⁴ Ho et al. also demonstrated that DOX complexed with ND and administered via convection-enhanced delivery (CED), was significantly more efficient at killing tumor cells than uncomplexed DOX and suggest that CED of ND–DOX is a promising approach for brain tumor treatment.²⁵ Our previous study also established the FND–DOX system and found slow and sustained drug release capability.²⁶ Despite research showing ND could improve antitumor activity of free DOX, the wide application in biology of ND has been limited due to its trend to aggregate.²⁷

Nanoparticles functionalized with polymer and protein have demonstrated enhanced circulation time and increased dispersibility.^28–30 Poly(ethylene glycol) (PEG) has been introduced onto ND, which can not only improve the dispersion, but also reduce the interaction with cells for reducing non-specific effects, and prolong the blood circulation time.^31,32 Our previous study verified that transferrin and PEG improved the dispersibility of ND and prolonged the cellular uptake half-life,^33–36 whereas the loading efficiency and treatment efficiency are still not so satisfactory.

Taking into account the enhanced drug loading and treatment efficiency for tumors, a novel nanodrug based on the physical adsorption of free DOX onto PEGylated ND in a pH 8.0 sodium borate buffer solution was designed. Specifically, carboxylated ND was conjugated by polyethylene glycol amine carboxyl (H₂N–PEG–COOH) to obtain the ND–PEG nanocarrier. In the following step, DOX was successfully adsorbed onto ND–PEG in a pH 8.0 sodium borate buffer solution via electrostatic interactions to obtain ND–PEG/DOX (NP/D) nanoparticles. We found that the addition of a pH 8.0 sodium borate buffer solution is an essential component of the loading process and it was shown to be able to promote the adsorption of DOX onto the PEGylated NDs, where nearly 50% wt adsorption of DOX on the ND–PEG was achieved in the pH 8.0 sodium borate buffer solution. The NP/D was found to move inside the cells quickly and was capable of ferrying the drug inside living cells efficiently. In addition, NP/D showed a higher increase of cytotoxicity with time than that of the free DOX. As such, this work convincingly demonstrates the potential of ND as a broad drug-functionalization platform.

2. Experimental section

2.1. Materials and instruments

Synthetic type 1b nanodiamond (ND) powders commercially available (sizes ≈ 140 nm, Element Six) were chosen here because the tumor blood vessels can retain 100–400 nm particles. Then the nanodiamond powders were carboxylated to use throughout the experiment according to previously reported method by us. Doxorubicin hydrochloride (DOX) was purchased from Shanxi pude Pharmaceutical Co., Ltd. (China). Polyethylene glycol amine carboxyl (H₂N–PEG–COOH, M_w: 2000) was bought from Shanghai seebio Biotech Inc, China. Fluorescamine was bought from TCI (Shanghai) Chemical Industry (China). N-Hydroxy succinimide (NHS), 2-(N-morphine) ethane sulfonic acid (MES) and 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDC) were purchased from Sigma. (M-β-CD) was purchased from Shanghai Aladdin Reagent Co., Ltd. (China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), penicillin–streptomycin combination and paraformaldehyde were purchased from Solarbio (Beijing, China). Dulbecco’s Minimum Essential Media (DMEM) was purchased from Thermo Fisher Biological and Chemical Product (Beijing, China). Fetal bovine serum was purchased from Hangzhou Sijiqing Biological Engineering Materials Co., Ltd (China). Trypsin was purchased from Sino-American Biotechnology Company. Hoechst 33258 was purchased from Beyotime Biotechnology (in China). Sucrose was purchased from Amresco Co. (USA). HeLa, HepG2 and MCF-7 cells were provided by the Gene Engineering Center of Shanxi University. All other chemicals and solvents were of analytical grade and procured from local suppliers unless otherwise mentioned. Millipore filtered water was used for all aqueous solutions.

A fluorescence spectrophotometer (970CRT, Shanghai Analytical Instruments Factory), UV-visible spectrophotometer (UV1901, Beijing spectral analysis of GM), ultrasonic cleaner (KQ-100DE, Kunshan Ultrasonic Instrument Co., Ltd.), high speed micro-centrifuges (TG16-W, Hunan Instrument Laboratory Instrument Development Co., Ltd.), flip shake instrument (DR-MIX, Beijing Hao North Biotechnology Co., Ltd.), air oscillator (THZ-22, Taicang City Experimental Equipment Factory), fourier transform infrared spectrometer (FTIR-8400S, Shimadzu Corporation, Kyoto, Japan), Malvern Nano Particle Sizer (ZETA Sizer, Nano-ZS90, England), Autoclave (YX280B, Shanghai Medical Devices Co., Ltd.), transmission electron microscope (JEM-2100, JEOL, Japan), vacuum drying oven (DZF-6020, Shanghai Yiheng Scientific Instrument Co., Ltd.), Thermostat water bath (ZDKW-4, Beijing Zhongxing Weiye Instrument Co., Ltd.), inverted microscope (MI11, Guangzhou Mingmei Technology Co., Ltd.), optical microscope (XSP-8CA, Shanghai Optical Instrument Factory), clean bench (YT-CG-1ND, Beijing Both Cologne Experiment Technology Co., Ltd.), full automatic microplate reader (Model 550, Bio-Rad, USA), flow cytometer (FACS Calibur, BD, USA), and laser scanning confocal microscope (Leica TCSSP5, Germany) were used.

2.2. Optimization and preparation of ND–PEG/DOX

2.2.1. The pH effect on DOX adsorption. First, ND–PEG was prepared following a previously reported procedure.³⁶ Then six 1 mg aliquots of ND–PEG nanoparticles were sonicated at 100 W for 30 minutes in 1 mL of different buffer solutions (4.0, 6.0, 7.0, 8.0, 9.0 and 10.0). Next, 100 μg of doxorubicin was added, and the suspension was shaken and protected from light at room temperature for 6 h. Finally, the suspension was centrifuged at 15 000 rpm for 5 minutes, and the precipitate was washed three times with the corresponding buffer solution to remove the unadsorbed DOX. The amount of DOX adsorbed was determined by calculating the change in DOX concentration before and after adsorption using an ultraviolet-visible spectrophotometer at 495 nm.

2.2.2. The time effect on DOX adsorption. Based on the optimum pH effect between ND–PEG and DOX, the optimum reaction time between ND–PEG and DOX was also investigated in a pH 8.0 sodium borate buffer solution using the technique as previously reported by us.³⁶

2.2.3. Drug loading efficiency. Based on the optimum pH effect and reaction time between ND–PEG and DOX, the amount of drug loading was further researched, where the concentrations of DOX were 100, 200, 500, 600, and 1000 μg mL⁻¹ and the concentration of ND–PEG was 1 mg mL⁻¹ for all solutions in pH 8.0 sodium borate buffer solution for 6 h. Then the solutions were centrifuged at 15 000 rpm for 5 min to remove excessive DOX. As previously reported,³⁷ briefly, the supernatants were removed, and their absorbance was read from 200 to 800 nm at ambient temperature with a UV-visible spectrometer. The peak absorbance of DOX was found at 495 nm. The difference in added DOX and DOX in the supernatant yielded the amount of DOX adsorbed. The drug loading efficiency and drug loading percentage on ND–PEG were assessed using the following eqn (1) and (2), respectively:

2.2.4. Preparation of ND–PEG/DOX. In the subsequent experiments, the as-prepared NP/D nanoparticles with a DOX loading of 99.5 μg mg⁻¹ were characterized and studied. ND–PEG (1 mg) was dispersed in the pH 8.0 sodium borate buffer and sonicated for 30 min. DOX (200 μg) was then added and the mixture was shaken up at room temperature for 6 h. ND–PEG/DOX (NP/D) was thus obtained by centrifugation at 15 000 rpm, rinsed with pH 8.0 sodium borate buffer three times and then placed in a vacuum drying oven and protected from light. The amount of absorbed DOX was measured by calculating the difference value of the total DOX and the supernatant DOX.

2.3. Characterization of ND–PEG/DOX

Fourier transform infrared spectroscopy (FTIR) and UV-visible spectroscopy were used to confirm the presence of DOX and PEG on the surface of ND. A Zeta-sizer Nano ZS90 was used to monitor the zeta potential, polydispersity index (PDI) and hydrodynamic size of the ND before and after modification. Measurements of the nanoparticle size were performed at 25 °C and a scattering angle of 90°. The mean hydrodynamic diameter was determined by cumulative analysis. Determination of the zeta potential was based on the electrophoretic mobility of the nanoparticles in aqueous medium, and was performed using folded capillary cells in automatic mode. The dispersion of ND, ND–PEG and ND–PEG/DOX in distilled water was observed by a digital camera.

2.4. In vitro drug release

The DOX release study was carried out in dialysis bags at 37 °C in phosphate buffer solutions (pH 7.4, 6.5 and 5.0) with an air oscillator. Firstly, 1 mL of the ND–PEG/DOX suspension that was well dispersed was placed in a dialysis tube. Then the dialysis bag was quickly immersed in 9 mL of the corresponding PBS solution in an air oscillator continually shaking (150 rpm) and a changeless temperature (37 °C). At the scheduled time, 2 mL of the sample was withdrawn and the same amount of fresh release medium was replenished. The amount of released DOX was measured with ultraviolet measurements at a wavelength of 480 nm.

2.5. Cell culture

HepG2, HeLa and MCF-7 cells were obtained from the Gene Engineering Center of Shanxi University. All cells were cultured in plastic dishes containing DMEM supplemented with 10% FBS and 1% penicillin–streptomycin in a CO₂ incubator at 37 °C and passaged every 2 days using trypsin containing EDTA and PBS (pH 7.4).

2.6. In vitro viability

The in vitro viability assay was performed as described previously.³⁶ Briefly, 5000 cells per well in the case of HepG2, HeLa and MCF-7 cells were seeded into 96-well plates with 200 μL per well and treated with ND–PEG, NP/D or free DOX at a series of various concentrations for 72 h. After 72 h, a stocked MTT solution (5 μg mL⁻¹) was added and incubated for another 4 h. Thereafter, the medium was discarded, and 150 μL of dimethyl sulfoxide was added. After 10 min of vibration, absorbance at 490 nm was read by a microplate reader.

2.7. In vitro wound scratch assay

The scratch assay was performed as previously reported.³⁸ Firstly, the MCF-7 cells were plated onto each well of a six-well plate at a density of 2 × 10⁵ to create a confluent monolayer, and then the cells were incubated properly for approximately 20 h at 37 °C, allowing the cells to adhere and spread on the substrate completely. After that, the cell monolayer was scraped in a straight line to create a wound scratch with a sterile pipette of 10 μL, the injured cells were removed by washing thrice with PBS (pH 7.4), followed by adding control fresh medium or medium with ND–PEG, NP/D and free DOX and continued to develop for a period of time. Finally, the six-well plate was placed under a phase-contrast microscope and images of each sample were taken at the predetermined time. The images can be analyzed quantitatively by using a computing software of choice. The wound closure rate and migration inhibiting rate was calculated by the following equation:
where sw₀ and sw_t stand for the scratch width at 0 h and a given time, respectively, wc_treatment and wc_control stand for the wound closure rate of treatment and control groups.

2.8. Cellular uptake

MCF-7 cells were sowed into a confocal laser special dish at a density of 1 × 10³ per dish overnight at 37 °C under a 5% CO₂ atmosphere. Then the culture medium was withdrawn and replaced with fresh medium containing 5 μg mL⁻¹ NP/D. After incubation for 2 h and 5 h, respectively, the cells were fixed with 4% paraformaldehyde and then stained with Hoechst 33258. At last, the cells were observed by confocal laser scanning microscopy (CLSM). Samples stained with H33258 and DOX were visualized with excitation wavelengths of 405 nm and 488 nm, respectively.

2.9. Cell apoptosis and cell cycle assay

MCF-7 cells in fresh complete medium were cultured (1 × 10⁵) in 35 mm Petri dishes for 16 h. After complete adhesion, the cells were treated with NP/D (5 μg mL⁻¹) for another 48 h. The cells were then collected with trypsin and washed twice with cold PBS (pH 7.4). After that, the cells were resuspended in PBS and stained by both Annexin V-FITC (5 μL) and PI (5 μL) in a dark condition for 15–20 min. Finally, the cells were analyzed by a FACS Calibur flow cytometer.

MCF-7 cells were cultured in a 60 mm culture dish at a density of 1 × 10⁶ and incubated for 16–20 h to make the cells adhere completely. After incubation, the cells were washed with PBS (pH 7.4) three times and cultured with NP/D or free DOX at a concentration of 5 μg mL⁻¹ for another period of time. When the treatment was over, the cells were collected, washed twice with cold PBS (pH 7.4) and fixed with ice-cold 70% ethanol overnight at 4 °C. Sample cells prepared for flow cytometry analysis were treated with RNase and propidium iodide (PI) for 30 min at room temperature in the dark.

3. Results and discussion

3.1. Synthesis and characterization of ND–PEG/DOX

To increase the antitumor activity of DOX, simultaneously decrease the side effects of DOX in normal tissue and improve the dispersity of nanodiamond, a relatively stable amide bond was introduced to conjugate H₂N–PEG–COOH and ND, to produce ND–PEG by one step activation of carboxyl groups and the other step esterification of amine groups as previously reported.^36,39 The synthesis route is presented in Scheme 1. Meanwhile, a promising drug delivery system based on PEGylated nanodiamond bearing the anticancer drug DOX was also obtained in the presence of a sodium borate buffer solution with a pH value of 8.0. In this work, we achieved a high DOX loading content near 50 wt% adsorption of DOX on the PEGylated nanodiamond. The loading mechanism of adsorption of DOX onto the PEGylated nanodiamond can be explained as follows: because doxorubicin is a weak base with a pK_b of 8.3, doxorubicin in a buffer solution with a pH value of 8.0 would result in ionized NH³⁺ and would balance with the carboxyl groups on ND–PEG via electrostatic interaction. In addition, due to the grafting amount of PEG onto nanodiamond is 7.5 × 10⁻⁸ μmol mg​⁻¹ (Fig. S1†), the amount of carboxyl on the ND is redundant (the total amount of carboxyl on the ND is 1.26 × 10⁻⁷mol mg⁻¹ (ref. 40)), so DOX can also be adsorbed onto the carboxylic groups of the nanodiamond. Moreover, the ND–PEG/DOX (NP/D) complexes may be the result of van der Waals' forces between doxorubicin molecules and the carboxylic groups on ND–PEG or ND as previously described.⁴¹

Scheme 1 Synthesis of ND–PEG/DOX. The succession of synthetic processes is as follows: (a) EDC, NHS, MES (0.1 M, pH 5.8), rt, 6 h; (b) H₂N–PEG–COOH, BBS (pH 8.4), rt, overnight; (c) DOX, BBS (pH 8.0), rt, 6 h.

To improve the amount of drug loaded onto the ND–PEG nanoparticles, first, the adsorption of DOX with different pH values was explored as shown in Fig. 1A. We can observe that the maximum amount of DOX adsorption occurred at pH 8.0. Subsequently, the amount of DOX loaded as a function of time is displayed in Fig. 1B. One can observe that a dynamic equilibrium process of adsorption and dissociation exists between DOX and ND–PEG as we previously reported.³⁶ Specifically, (1) within 2.5 h, the dissociation rate accounts for the main control position, resulting in the amount of DOX adsorbed on nanoparticles decreasing with time; (2) within 3–6 hours, the adsorption rate is greater than the dissociation rate, resulting in the amount of DOX adsorbed on nanoparticles increasing with time; however, after six hours, a dynamic equilibrium process between dissociation and adsorption occurred, and thus the amount of drug adsorbed on the nanoparticles no longer changed. This behavior follows first-order kinetics. In the system, the amount of DOX adsorbed was determined through converting UV-vis absorbance to concentration using a linear regression equation (Fig. S2†).

Fig. 1 Optimal conditions for preparing the NP/D system. (A) Determination of the best pH for adsorption of DOX. (B) Determination of the best time for adsorption of DOX.

Table 1 summarizes the adsorbed DOX, DOX loading efficiency, and DOX loading on ND–PEG. NDs adsorbed 99.5 μg of the added 200 μg DOX, which yielded a 49.8% loading efficiency. When 1000 μg DOX was added, the ND–PEG adsorbed approximately 498.5 μg DOX corresponding to a DOX loading efficiency of about 49.9%. Since 1.0 mg of ND–PEG was used, loadings of DOX on the ND–PEG can up to 49.9% for the added 1000 μg DOX. The DOX loading on ND–PEG and drug loading efficiency achieved with our adsorption technique were much higher than that of our reported for ND–PEG/DOX complexes,³⁵ which was almost sixteen fold increase in DOX loading onto the ND–PEG in the best conditions.

Table 1 Summary of added DOX, absorbed DOX, DOX loading efficiency and DOX loaded on ND–PEG

DOX added μg	DOX adsorbed μg	DOX loading efficiency (%)	DOX loaded on ND–PEG (%)
100	46.9 ± 1.2	46.2 ± 0.7	4.7 ± 0.2
200	99.5 ± 0.9	49.8 ± 0.9	10.0 ± 0.3
500	249.2 ± 1.3	49.8 ± 0.6	24.9 ± 0.3
600	301.6 ± 1.1	50.3 ± 0.4	30.2 ± 0.4
1000	498.5 ± 1.0	49.9 ± 1.0	49.9 ± 0.6

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To confirm the synthesis, the surface chemistry, particle size, polydispersity index (PDI) and zeta potential of the NP/D were characterized. Fig. 2A shows the FTIR of ND, ND–PEG, NP/D, H₂N–PEG–COOH and DOX. Compared to the spectrum of ND, the peak at 1656 cm⁻¹ and 1621 cm⁻¹ correspond to the C=O stretching and N–H bending, respectively, which represent the characteristic peaks of amide linkage, confirming that H₂N–PEG–COOH was coupled onto ND successfully. In the spectrum of NP/D, the peaks at 1402 cm⁻¹, 1087 cm⁻¹ and 803 cm⁻¹ are attributed to the characteristic peaks of DOX, indicated by black arrows, therefore, the FTIR results support that DOX was also absorbed onto ND–PEG.

Fig. 2 Characterization of ND–PEG/DOX. (A) FTIR spectra of various materials. (B) UV-vis absorption of DOX, ND, ND–PEG and ND–PEG/DOX in PBS (pH 7.4). (C) The dispersity of different nanoparticles in PBS 7.4 with time (the left is ND, the middle is ND–PEG, and the right is NP/D).

Fig. 2B depicts the absorption spectra measured by a UV-vis spectrometer for ND, ND–PEG, NP/D, and free DOX. The ND and ND–PEG have no absorption peak, while NP/D revealed a pronounced absorption with a peak at 480 nm just as the free DOX, which suggested the successful loading of DOX onto the surface of ND–PEG.

To further estimate the potential of using NP/D as an anti-tumor therapeutic agent, the dispersity of NP/D was also investigated. Due to the easy aggregation of ND, H₂N–PEG–COOH was introduced to functionalize the ND through the covalent interaction of an amido bond, forming PEGylated ND with good dispersibility. The test results are shown in Fig. 2C. As the photos show, after stewing for 69 h, the ND almost settled entirely, while ND–PEG and NP/D still kept a good dispersibility, which verify that H₂N–PEG–COOH can confer the NP/D with favorable dispersity, better for proving that NP/D can act as an antitumor agent.

The ND–PEG and NP/D were further analyzed by the DLS and zeta potential technique, and the data is given in Table 2. As shown in Table 2, DLS results showed that the average size of the unmodified ND is 166.0 ± 1.6 nm while, after coupling with H₂N–PEG–COOH, the size increased to 184.6 ± 6.4 nm, which further confirmed that H₂N–PEG–COOH was connected to ND as the FTIR spectrum verified. And the size of NP/D is about 195.0 ± 1.3 nm with a PDI of 0.089, which indicated that it is very suitable for drug delivery. In addition, the zeta potential of the above mentioned nanoparticles was measured (Table 2). The ND has a relatively negative initial zeta potential of −30.2 ± 1.0 mV, along with the successfully synthesized ND–PEG (−24.7 ± 1.9 mV) and ND–PEG/DOX (−20.8 ± 0.4 mV) with their zeta potential slowly increasing, which indicated that PEG and DOX were successfully coated onto the surface of the ND.

Table 2 PDI, particle size and zeta potential of ND, ND–PEG and ND–PEG/DOX

	Diameter (nm)	Zeta potential (mV)	PDI
ND	166.0 ± 1.6	−30.2 ± 1.0	0.164
ND–PEG	184.6 ± 6.4	−24.7 ± 1.9	0.126
NP/D	195.0 ± 1.3	−20.8 ± 0.4	0.089

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3.2. In vitro drug release

The ND–PEG/DOX drug delivery system is designed for tumor micro-environment release of DOX. Since the pH of blood and normal tissue is around 7.4, and the pH values of the tumor micro-environment are much lower (at pH 5.0–6.5),⁴² the pH-sensitive releasing property of the NP/D nanocomposite will facilitate drug delivery. To verify the pH-sensitive release of DOX in vitro, NP/D samples were incubated at a simulated physiological condition (pH 7.4) and in an acidic tumor micro-environment (pH 5.0 and 6.5) at 37 °C. From Fig. 3, the rate and amount of DOX released from the NP/D nanocomposite strongly depended on the pH value and release time. NP/D showed a much faster DOX release at pH 5.0 and 6.5 than at pH 7.4. As can be seen from Fig. 3, the cumulative release of DOX from NP/D is less than 18% after 35 h at pH 7.4, which indicates that NP/D kept a rather good stability in the physiological condition. By comparison, a noticeably increased release of DOX is observed at pH 6.5 up to 40% and at pH 5.0 up to 65%, which demonstrated that the release rate of DOX from NP/D in an acidic tumor micro-environment is much faster than that at pH 7.4. The release of DOX from the NP/D in an acidic tumor micro-environment is likely due to the relief of electrostatic interaction characteristics between the DOX molecules and the ND–PEG nanoparticles. It can be inferred that the NP/D nanoparticle exhibits relatively high stability at physiological conditions, whereas when NP/D reach tumor cells, a large amount of DOX is released to inhibit the proliferation of the cancer cells. This pH-dependent releasing behaviour of the drug delivery system is highly desirable for achieving the tumor-targeted programmable delivery.

Fig. 3 Cumulative release of DOX from NP/D nanoparticles in different pH environments (PBS, pH 5.0 (▲), 6.5 (●), and 7.4 (■)) with time in vitro.

3.3. In vitro cytotoxicity

The NP/D was further investigated to evaluate its potential therapeutic efficacy. Here, the in vitro cytotoxicity of NP/D against HepG2, HeLa and MCF-7 cells was investigated using the MTT assay, where the cells were cultured in suspensions of NP/D and free DOX solutions at a series of equivalent DOX dose for 72 h, respectively. As shown in Fig. 4, the ND–PEG has no cytotoxic effect on cell viability, thus indicating that the carrier exhibits good biocompatibility, while both free DOX and NP/D were found to inhibit HepG2, HeLa and MCF-7 cells, and the relative cell viability declined monotonously with ascending concentration of DOX. When the concentration of DOX was very low (1 μg mL⁻¹), both NP/D and free DOX had similar cytotoxicity effects on both HepG2 and HeLa cells, nevertheless, NP/D obviously exhibits superior cytotoxicity on HepG2 cells than that of free DOX when the concentration increased. For example, the viability of HepG2 cells was 20% for NP/D and 27% for free DOX with 5 μg mL⁻¹ of DOX. Furthermore, it was worthy to note that NP/D with a low concentration exhibited a similar cytotoxicity to free DOX with a high concentration. For instance, the viability of the HepG2 cells was 24% for free DOX with a concentration of 9 μg mL⁻¹, while it was 20% for NP/D with just 5 μg mL⁻¹ of DOX, which indicated that NP/D nanoparticles can significantly improve the drug efficacy of DOX.

Fig. 4 Effect of ND–PEG, free DOX and NP/D on HepG2, HeLa and MCF-7 cell viability for 72 h was measured by the MTT assay. (A) HepG2 cells, (B) HeLa cells and (C) MCF-7 cells. Experiments were repeated three times and data are presented as the mean ± SD (for each group, n = 6).

In addition, the cytotoxicity of both NP/D and free DOX with incubation time was also investigated (Fig. S3†). The result displayed that with prolonged incubation time, the cancer cell viability when treated with ND–PEG had not been significantly affected, which indicated that ND–PEG can be a nanocarrier for cancer therapy. However, the cell viability when treated with NP/D decreased as the incubation time went on and gradually over the free DOX, which suggested that the slow and prolonged cellular uptake of the NP/D may delay the killing of the cancer cells.

3.4. In vitro scratch assay

As DOX significantly influenced the proliferation of cancer cells, we wondered whether the NP/D has the same effect on tumor cell migration, and thus scratch assays were performed in the presence of 5 μg mL⁻¹ of NP/D. As shown in Fig. 5A, the scratch width is evidently narrowed when MCF-7 cells were treated by ND–PEG, even over time, which is similar to the control group, indicating that ND–PEG has almost no affect on cell migration and can be applied as a nanocarrier for drug delivery however the scratch width is nearly essentially unchanged. Meanwhile in MCF-7 cells that were treated with NP/D and DOX, the migration inhibition rate of NP/D and DOX were up to 85% and 86% as shown in Fig. 5B, respectively, which verified our conjecture. Therefore, we learned that the NP/D can inhibit cell migration effectively.

Fig. 5 The scratch assay of ND–PEG, NP/D and free DOX with time. (A) Representative images of MCF-7 cell treatment by ND–PEG, NP/D and free DOX, and untreated MCF-7 cells as controls. (B) The migration inhibiting rate, where the dates were adopted from (A).

3.5. Intracellular accumulation

Although NP/D nanoparticles were found to be active, whether they have entered the cells or remained in the extracellular fluid was still unknown. Moreover, the therapeutic effect of DOX was dependent on its ability to inhibit the synthesis of nucleic acid through intercalation after entrance into the nucleus. Therefore, to study whether the nanoparticles can enter living cells and DOX can dissociate from NP/D nanoparticles, we used laser scanning confocal microscopy with an excitation wavelength of 488 nm and emission wavelength at 560–590 nm. As shown in Fig. 6A, free DOX efficiently entered into the cells and was located in the cell nucleus after treatment for 1 h since free DOX accumulates in the nucleus by simple passive diffusion between the extracellular and intracellular surroundings. In Fig. 6B, the weak green fluorescence intensity is mainly located in the cytoplasm but no signal was seen in the nucleus after incubating for 2 h. When the incubation time was extended to 5 h, the higher green fluorescence intensity is located in the nucleus of MCF-7 cells. Such a result implied that the NP/D moved inside the cells and was capable of ferrying the drug inside living cells efficiently. Then DOX molecules were detached from the NP/D and continued migrating into the nucleus. This would lead to a sustained functional drug release compared to free DOX, which is consistent with the gradual DOX release from NP/D complexes in PBS at 37 °C (Fig. 3). The result was also accordant with the previous report by us,^26,34–36 which indicated the potential applications of NP/D for controlled drug delivery.

Fig. 6 Cellular uptake and distribution of NP/D nanoparticles. (A) Confocal laser scanning microscopy (CLSM) images of MCF-7 cells treated with free DOX for 1 h. (B) Confocal laser scanning microscopy (CLSM) images of MCF-7 cells treated with NP/D for 2 h and 5 h, respectively. The images (A and B) in the left column show the cell nucleus dyed with DAPI for free DOX and Hoechst 33258 for NP/D; the middle column shows DOX fluorescence, and the right column shows the merge of the two previous images. (C) The variations of side scatter (SS) after MCF-7 cells were treated with NP/D for 1 h and 7 h was quantified from a minimum of 10 000 cells by Cell Quest software using flow cytometry analysis, respectively. Y-Axis is side scatter (SS), which indicates intracellular particle complexity; X-axis is forward scatter (FSC).

Due to the fact that side scatter (SS) by flow cytometry analysis can indicate the particle complexities within the cells, the cellular uptake of NP/D can be indirectly demonstrated by SS. Fig. 6C showed that the SS of NP/D uptake by MCF-7 cells after 1 h and 7 h incubation, respectively. After co-culture with NP/D for 1 h, the value of SS only slightly increased, but it greatly increased after incubation for 7 h. The result suggests that the NP/D nanoparticles indeed enter the cells and the cell uptake is a time-dependent process.

To further examine the uptake kinetics of NP/D in cancer cells, MCF-7 cells were treated with 5 μg mL⁻¹ of NP/D for different times and analyzed by flow cytometry. As shown in Fig. S4,† the cellular uptake of NP/D and free DOX was greatly strengthened with the increase in time and the fluorescence intensity reached a plateau at about 7 h for NP/D and 3 h for free DOX. Curve fitting by single exponent function showed that the rate constants (k) of endocytosis for NP/D was about 0.232 h⁻¹, namely, the value of uptake half-life was near 2.99 h, while the value of uptake half-life of free DOX was about 0.93 h, which indicated that the uptake rate of NP/D was approximately three times slower than free DOX. These results are related to cellular uptake pathways, where the uptake of DOX occurs through an energy-independent passive diffusion mechanism,²⁶ while NP/D nanoparticles can efficiently deliver the drug inside living cells via the clathrin-dependent endocytosis pathway (Fig. S5†).

3.6. Cell apoptosis and cell cycle assay

As the above results revealed, NP/D has an efficient antitumor activity, so we further investigated the cell apoptosis behaviour of NP/D. From the qualitative analysis (Fig. S6†), after treating cancer cells with 5 μg mL⁻¹ of NP/D for 48 h and 72 h, the cell morphology was examined under a microscope. In the control group, the cells were flat and adhered to the culture dish with high cell density. However, the cells treated with NP/D became long and slender. In addition, the cells turned into fragments and the cell density decreased. Thus, NP/D inhibited cancer cell proliferation and may have induced cancer cell apoptosis.

Soon afterwards, MCF-7 cells treated with 5 μg mL⁻¹ of NP/D were further double-stained with Annexin V/PI for flow cytometry for quantitative analysis of cell apoptosis (Fig. 7A). In the apoptosis quadrant diagram, each quadrant represents living cells, early apoptotic cells, late apoptotic cells and cell debris in a counter clockwise direction from the bottom left. The results showed that about 33% (the sum of late and early apoptosis) of cells were apoptotic after NP/D treatment and approximately 38% of cells was apoptotic after free DOX treatment. The results again confirmed that DOX could efficiently release from NP/D in cells and resulted in cell apoptosis, and that mainly induced late cell apoptosis.

Fig. 7 Flow cytometry analysis. (A) Apoptosis of MCF-7 cells induced by ND–PEG (b), NP/D (c) and DOX (d) for 48 h with untreated MCF-7 cells as the control (a); the number of apoptotic cells (e), where dates were adopted from (a–d). (B) Cell cycle of MCF-7 cells induced by ND–PEG (b), NP/D (c) and DOX (d), with untreated MCF-7 cells as the control (a); the cell cycle histogram (e), where dates were adopted from (a–d).

From the above results, it can be seen that NP/D apparently induced cell apoptosis and inhibited cell migration. We made a hypothesis that the effect of NP/D knockdown on cell proliferation was regulated by the influence of the cell cycle. We assessed the cell cycle phases in MCF-7 cells by flow cytometry. As shown in Fig. 7B, the cell distribution number after NP/D knockdown was significantly increased in the G2/M phase and concomitantly the cell distribution number in the G0/G1 phase was significantly decreased, while the cell distribution number after free DOX knockdown was also significantly increased in the G2/M phase and concomitantly the cell distribution number in S phase was significantly decreased (Fig. 7B). Therefore, out data indicated that both the inhibition of knockdown NP/D and free DOX on cell proliferation was achieved by arresting the cells in the G2/M phase. In conclusion, the results indicated that NP/D could inhibit the growth and migration of MCF-7 cells and the mechanism was the regulation of cell cycle to arrest the cells in the G2/M phase.

4. Conclusions

In this work, a novel technique was presented for preparing ND–PEG/DOX nanoparticles and achieving almost sixteen fold improvement in DOX loading in comparison to previously reported techniques. The NP/D system exhibited excellent physiological stability and pH controlled drug release with high cytotoxicity compared to free drug. What is more, flow cytometry and confocal microscopy analysis proved that NP/D can easily pass into cells and undergo constant sustained release of DOX to the nucleus, which indicated that the NP/D system has a slow and sustained drug release capability. It was also illustrated that NP/D could markedly inhibit the migration of cells. Finally, we obtained that NP/D could induce cell apoptosis, which is mainly late apoptosis-dependence, and NP/D could change the cell cycle compared to free drug. These studies will provide guidelines for developing smart nanoparticles for drug delivery applications.

Acknowledgements

This work is supported by the National Natural Science Foundation of China (Grant No. 21071091), Shanxi Science and Technology Development Program (Grant No. 20130313021-1).

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Footnote

† Electronic supplementary information (ESI) available: Cell viability with time, cell morphology and cellular uptake kinetics. See DOI: 10.1039/c6ra04141h

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