Novel targeted pH-responsive drug delivery systems based on PEGMA-modified bimetallic Prussian blue analogs for breast cancer chemotherapy

Abstract

The development of novel nanoparticle-based drug delivery systems (nano-DDSs) with high loading capacity, low toxicity, precise targeting, and excellent biocompatibility remains urgent and important for the treatment of breast cancer (BC). Herein, novel BC-targeted nano-DDSs based on bimetallic Prussian blue analogs (PBA-DDSs) for intracellular doxorubicin (DOX) delivery and pH-responsive release were developed. Two kinds of bimetallic PBA, namely CuFe (copper–iron) PBA and CoFe (cobalt–iron) PBA, were synthesized by a coprecipitation method, followed by modification with polyethyleneglycol methacrylate (PEGMA) via surface-initiated atom transfer radical polymerization and immobilization with the AS1411 aptamer to obtain two kinds of novel BC-targeted nano-DDS. CuFePBA@PEGMA@AS1411 and CoFePBA@PEGMA@AS1411 showed high drug loading efficiency of 80% and 84%, respectively, for DOX, while 56.0% and 75.9% DOX release could be achieved under acidic pH conditions. In vitro cell viability and in vivo experiments proved the good biocompatibility of both PBA-DDSs. Cellular uptake and in vivo distribution suggested that both PBA-DDSs had efficient nucleolin-targeting capability, indicating the targeted delivery of DOX in tumor tissues. In vivo evaluation of anti-BC efficacy further confirmed that the obtained PBA-DDSs exhibited excellent therapeutic efficacy with limited side-effects. Therefore, the proposed novel PBA-DDSs can be used as secure and effective drug nano-DDSs for BC chemotherapy.

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1. Introduction

Breast cancer (BC) is the most commonly diagnosed cancer among women and is also the leading cause of female mortality.¹ In Asia, the overall age-standardized BC incidence rates have rapidly increased to approximately 39 per 100 000 women; the major issue in BC epidemiology is the high proportion of women younger than 50 years old.² Traditional treatments for BC, including surgery, radiotherapy, chemotherapy, and hormone therapy, remain unsatisfactory, especially for high-grade, late-stage, or aggressive types of BC, which exhibit a high rate of metastasis, deteriorated relapse, or death.³ Among these treatments, chemotherapy has played an essential role in the treatment of BC, whereas the lack of specificity induces low bioavailability of drug and multiple severe side-effects.⁴ Nanoparticle (NP)-based drug delivery systems (nano-DDSs) with enhanced permeability and retention effect have become a promising therapeutic option for BC.⁵ Several nanomedicine products, such as Doxil® (PEGylated liposomal/doxorubicin (DOX) hydrochloride) and Abraxane® (albumin-bound paclitaxel NP), have been extensively used for BC adjuvant therapy with favorable clinical outcomes.⁶ Several nanomaterials have been commonly used as anti-BC drug carriers; these nanomaterials include polymeric dendrimers,⁷ micelles,⁸ quantum dots,⁹ nanoemulsions,¹⁰ gold NPs,¹¹ carbon nanotubes,¹² metal oxide NPs,¹³ metal–organic frameworks (MOFs),^14,15 and covalent organic frameworks.¹⁶ However, the development of novel nano-DDSs with high loading capacity, low toxicity, precise targeting, multifunction, excellent biocompatibility and stability is still urgent and important.¹⁷

MOFs, an emerging class of hybrid porous nanomaterials originating from metal ions or clusters bridged by organic linkers, have drawn increasing attention in recent years.¹⁸ MOFs exhibit remarkable advantages, such as controllable size, well-defined pore aperture and high porosities, tailorable composition and structure, versatile functionality, large surface areas, high loading capacity, improved biocompatibility and biodegradability, and have become promising candidates as nano-DDSs.¹⁹ Prussian blue (PB), as a subclass of MOFs, is a typical hexacyanoferrate coordination compound approved by U.S. Food and Drug Administration for clinical radioactive exposure treatment due to its excellent biosafety in the human body.²⁰ However, the original NPs of PB (PBNPs) show low specific surface area with poor drug loading capability. Two approaches, including hollow mesoporous^21,22 and surface modifications,²³ are used to improve their surface area and drug loading capacity. Tian et al. developed a periodic mesoporous organosilica-coated PB with high surface area (866 m² g⁻¹) and high drug loading power (260 μg mg⁻¹), showing effective inhibition against BC by inducing the apoptosis and necrosis of tumor cells.²⁴ Among PBs, PB analogs (PBAs) or PB-based derivatives as nano-DDSs with high drug loading capacity have been widely investigated;²⁵ PB-based nanocarriers with tumor-targeting and controllable release capabilities are rarely reported.²⁶ Outfield-responsive nano-DDSs can effectively control drug release in response to inherent (e.g., pH, redox reagents, and enzymes) or external stimuli (e.g., temperature, ionic strength, light irradiation, ultrasound, and magnetic field).²⁷ Hence, PB-based “smart” nanocarriers with high drug loading capacity, active tumor-targeting capability, and stimuli-responsive controllable release are promising candidates of nano-DDSs that can alleviate toxicity of anti-tumor drugs and reduce side effects on normal tissues. Cai et al. reported that Mn PBAs were facilely coated onto the outer surface and inner mesoporous channels of hollow mesoporous PBNPs to prepare HMPB-Mn, which released Mn²⁺ and DOX in a pH-triggering manner.²¹ To make bimetallic MnFe-PBA/DOX which showed high pH-responsive drug release and efficiency in Michigan cancer foundation-7 (MCF-7) cells,²⁸ sodium citrate (SC) was used as a structure modifier and folic acid (FA) as a tumor target. However, bimetallic PBAs still show evident cytotoxicity at high doses, which restricts their bio-related application, especially in vivo anti-cancer therapy. Therefore, good biosafety, low cost, easy synthesis, and environment-friendly PBA alternatives are necessary for the treatment of BC.

The hydrophilic comb-like structure of poly(ethylene glycol)methacrylate (PEGMA) cross-section had been used to enhance the biocompatibility of nanocarriers.²⁹ Lale et al. developed an atom-transfer radical polymerization (ATRP)-based polyPEGMA–poly(caprolactone)–polyPEGMA for targeted delivery of DOX to cancer cells.³⁰ Huang et al. applied ATRP to graft superparamagnetic iron oxide NPs (SPIONs) with poly(glycidyl methacrylate)-co-PEGMA, and FA was conjugated with SPION–P(GMA-co-PEGMA) via “click” chemistry to improve the targeting efficiency and biocompatibility of these NPs.³¹ More importantly, PEGMA is sensitive to pH and commonly prepared by controlling polymerization via surface-initiated ATRP (SI-ATRP). This is one of the most effective ways to introduce polymer layers onto inorganic surface.^32,33 Among various stimuli, pH gradients have been extensively applied to design novel stimuli-responsive delivery systems for anticancer therapies, which are substantially critical due to the intracellular organelles and special acidic extracellular microenvironment of solid tumors.³⁴ Therefore, bimetallic PBAs are modified by PEGMA via surface initiated ATRP (SI-ATRP), they are hopeful to develop novel stimuli-responsive nano-DDSs for BC therapy.

In this study, we developed a BC-targeted nano-DDS based on PBA (PBA-DDS) for DOX intracellular delivery and pH-responsive release. Two kinds of bimetallic PBA (CuFe PBA and CoFe PBA) were modified with PEGMA via SI-ATRP (denoted as CuFePBA@PEGMA and CoFePBA@PEGMA, respectively), followed by immobilization with AS1411 to improve their BC-targeted delivery capability (denoted as CuFePBA@PEGMA@AS1411 and CoFePBA@PEGMA@AS1411). Subsequently, DOX was loaded onto these PBAs (denoted as CuFePBA@PEGMA@AS1411/DOX and CoFePBA@PEGMA@AS1411/DOX). Scheme 1 illustrates the synthesis procedure used in this study. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to measure cell viability of various cell types, such as human BC cell (MCF-7), mouse BC cell (4T1), and normal mouse fibroblast (L929), to investigate the in vitro biocompatibility of the analogs. Tissue sections and ex vivo images of vital organs, including heart, liver, spleen, lung, and kidney, were obtained to detect the in vivo biocompatibility and biodistribution of the analogs. Cell imaging and cellular behavior study were performed by confocal laser scanning microscopy and transmission electron microscopy (TEM). In vivo imaging and tumor sections of 4T1 xenograft tumor model were used to further investigate the efficacy of anti-BC treatment. PBA-DDS exhibited high DOX loading efficiency, good biocompatibility, excellent pH-responsive ability, excellent pH-responsive capability, well-controlled release of DOX in BC cells and thus less DOX-induced side-effects.

Scheme 1 Schematic illustration of the synthetic procedure for core–shell-structured PBA@PEGMA@AS1411-based DOX loading and pH-responsive controlled release system.

2. Experimental section

The S1 section (see the ESI†) lists the parts of chemicals and materials, synthesis and cytotoxicity assays of CuFe PBA, CoFe PBA, CuFePBA@PEGMA and CoFePBA@PEGMA, and their basic characterizations.

2.1 Preparation of CuFe PBA-DDS and CoFe PBA-DDS

CuFePBA@PEGMA and CoFePBA@PEGMA powders (100 mg) were separately dispersed in 100 mL of Milli-Q water to produce a homogenous suspension (1 mg mL⁻¹). The aptamer AS1411 solution (1 mL, 1 μM) was added into CuFePBA@PEGMA or CoFePBA@PEGMA suspensions (4 mL, 1 mg mL⁻¹) below stirring in the darkness for one day at room temperature. The suspension was then dialyzed against DI water for two days at room temperature to clear physically adsorbed or unloaded AS1411 molecules away, the dialysate was replaced every 4 h during this process.

5 mg of CuFePBA@PEGMA@AS1411 and CoFePBA@PEGMA@AS1411 were separately added into the DOX solution (5 mL, 1 mg mL⁻¹) and then stirred for one day in the darkness to achieve adequate adsorption. After, the solutions were washed with deionized water trice after centrifugation to remove physically adsorbed or unloaded DOX.

2.2 In vitro pH-responsive DOX release behavior

The CuFePBA@PEGMA@AS1411/DOX or CoFePBA@PEGMA@AS1411/DOX suspension (5.0 mL, 1 mg mL⁻¹) was put into the dialysis bag (MWCO 3500 Da) against 1× PBS (100 mL) with pH at 5.0 or 7.4. The dialysis bags were placed in the thermostatic shaker with 180 rpm at 37 °C. 3 mL dialysate was then collected at the desired intervals (0.5, 2, 4, 6, 8, 10, 12, 14, 25, 30, 36, 54, 80, 108, and 120 h), followed by adding 3 mL of fresh 1× PBS.

2.3 In vitro anti-BC efficacy assessment

As for in vitro anti-BC efficacy, the MTT assay (see the S1.5 for details†) was performed to detect the cell viability. 1 × 10⁴ cells were typically seeded onto each well of the 96 well-plates overnight. Then, the cells were dealt with distinct densities (5, 10, 20, 40, 80, and 160 μg mL⁻¹) of DOX, CuFePBA@PEGMA@AS1411, CuFePBA@PEGMA@AS1411/DOX, CoFePBA@PEGMA@AS1411 and CoFePBA@PEGMA@AS1411/DOX for 12 hours, respectively. Afterward, the cells were cleaned and further cultured with DMEM for another 12 h. Then, 20 μL MTT solution (5.0 mg mL⁻¹) was then added to per well and cultured for 4 h. Finally, the medium was removed and dimethyl sulfoxide (150 μL) was added into each well. After sufficient shaking, the absorbance of each well was observed at 490 and 630 nm using a microplate reader, and the anti-BC efficacy was calculated accordingly.

2.4 In vitro imaging and intracellular distribution

As for in vitro imaging, cellular uptake of CuFePBA@PEGMA@AS1411/DOX and CoFePBA@PEGMA@AS1411/DOX were observed in MCF-7, 4T1, and L929 cells via CLSM (LSM710, ZEISS, Germany). Briefly, cells were seeded in the glass-bottom dish (1 × 10⁵ cells) and cultured at 5% CO₂ and 37 °C overnight. Then, CuFePBA@PEGMA@AS1411/DOX and CoFePBA@PEGMA@AS1411/DOX (20 μL, 5 mg mL⁻¹) were added and cultured for 2 hours. After washing with PBS thrice, the cells were fixed with paraformaldehyde (4%) at 37 °C for 10 min in dark. Subsequently, the cells were rinsed and incubated with Hoechst 33342 (nucleus-staining, 300 nM) at 37 °C for 15 min. After washing three times, the cells were observed by the CLSM with a 20× objective lens (E_x/E_m of Hoechst 33342: 405/450–480 nm; E_x/E_m of Cy3-AS1411: 515/550–570 nm; E_x/E_m of DOX 488/580–620 nm).

To observe the cellular distribution of CuFePBA@PEGMA@AS1411, CuFePBA@PEGMA@AS1411/DOX, CoFePBA@PEGMA@AS1411 and CoFePBA@PEGMA@AS1411/DOX, the transmission electron microscopy (TEM) was applied. 4T1 cells (1 × 10⁵ cells each well) were seeded in 6-well plates and incubated for one day, then 20 μL of CuFePBA@PEGMA@AS1411, CuFePBA@PEGMA@AS1411/DOX, CoFePBA@PEGMA@AS1411 or CoFePBA@PEGMA@AS1411/DOX (1 mg mL⁻¹) was added into each well and incubated for 30 min. After washing by cold PBS thrice, the fresh cell samples were obtained by trypsin digestion and centrifugation. Subsequently, the samples were first fixed with 2% glutaraldehyde solution overnight at 4 °C and followed by the secondary fixation with 1% osmium tetroxide for 2 hours. The samples were dehydrated in a graded series of ethanol and finally with propylene oxide. Then, the samples were implanted in Epon resin. 70 nm or so slight sections were made ready with the PowerTome-XL ultramicrotome (RMC, USA). Finally, the JEM-2100F high-resolution transmission electron microscope (HR-TEM, JEOL, Japan) were used to observe the sections with a voltage of 120 kV.

2.5 In vivo biodistribution

Healthy female BALB/c mice of 14–16 g body weight were procured from the Experimental Animal Center of Zhengzhou University with free access to food and water in standard animal equipment and authorized by Zhengzhou University's Animal Ethics Committee. After one-week adaptive feeding, mice were injected with CuFePBA@PEGMA@AS1411/DOX and CoFePBA@PEGMA@AS1411/DOX (10.0 mg kg⁻¹) through tail vein. Afterward, mice were sacrificed at various time-points (0, 1, 4, and 24 h post-injection), and the crucial organs (heart, lung, spleen, liver, and kidney) were collected to perform in vitro imaging by adopting the IVIS Lumina III system (PerkinElmer) with an appropriate E_x/E_m of 480/590 nm.

2.6 In vivo imaging

BALB/c nude mice (female, 18 g body weight) were acquired from Hunan Slaccas Jingda Laboratory Animal Co., Ltd (Hunan, China) and housed in the specific pathogen free (SPF) animal facility with free access to food and water. After one-week adaptive feeding, 4T1 cells suspended in PBS (1 × 10⁶ cells per 100 μL per mouse) were subcutaneously injected into the right axillary region. To calculate the volume (V) of tumor, the width (W) and length (L) was measured by electronic vernier caliper. Then, the volume of tumor was estimated according to the formula V = (W² × L)/2. When the tumor volumes reached approximately 100 mm³, the 4T1 tumor-bearing nude mice were randomly divided into 4 groups (n = 4), and were separately injected with 100 μL 0.9% saline, 100 μL DOX (0.5 mg mL⁻¹), 100 μL CuFePBA@PEGMA@AS1411/DOX (0.5 mg mL⁻¹) and 100 μL CoFePBA@PEGMA@AS1411/DOX (0.5 mg mL⁻¹). After treating for 30 min, the mice were imaged at IVIS Lumina III system (PerkinElmer) with an appropriate excitation/emission wavelength of 480/590 nm.

2.7 In vivo anti-BC assessment

Firstly, the animal model of BC was established by subcutaneously injecting 4T1 cells into the right axillary region of healthy female BALB/c mice (procured from Zhengzhou University's Lab Animal Center). The 4T1 tumor-bearing mice were separated to five groups (three mice for each group) when the tumor volumes achieved about 150–200 mm³ randomly, and administered with 200 μL 0.9% saline, 200 μL DOX (0.5 mg mL⁻¹), 200 μL CuFePBA@PEGMA@AS1411/DOX (0.25, 0.5, and 1.0 mg mL⁻¹) via tail vein injection every two days. The body weight and tumor volume were noted every two days until 14 days post-injection. After a survival period of 14 days, the 4T1 xenograft-bearing mice were anesthetized by injecting 10% chloral hydrate (0.1 mL g⁻¹) into abdominal cavity. The tumors and vital organs including hearts, livers, spleens, lungs and kidneys were excised, harvested, fixed in 4% paraformaldehyde for 24 h, dehydrated, embedded in paraffin, sliced at 4 μm for hematoxylin and eosin (H&E) staining, and examined by using CLSM. The similar procedures were performed for CoFePBA@PEGMA@AS1411/DOX. The tumor inhibition rate (TIR) was accessed as TIR (%) = (V_control − V_drug)/V_control × 100%, where V_control and V_drug represented the tumor volumes in control and drug treated groups for 14 days, respectively.

2.8 Live subjects

The animal experiments have obtained the approvals from the Animal Care and Ethics Committee (ACEC) of Zhengzhou University in China. All animal experiments were performed in accordance with the statutory requirements of People's Republic of China (GB14925-2010).

3. Results

3.1 Crystal and chemical structures of CoFe PBA, CuFe PBA, CoFePBA@PEGMA, and CuFePBA@PEGMA

The chemical and crystal structures of CoFe PBA, CuFe PBA, CoFePBA@PEGMA, and CuFePBA@PEGMA were analyzed; their detailed description and discussion are shown in Section S2 (ESI).† The results verified that the crystallinity of PBA nanocubes decreased due to the coverage by the PEGMA layer, in which the chemical structures of PEGMA were determined by the Fourier transform infrared spectrum analysis of CoFePBA@PEGMA and CuFePBA@PEGMA. Moreover, variations in the chemical components and environments of CoFe PBA and CuFe PBA before and after modification by PEGMA were characterized by X-ray photoelectron spectroscopy (XPS). Fig. S2† shows the XPS survey spectra of all samples. The detailed expansions are supplied in Section S2.1 (ESI).† In addition, changes in the chemical environments and valence states of each element containing in CoFe PBA and CoFePBA@PEGMA were probed by their high-resolution XPS spectra. The high-resolution Co 2p, Fe 2p, C 1s, and N 1s XPS spectra of CoFe PBA and CoFePBA@PEGMA were deconvoluted and analyzed by the XPSPEAK41 software (Fig. S3†). As shown in Fig. S3a1,† the Co 2p XPS spectrum of CoFe PBA was separated into two spin–orbit doublets at the binding energies (BEs) of 782 (Co 2p_3/2) and 797.24 eV (Co 2p_1/2) with a difference of 15.2 eV. The average BEs of Co 2p_3/2 were recorded at 781.5 and 782.6 eV for Co²⁺ and Co³⁺, respectively.³⁵ The peaks at 784.1 and 787.7 eV were associated with the shake-up satellite of Co 2p_3/2.³⁶ These findings suggest the mixed chemical valences of Co²⁺ and Co³⁺ in CoFe PBA. As for Fe 2p XPS spectrum, the peaks at the BEs of 710.2 and 723.5 eV were assigned to Fe³⁺ 2p_3/2 and Fe³⁺ 2p_1/2, respectively. The peaks at 715.2, 719.0, and 735.6 eV were satellite peaks of Fe 2p_3/2 and Fe 2p_1/2. Above results indicate that Fe and Co existed in CoFe PBA nanocubes as mixed valances. However, the signals of Co 2p and Fe 2p remarkably decreased in CoFePBA@PEGMA (Fig. S3a2 and b2†). The CoFe PBA nanocubes were totally covered by the PEGMA layer. As such, the chemical components of CoFe PBA cannot be determined by XPS because of the limitation of its test thickness.³⁷ As for the high-resolution C 1s XPS spectrum of CoFe PBA and CoFePBA@PEGMA (Fig. S3c1 and c2,† respectively), four peaks at 284.5 (C–C), 285.0 (C–N), 285.6 (C–O), and 288.3 (N–C=O) eV were fitted out. As displayed in Fig. S3d1 and d2,† the peaks at the BEs of 399.7 and 397.5 eV were ascribed to N–C=O and C≡N, respectively.³⁸ After the modification of PEGMA, the peak area of C–O increased, and that of C≡N decreased, consistent with the external structure of CoFePBA@PEGMA.

The high-resolution XPS spectra for CuFe PBA and CuFePBA@PEGMA were also deconvoluted. As shown in Fig. S4a1 and a2,† the Cu 2p XPS spectrum of CuFe PBA exhibited two main peaks at the BEs of 935.3 and 955.6 eV, which were assigned to Cu 2p_3/2 and Cu 2p_1/2 of Cu²⁺, respectively.³⁹ The BE of 932.6 eV was ascribed to Cu⁺,⁴⁰ and the BE of 935.2 eV was due to Cu(II) species.⁴¹ After modification with PEGMA, a new peak of CuFePBA@PEGMA appeared at 933.7 eV (Fig. S4a2†), and it may be ascribed to the existence of low amount of CuO.⁴² Moreover, two satellite peaks, namely, the “shake-up” peaks, were observed, indicating the existence of Cu²⁺. As for the Fe 2p XPS spectra, two peaks at the BEs of 710 and 723 eV were fitted out in CuFe PBA, corresponding to Fe 2p_3/2 and Fe 2p_1/2, respectively.⁴³ The Fe 2p_3/2 spectrum was deconvoluted into two peaks at the BEs of 708.2 and 709.8 eV, which were attributed to Fe²⁺ and Fe³⁺, respectively. The Fe 2p signal (Fig. S4b2†) was weakened due to the PEGMA layer. Similar analysis results were observed in the N 1s XPS spectra of CuFe PBA and CuFePBA@PEGMA (Fig. S4c1 and c2,† respectively) and those of CoFe PBA and CoFePBA@PEGMA. Additionally, a strong Br 3d XPS signal was obtained with CoFePBA@PEGMA and CuFePBA@PEGMA (Fig. S5†), and this finding resulted from the anchoring of SI-ATRP initiator, that is, 2-bromoisobutyl bromide, which was applied to initiate monomer polymerization over the PBA surface.⁴⁴ Over all, the schemes of chemical structures and synthesis steps for CuFePBA@PEGMA@AS1411 and CoFePBA@PEGMA@AS1411 have shown in Fig. S6.†

3.2 Surface morphologies and nanostructures of CoFe PBA, CuFe PBA, CoFePBA@PEGMA, and CuFePBA@PEGMA

The surface morphologies and nanostructures of CoFe PBA and CoFePBA@PEGMA were characterized by field-emission scanning electron microscopy (SEM) and high-resolution TEM (HR-TEM) (Fig. 1). Fig. 1a shows that CoFe PBA demonstrated a nanocube shape form with the inhomogeneous size (80–120 nm) and coarse surface. The high-magnification SEM image of CoFe PBA (Fig. 1b) revealed that the CoFe PBA nanocube displayed a porous structure, which greatly boosted the adsorption of DOX.⁴⁵ The TEM image of CoFe PBA (Fig. 1c) confirmed the nanocube structure. No clear lattice spacing was observed in the HR-TEM image of CoFe PBA (Fig. 1e), suggesting its low crystallinity.

Fig. 1 (a and b) Low- and high-magnification SEM images and (c–e) TEM and HR-TEM images of CoFe PBA. (f and g) Low- and high-magnification SEM images and (h–j) TEM and HR-TEM images of CoFePBA@PEGMA composite.

In terms of CoFePBA@PEGMA, the CoFe PBA nanocubes were inserted with a polymer layer, resulting in the loss of the nanocube shape (Fig. 1f and g). As shown in its TEM image (Fig. 1h), the CoFePBA@PEGMA composite was dispersed into small NPs with sizes of 5–20 nm (Fig. 1i). The HR-TEM image of the CoFePBA@PEGMA composite (Fig. 1j) showed three types of lattice fringes with spacings of 0.208, 0.228, and 0.253 nm, which were attributed to the (422), (420), and (400) planes of CoFe PBA, respectively.

A nanocube shape was also obtained for CuFe PBA, as observed from its SEM image (Fig. 2a). The nanocube size of CuFe PBA was uneven and ranged from 100–180 nm, as shown in the high-magnification SEM image (Fig. 2b). The TEM image of CuFe PBA (Fig. 2c) also indicated its inhomogeneous nanocube size, and the deep black color further revealed its solid structure (Fig. 2d). No clear lattice fringe was obtained in the HR-TEM of CuFe PBA (Fig. 2e), hinting its low crystallinity. After the modification with PEGMA, the nanocubic particles were accumulated by PEGMA, as indicated in the SEM image of CuFePBA@PEGMA (Fig. 2f and g). The similarly small NPs of CuFePBA@PEGMA were observed in the TEM image (Fig. 2h), also suggesting its excellent dispersion capability in aqueous solutions (Fig. 2i). In the HR-TEM image of CuFePBA@PEGMA (Fig. 2j), clear lattices spacing of 0.251, 0.223, 0.204, and 0.302 nm were contributed by the (400), (420), (422), and (311) planes of Cu₂Fe(CN)₆ nanocube, respectively.⁴⁶

Fig. 2 (a and b) Low- and high-magnification SEM images and (c–e) TEM and HR-TEM images of CuFe PBA. (f and g) Low- and high-magnification SEM images and (h–j) TEM and HR-TEM images of CuFePBA@PEGMA composite.

3.3 DOX loading and in vitro pH-responsive drug release

Herein, DOX was selected as a model drug to explore the drug loading and release of PBA-based nano-DDSs. The DOX-loading efficiency was 33% for both CuFe PBA and CoFe PBA, whereas CuFePBA@PEGMA and CoFePBA@PEGMA exhibited remarkably high DOX-loading efficiencies of 80% and 84%, respectively, after the modification with PEGMA. The results confirmed that the high drug loading capacities of CuFePBA@PEGMA and CoFePBA@PEGMA were attributed to the strong hydrophobic interaction between PEGMA and DOX.⁴⁷ To further investigate the stimuli-responsive drug release behavior of PBA-DDS, we dispersed CuFePBA@PEGMA/DOX, CuFePBA@PEGMA@AS1411/DOX, CoFePBA@PEGMA/DOX, and CoFePBA@PEGMA@AS1411/DOX in 1× phosphate buffered saline (PBS) with different pH. As shown in Fig. 3A and C, CuFe PBA and CoFe PBA barely showed pH-dependent DOX release behavior. However, PEGMA-modified CuFe PBA and CoFe PBA exhibited pH-dependent DOX release behavior. As for CuFePBA@PEGMA@AS1411, the DOX maximum release was achieved at 48 h, with 56% and 23% released at pH 5.0 and 7.4, separately (Fig. 3B). Similar behaviors can be observed for CoFePBA@PEGMA@AS1411 (Fig. 3D). DOX release increased to 75% at pH 5.0. Moreover, the pH-responsive DOX release profiles of CuFePBA@PEGMA and CoFePBA@PEGMA were investigated in PBS solutions at pH of 5.0 and 7.4. A total of 58.2% and 65.8% DOX releases were observed in the original 24 h at pH = 5.0 and leveled off at 48 h with values of 68% and 75%, respectively (Fig. S7†). Thus, PEGMA modification endowed the nano-DDSs with pH-triggered drug-release behaviors due to the increased solubility and surface modification with PEGMA,^48,49 and the modification of targeted aptamer AS1411 showed no influence on the pH-controlled releasing property. Therefore, the pH-responsive PBA-DDSs were favorable for improving the antitumor therapeutic effects.

Fig. 3 The pH-dependent DOX release of (A) CuFePBA/DOX, (B) CuFePBA@PEGMA@AS1411/DOX, (C) CoFePBA/DOX, and (D) CoFePBA@PEGMA@AS1411/DOX.

3.4 In vitro biocompatibility and in vitro anti-BC efficacy

The cell viabilities of the four samples, including CuFe PBA, CuFePBA@PEGMA, CoFe PBA, and CoFePBA@PEGMA, were evaluated by MTT assay to determine their in vitro biocompatibility. The samples of CuFe PBA and CuFePBA@PEGMA exhibited fair cell viability (ca. 70%) toward MCF-7, 4T1, and L929 cells when the concentration was 80 μg mL⁻¹ (Fig. 4A–C), which suggest the good biocompatibility of the designed nano-DDSs. CoFe PBA and CoFePBA@PEGMA exhibited high cell viability (ca. 80%) of MCF-7, 4T1, and L929 cells (Fig. 4D–F, respectively) when the concentration was 160 μg mL⁻¹.

Fig. 4 Cytotoxicity assays. In vitro biocompatibility of CuFe PBA and CuFePBA@PEGMA in MCF-7 (A), 4T1 (B) and L929 cells (C); in vitro biocompatibility of CoFe PBA and CoFePBA@PEGMA in MCF-7 (D), 4T1 (E) and L929 cells (F); in vitro anti-BC efficacy of DOX and CuFePBA@PEGMA@AS1411/DOX in MCF-7 (G), 4T1 (H) and L929 cells (I); in vitro anti-BC efficacy of DOX and CoFePBA@PEGMA@AS1411/DOX in MCF-7 (J), 4T1 (K) and L929 cells (L).

Subsequently, the in vitro anti-BC efficacies of DOX, CuFePBA@PEGMA@AS1411, CuFePBA@PEGMA@AS1411/DOX, CoFePBA@PEGMA@AS1411, and CoFePBA@PEGMA@AS1411/DOX were measured by MTT assay. As shown in Fig. 4G and J, the cell viability of MCF-7 cells treated with DOX, CuFePBA@PEGMA@AS1411/DOX, and CoFePBA@PEGMA@AS1411/DOX significantly decreased to 44.2%, 45.3%, and 48.0% when the concentration was 20 μg mL⁻¹, respectively. Thus, CuFePBA@PEGMA@AS1411/DOX and CoFePBA@PEGMA@AS1411/DOX showed equivalent cytotoxicity as free DOX when the low concentration was 20 μg mL⁻¹. As the concentrations increased, the cell viability further reduced, suggesting a significant anti-tumor efficacy. Similar results can be observed in mouse 4T1 cells (Fig. 4H and K). The cytotoxicity of DOX, CuFePBA@PEGMA@AS1411/DOX, and CoFePBA@PEGMA@AS1411/DOX against normal L929 cells showed remarkably different behaviors (Fig. 4I and L). Free DOX exhibited significant toxicity to L929 cells at the low density of 5 μg mL⁻¹, CuFePBA@PEGMA@AS1411/DOX and CoFePBA@PEGMA@AS1411/DOX presented mild toxicity, and above 60% of L929 cells were alive at the dose of 10 μg mL⁻¹. The cell viability of normal L929 cells was still ca. 70–80% at the highest concentration of CuFePBA@PEGMA or CoFePBA@PEGMA (160 μg mL⁻¹), further indicating their good in vitro biocompatibility. In our previous study, about 30–60% of L929 cells were alive when dealt with a series of MnFe PBAs at high dosage (80 μg mL⁻¹), and the MnFe PBA drug nanocarriers demonstrated greater cytotoxicity to normal L929 cells than to tumor MCF-7 cells.²⁸ These results indicate that the novel PBA-DDS nano-carrier exhibited improved in vitro biocompatibility and good anti-BC efficacy.

3.5 Cellular uptake and intracellular distribution

AS1411 is a synthetic 26-nucleotide phosphodiester oligodeoxynucleotide (i.e., unmodified DNA) with the sequence, 5′-GGTGGTGGTGGTTGTGGTGGTGGTGG, and specifically binds to nucleolin, which is highly expressed on the surface of BC cell membrane.^50–52 To investigate the cellular behavior, Cy3-AS1411 with yellow fluorescence was adopted. As shown in Fig. S8,† the yellow fluorescence intensity in MCF-7 and 4T1 cells treated by CuFePBA@PEGMA@AS1411/DOX and CoFePBA@PEGMA@AS1411/DOX was stronger than that in L929 cells, which indicates that CuFePBA@PEGMA@AS1411/DOX and CoFePBA@PEGMA@AS1411/DOX were internalized by BC cells (MCF-7 and 4T1) via AS1411-mediated cell uptake. The nano-DDSs were mainly distributed in the cytoplasm. To further confirm the AS1411 specifically mediated endocytosis, we analyzed the mean fluorescence intensity (MFI) of the entire cell by selecting three different visual fields containing 10–20 cells in three parallel experiments. The MFI of the entire cell was analyzed in all nine images by using ImageJ software. The results showed that the MFI of AS1411 in MCF-7 cells was significantly higher than that in L929 cells (P < 0.05), and no significant difference was observed between MFI of AS1411 in 4T1 cells and that in L929 cells (Fig. S9†). Liao et al. reported that AS1411-functionalized NPs remarkably promoted the affinity and binding to nucleolin receptors, enhancing the subsequent intracellular uptake and trafficking.⁵³ The underlying mechanism of internalization of AS1411-functionalized NPs can be related to micropinocytosis in a nucleolin expression-dependent manner.⁵⁴ Furthermore, the cellular uptake behaviors of CuFePBA@PEGMA@AS1411/DOX and CoFePBA@PEGMA@AS1411/DOX were investigated at pH = 6.0 to simulate the tumor acid microenvironment (Fig. 5). The MFI analysis of DOX exhibited that the MFI of nano-DDS in MCF-7 and 4T1 cells was significantly higher than that in L929 cells (P < 0.05). Thus, more DOX can be released in MCF-7 and 4T1 cells (Fig. S10†).

Fig. 5 Cellular uptake behaviors of CuFePBA@PEGMA@AS1411/DOX in L929 (A), MCF-7 (B), and 4T1 cells (C) and CoFePBA@PEGMA@AS1411/DOX in L929 (D), MCF-7 (E), and 4T1 cells (F) at pH = 6.0. (The scale bar is 40 μm.)

For further investigation of the intracellular distribution of the designed nano-DDSs, the cryo-TEM technique was used to observe the cellular ultrastructure. CuFePBA@PEGMA@AS1411, CuFePBA@PEGMA@AS1411/DOX, CoFePBA@PEGMA@AS1411, and CoFePBA@PEGMA@AS1411/DOX were mainly distributed within the endosomes and lysosomes, further confirming the AS1411-mediated cellular endocytosis (Fig. 6). Moreover, endocytic structural collapse was observed in the 4T1 cells treated by CuFePBA@PEGMA@AS1411/DOX and CoFePBA@PEGMA@AS1411/DOX, hinting the intracellular drug release and anti-tumor effect. The difficulty of controlling drug release at an intracellular level continues to be a key challenge for improving the efficacy and safety of anti-tumor therapy.⁵⁵ The current results suggest that the designed CuFePBA@PEGMA@AS1411/DOX and CoFePBA@PEGMA@AS1411/DOX have great potential as controlled intracellular drug-release nano-carriers for BC nano-therapy.

Fig. 6 Intracellular distribution of CuFePBA@PEGMA@AS1411 (A–D), CuFePBA@PEGMA@AS1411/DOX (E–H), CoFePBA@PEGMA@AS1411 (I–L) and CoFePBA@PEGMA@AS1411/DOX (M–P) in 4T1 cells. Arrows indicated that internalized CuFePBA@PEGMA@AS1411 (red arrows), CuFePBA@PEGMA@AS1411/DOX (blue arrows), CoFePBA@PEGMA@AS1411 (yellow arrows), CoFePBA@PEGMA@AS1411/DOX (green arrows) resided within endosomes and lysosomes. Blue circle indicated the structural changes of CuFePBA@PEGMA@AS1411/DOX and yellow circle indicated the apoptotic body.

3.6 In vivo biodistribution

To directly observe the in vivo biodistribution of CuFePBA@PEGMA@AS1411/DOX and CoFePBA@PEGMA@AS1411/DOX, healthy mice were used as the model. The in vivo fluorescence images of crucial organs (heart, lung, spleen, liver, and kidney) were collected after intravenous injection for different durations (Fig. 7). As shown in Fig. 7A–E, CuFePBA@PEGMA@AS1411 was mostly distributed in the kidney and liver 1 h post-injection. A small part of CuFePBA@PEGMA@AS1411 was distributed in the lung. At 4 h post-injection, CuFePBA@PEGMA@AS1411 only resided within kidney and liver. After 24 h, almost no fluorescence was observed, indicating that CuFePBA@PEGMA@AS1411 was metabolized within 24 h. The renal clearable NPs can accelerate body clearance of an off-target drug and reduce its side effects during anti-tumor therapy.⁵⁶ As shown in Fig. 7F–J, CuFePBA@PEGMA@AS1411/DOX was mostly dispersed in the kidney, liver, and lung post-injection. A small part of CuFePBA@PEGMA@AS1411/DOX was distributed in the heart. After 24 h post-injection, the fluorescence signals decreased in the kidney, liver, and lung, which might indicate the possible metallization by the immune response of the mice. Compared with CuFePBA@PEGMA@AS1411/DOX, CoFePBA@PEGMA@AS1411/DOX showed a similar in vivo biodistribution. After 24 h, the CoFePBA@PEGMA@AS1411 was still distributed in the liver and kidney, indicating a low excretion rate (Fig. 7O).

Fig. 7 In vivo biodistribution of CuFePBA@PEGMA@AS1411 (A–E), CuFePBA@PEGMA@AS1411/DOX (F–J), CoFePBA@PEGMA@AS1411 (K–O), and CoFePBA@PEGMA@AS1411/DOX (P–T).

3.7 In vivo anti-BC assessment

The in vivo anti-BC efficacy of CuFePBA@PEGMA@AS1411/DOX and CoFePBA@PEGMA@AS1411/DOX was assessed by using 4T1 tumor-bearing mice. As shown in Fig. 8A, 1.0 mg mL⁻¹ CuFePBA@PEGMA@AS1411/DOX-treated mice showed the smallest tumor size compared with the control and other treatment groups. Furthermore, the tumor volumes of mice were monitored during the whole treatment periods (Fig. S11A and C†). The tumor volume of mice treated with 0.9% saline reached almost 1239.3 ± 61.9 mm³ after 15 days, whereas that of mice treated with 0.5 mg mL⁻¹ free DOX decreased to 638.0 ± 31.9 mm³. The tumor volumes of mice treated with 0.5 and 1.0 mg mL⁻¹ CuFePBA@PEGMA@AS1411/DOX and CoFePBA@PEGMA@AS1411/DOX were approximately 450 and 380 mm³, respectively, which are lower than that of mice treated with 0.5 mg mL⁻¹ free DOX (P < 0.05), indicating the improved anti-BC efficacy. Mice in the DOX-treated group showed a significant decrease in body weight, whereas mice in the 0.9% saline and CuFePBA@PEGMA@AS1411/DOX-treated groups showed almost no decrease in body weight (P < 0.05) (Fig. S11B and D†). Similar results were observed in the CoFePBA@PEGMA@AS1411/DOX-treated groups (Fig. 8B). The tumor inhibition rates of CuFePBA@PEGMA@AS1411/DOX and CoFePBA@PEGMA@AS1411/DOX were calculated and reached approximately 70.1% and 70.5% of the concentration of 1.0 mg mL⁻¹, respectively, which were remarkably higher than that of free DOX (P < 0.05, Fig. S12†). Thus, CuFePBA@PEGMA@AS1411/DOX and CoFePBA@PEGMA@AS1411/DOX exhibited excellent anti-BC efficacy and limited side effects. The tumor accumulation effects were investigated by in vivo fluorescence imaging of 4T1 tumor-bearing BALB/c mice treated by CuFePBA@PEGMA@AS1411/DOX and CoFePBA@PEGMA@AS1411/DOX for 30 min. As shown in Fig. 8C–E, the photon counts at the region of interest (ROI) within the 4T1 tumor-bearing nude mice treated with 0.9% saline, 0.5 mg mL⁻¹ CuFePBA@PEGMA@AS1411/DOX, and 0.5% DOX were 2.640 × 10⁴, 1.262 × 10⁵, and 7.193 × 10⁴, respectively. Meanwhile, the photon counts at the ROI within 4T1 tumor-bearing nude mice treated with 0.9% saline, 0.5 mg mL⁻¹ CuFePBA@PEGMA@AS1411/DOX, and 0.5% DOX were 4.888 × 10⁴, 2.116 × 10⁵, and 9.426 × 10⁴, respectively (Fig. 8F–H). These results show that the designed nano-DDSs accumulated in the tumor tissue, and this result can be ascribed to the AS1411-mediated recognition of DDSs and tumor cells, confirming their BC-targeting effect. Furthermore, the histological analysis of BC tumor tissues was performed in different groups (Fig. S13†). Compared with the other treatment groups, tumor cell proliferation was significantly inhibited by 1.0 mg mL⁻¹ CuFePBA@PEGMA@AS1411/DOX, and tumor cells were sparsely dispersed.⁵⁷

Fig. 8 In vivo anti-BC efficacy of CuFePBA@PEGMA@AS1411/DOX and CoFePBA@PEGMA@AS1411/DOX in 4T1 tumor-bearing mice. (A and B) Photographs of tumor tissues; (C–E) in vivo imaging of 4T1 tumor-bearing mice treated with 0.9% saline, 0.5 mg mL⁻¹ CuFePBA@PEGMA@AS1411/DOX, and 0.5 mg mL⁻¹ DOX; (F–H) in vivo imaging of 4T1 tumor-bearing mice treated with 0.9% saline, 0.5 mg mL⁻¹ CoFePBA@PEGMA@AS1411/DOX (H) and 0.5 mg mL⁻¹ DOX.

Subsequently, the histological examinations of crucial organs (heart, lung, spleen, liver, and kidney) from mice with different treatments were performed. Free DOX treatment caused typical histopathological changes, such as vacuolar degeneration (hepatic lesions). DOX-induced cardiotoxicity (small vacuoles in myocytes) was also observed in the DOX-treated mice (Fig. 9G). Similarly, silk sericin-based DOX-loaded NPs can mitigate DOX-induced cardiotoxicity and hepatotoxicity.⁵⁸ However, no significant histopathologic change was recorded in mice treated with CuFePBA@PEGMA@AS1411/DOX and CoFePBA@PEGMA@AS1411/DOX. Fig. S14† shows the histological examination of vital organs from healthy mice as the negative control. These results further suggest that CuFePBA@PEGMA@AS1411/DOX and CoFePBA@PEGMA@AS1411/DOX possess good biocompatibility and limited side effects.

Fig. 9 Histological examination of vital organs from mice treated with (A–E) 0.9% saline, (F–J) 0.5 mg mL⁻¹ DOX, (K–O) 0.5 mg mL⁻¹ CuFePBA@PEGMA@AS1411/DOX, and (P–T) 0.5 mg mL⁻¹ CoFePBA@PEGMA@AS1411/DOX. Black arrows indicated vacuolar degeneration, and red arrows indicated small vacuoles in myocytes. Scale bar = 100 μm.

4. Discussion

BC is the most common malignancy among women.⁵⁹ It is currently treated by conventional chemotherapy, but the toxic side effects are high and therefore more optimal treatment modalities are needed.⁶⁰ Nanotechnology has developed rapidly in recent years and provides an excellent means to overcome the bottleneck of conventional therapies that cause non-specific effects on body tissues, improve the effectiveness of drug and radiation therapy, and greatly reduce adverse effects.^61–63 These materials can be used as drug carriers, imaging contrast agents, photothermal materials, photoacoustic compounds and radiation dose enhancers.^64,65

Recently, with the increased understanding of acidic TME, it has been recognized as a new target for tumor diagnosis and treatment, which has important implications for the design of pH-responsive nanomedicines.⁶⁶ Gong et al. developed a pH-triggered carbon dioxide gas-generating nanoplatform based on metformin to promote T cell-mediated immune responses and enhance antitumor effects.⁶⁷ Shen et al. developed a robust nanoplatform made of a TME pH-responsive polymer (MEO-PEG-BPPMEMa) and a cationic lipid compound (G0–C14) that significantly enhanced the cytoplasmic uptake and subsequent intracellular escape of saponin from tumor cells, resulting in effective inhibition of tumor growth.⁶⁸ In this study, PEGMA modified CuFe-PBA and CoFe-PBA by SI-ATRP to impart good biocompatibility and excellent pH responsiveness to the nano-DDS. The designed nano-DDS showed up to 80% and 84% DOX loading efficiency, releasing about 56.0% and 75.9% DOX under acidic conditions (Fig. 3). In summary, we designed two nano-platforms with extraordinary pH responsiveness.

Aptamers are synthetically designed oligonucleotides that have been screened to bind specific targets. The process of developing aptamers is known as SELEX (systematic evolution of ligands by exponential enrichment).^69,70 Aptamers have recently been used as biomarkers to identify tumor markers and mobile tumor cells, prognostic monitoring and tumor therapy.⁷¹ The AS1411 aptamer was shown to bind the Bcl-2 mRNA attachment protein nucleolin, which was evaluated to stimulate cytotoxicity and instability of the Bcl-2 gene in MDA-MB-231 and MCF-7 breast tumors, thereby blocking nucleolin attachment to the Bcl-2 gene and ultimately triggering the apoptotic pathway.⁷² Liu et al. designed an AS1411–AuNSs that exhibited high durability in both serum and solution and was simply internalized by target cells at higher doses and resulted in higher anti-cytotoxic/proliferative effects compared to pure AS1411 aptamers or AuNSs.⁷³ In addition, in vivo injection of AS1411–AuNSs significantly inhibited the growth of xenograft tumors in mice without any toxic side effects.⁷⁴ Taken together, AS1411–AuNSs are suitable therapeutic candidates for clinical application in the treatment of cancer in the breast. In this study, AS1411 was immobilized in the novel PBA-DDS improving its BC targeting ability and specific uptake by BC in vitro and in vivo (Fig. 5 and 6). In addition, the novel PBA-DDS has better fluorescence, allowing the in vivo distribution of the drug to be clearly seen (Fig. 7). The novel PBA-DDS nanocarriers greatly reduce the side effects caused by DOX. It exhibits extraordinary ex vivo biocompatibility (Fig. 9) and anti-BC efficacy (Fig. 4 and 8).

Much progress has been made in the development of smart NPs for payload of anti-BC drugs, specific enrichment of tumor cells and tissues, and controlled release and efficient delivery of drugs. However, there are limitations in subcellular targeting that require continuous efforts to overcome the main obstacles, namely the design of nanocarriers based on common pathological disease states, less consideration of tumor heterogeneity, and endocytic pathways of the same nanocarriers that limit their clinical translation. We believe that through the continuous efforts of researchers, by continuously optimizing nanoparticle properties and combining different drugs, the generated novel nanoparticles can definitely bring benefits to clinical cancer patients.

5. Conclusion

In summary, novel BC-targeted nano-DDSs based on bimetallic PBAs (CuFePBA@PEGMA@AS1411/DOX and CoFePBA@PEGMA@AS1411/DOX) were developed for intracellular DOX delivery and pH-responsive drug release. PEGMA was modified against CuFe PBA and CoFe PBA via SI-ATRP to endow the nano-DDSs with good biocompatibility and excellent pH response capability. The designed nano-DDSs showed as high as 80% and 84% DOX-loading efficiency, and in acidic condition, approximately 56.0% and 75.9% DOX can be released. The in vivo distribution results indicated that the nano-DDSs can be slowly metabolized out of the body. The in vivo and in vitro experiments showed that the novel nano-DDSs exhibited good biocompatibility, excellent tumor-targeting capability, and significant anti-BC efficacy without severe side effects. Therefore, the designed bimetallic PBA-based nano-DDSs can be secure and effective drug nano-carriers for BC chemotherapy.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by Medical Science and Technology Research Plan Project (SB201904005). We thank for the help of Prof. Zhihong Zhang from Zhengzhou University of Light Industry to nanoparticle synthesis.

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Footnotes

† Electronic supplementary information (ESI) available: Experimental section, basic characterizations of all samples, cellular uptake behaviors, MFI analysis, tumor inhibition rates and HE staining evaluation. See DOI: https://doi.org/10.1039/d2ra06631a

‡ These authors contributed equally to this work.

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