In vitro evaluation of a novel pH sensitive drug delivery system based cockle shell-derived aragonite nanoparticles against osteosarcoma

ABSTRACT Background: Osteosarcoma (OS) is a highly malignant primary bone cancer. Severe side effects and multidrug resistance are obstacles faced with chemotherapy against OS. With the hope to overcome the obstacles of the conventional chemotherapy, various targeted drug delivery systems using nanotechnology have been explored in the past few decades. Biogenic calcium carbonate (CaCO3) has great potential to be a smart drug delivery system. Results: In this study, cockle shells-derived aragonite nanoparticles (ANPs) were developed and loaded with doxorubicin (DOX). The physicochemical properties of the DOX-loaded ANPs (DOX-ANPs) were characterised by various techniques. The results of drug-loading study demonstrated that DOX was loaded onto ANPs at high loading and encapsulation efficiency (11.09% and 99.58%, respectively). The pH-sensitive release of DOX from DOX-ANPs was successful. At lower pH values (4.8), the release of DOX was much quicker than that at pH 7.4. Additionally, cellular uptake study using fluorescence microscopy showed obviously cellular uptake of DOX-ANPs through endocytosis. Moreover, the flow cytometric analysis revealed DOX-ANPs-induced cell cycle arrest, which was consistent with the mechanism of DOX. DOX-ANPs also showed an efficient cytotoxicity against OS cancer cells, close to the toxicity effect of free DOX at the same concentration. Morphological observations showed microvilli disappearance, chromatin condensation, cell shrinkage, membrane blebbing, and formation of apoptotic bodies, which confirmed both DOX-ANPs- and DOX-induced apoptosis of OS cancer cells in vitro. Conclusion: Our findings indicated that ANPs could act as a pH-sensitive drug delivery against OS.


Introduction
Osteosarcoma (OS) is a highly malignant primary bone cancer and most prevalent in children and young adults [1]. The long-term survival rate for patients with localised OS has improved to approximately 70% due to the clinical application of multiagent chemotherapy combined with surgery [2], while the outcome for patients with metastasis or relapsed OS is much worse with survival rate less than 20% [3][4][5]. Chemotherapy plays a major role in the treatment against OS. However, the chemotherapy is associated with severe side effects due to nonspecific tissue biodistribution, such as myelosuppression, alopecia, and cardiac failure [2]. In addition, another major clinical obstacle in the chemotherapy against OS is the development of multidrug resistance [6]. Consequently, there is an urgent requirement for improving the antitumour efficacy and minimising the adverse effects.
With the hope to overcome the obstacles of the conventional chemotherapy, various targeted drug delivery systems using nanotechnology have been explored in the past few decades [7][8][9]. These systems can advantageously enhance drug solubility, extend clearance time, offer higher selectivity targeting the tumour microenvironment and enhance accumulation in tumour tissues due to the enhanced permeability and retention (EPR) effect. In addition, drug delivery system can be designed to release the drug responding to a particular stimulus, such as temperature, magnetism, ultrasound intensity and pH [10,11]. Among these stimuli, pH-sensitive delivery is regarded as one of the most common strategies against cancer due to the considerable pH differences existing between normal physiological environment and acidic microenvironment of tumour or lysosomes [12][13][14][15]. Nanoparticles are taken up by the cells via endocytosis; consequently, the anticancer drug delivered by nanoparticles can bypass the efflux pumps localised in the cytoplasmic membrane, which leads to overcome multidrug resistance [8].
Compared to other nanoparticles, biogenic calcium carbonate (CaCO 3 ) has great potential to be a smart drug delivery system. Due to its biocompatibility, slow biodegradability, bioresorbability, pH-sensitive property and osteoconductivity [16,17], CaCO 3 is much more suitable to be controlled release systems for anticancer drugs against OS [18][19][20][21]. CaCO 3 has three polymorphs: calcite, aragonite and vaterite. Aragonite has osteoregenerative potential, and can be resolved and replaced by bone [22]. Aragonite form exists naturally in almost all mollusk shells. Cockle shell (Anadara granosa) can be used as a rich source of biogenic aragonite [23]. Cockle shell is the most abundant sea species cultured in Malaysia [24]. The production of cockle in Malaysia is great and keeps increasing year by year. However, the shells are treated as waste with an unpleasant smell, and are often just dumped in landfill or the sea.
In this study, waste cockle shells were used for the synthesis of aragonite nanoparticles (ANPs) through a top-down approach. Doxorubicin (DOX) as one of the most effective anticancer drugs against OS was loaded onto the ANPs. The DOX-loaded ANPs (DOX-ANPs) were characterised for morphology and chemical properties. We evaluated the drug loading efficiency and the drug release behaviours. Additionally, the G2/M cell cycle arrest and apoptosis induction of DOX-ANPs against OS cancer cells were also observed. The purpose of this study was to substantiate the potential of ANPs in the development of an efficient controlled drug release delivery system against OS.

Preparation of DOX-ANPs
ANPs were synthesised using a top-down method, as reported earlier [25] with some modifications. Briefly, cockle shells were washed thoroughly to remove dirt and dried at 50 C in an oven (Memmert UM500, Germany). The shells were finely grounded and sieved to obtain micron-sized powders. After that, 5 g micron-sized powders were mixed with 50 ml deionised water and stirred at 1300 rpm at 70 C for 60 min using a magnetic stirrer (Wise Stir SMHS-6, Germany). 1 ml of BS-12 was added into the mixture and vigorously stirred at 1000 rpm for 110 min at room temperature. The final ANPs suspension was filtered and dried in the oven at 80 C for 2 days. 50 mg ANPs were suspended in 10 ml double distilled water. 1 ml DOX solution (2, 4, 6 mg/ml) was added into the ANPs suspension and stirred gently overnight in a dark environment at room temperature. The DOX-ANPs were collected by centrifugation, washed and oven-dried at 50 C, and packed in a poly-ethylene plastic bag for subsequent experiments.

Drug-loading study
After incubation of DOX with ANPs overnight, the particles were centrifuged. The concentration of non-encapsulated DOX in the supernatant was assayed by reading the absorbance at 485 nm with a microplate reader (TECAN Safire, Austria). A standard calibration curve of the DOX concentrations was prepared (R 2 D 0.9993). Drug loading content and loading efficiency were calculated using Equations (1) and (2), respectively.
Loading efficiency ð%Þ ¼ where Wt is the total weight of DOX used, Wf is the weight of free DOX, and Wnp is the weight of the ANPs used.

Morphology and surface properties
Transmission electron microscopy (TEM) (Hitachi H-7100, Japan) measurements were carried out to investigate the shape and size distribution of ANPs and DOX-ANPs. The surface morphology of ANPs and DOX-ANPs were observed by field-emission scanning electron microscope (FESEM) (JEOL JSM-7600F, USA). FESEM measurements were operated at 5 kV. The zeta potential was measured using Malvern Zetasizer Nano (Malvern Instruments, UK). The freshly prepared suspension of nanoparticles was diluted with deionised water. Three measurements were made for each sample, and the mean § standard deviation was reported.

Crystallinity and chemical properties
The purity and crystallinity of blank ANPs, DOX-ANPs and free DOX were investigated with an X-ray powder diffractometer (XRD) (APD 2000, Italy) using CuKa (λ D 1.5406 A ). The samples were determined using diffraction angles from 2 to 80 . The chemical analyses were carried out using a Fourier transform infrared spectrometer (FTIR) (Nicolet 6700, Thermo Nicolet, USA) in the range of 4000 to 400 cm ¡1 .

In vitro drug release study
To evaluate the drug release profile, 6 mg DOX-ANPs was suspended in 3 ml of phosphate buffered saline (PBS) solution. Two different PBS solutions with pH values of 4.8 and 7.4 were used. The accumulated concentrations of DOX released into the PBS solution was determined at preset time intervals by automatic measuring the absorbance at 485 nm using the kinetics program of an ultraviolet-visible spectrophotometer (Lambda 35, PerkinElmer, USA). Each experiment was performed three times.

Cell culture
Rat OS cell line UMR-106 and human fetal osteoblastic cell line hFOB 1.19 were chosen for this study. UMR-106 cells were cultured in DMEM with 10% FBS and 100 mg/ml each of penicillin and streptomycin at 37 C in a 5% CO 2 incubator (Thermo Fisher Scientific, USA). The complete culture medium for hFOB 1.19 was DMEM-Ham's F-12 (1:1) with 10% FBS and 0.3 mg/ml G418. The hFOB 1.19 cells were cultured at 34 C in a 5% CO 2 incubator.

Cell viability assay
The cells were seeded in 96-well plates (hFOB 1.19 at 1 £ 10 4 cells/well; UMR-106 at 0.5 £ 10 4 cells/ well), and incubated for 24 h. For the biocompatibility assay of blank ANPs, hFOB 1.19 cells and UMR-106 cells were co-cultured with different concentrations of ANPs for 24 h. For the cytotoxicity assay of DOX-ANPs, UMR-106 cells were treated with different concentrations of DOX-ANPs and DOX for 24, 48 and 72 h. The nanoparticles were sterilised with UV irradiation for 30 min before use. Untreated cells acted as control. For each condition, three replicates were performed. Six wells without cell seeded were left as blank control. After the exposure was completed, the cells were washed with PBS. Subsequently, MTT reagent (5 mg/ml) was added and incubated at 37 C for 4 h. The medium was discarded, and 150 ml DMSO was added to dissolve the dark blue formazan crystals. The absorbance of 570 nm was measured using a microplate reader. Each assay was performed in triplicates. IC 50 values indicating the drug concentration that inhibits 50% of the tested cells were calculated by the probit analysis using SPSS software. The cell viability was calculated using the following equation: where OD treated is the absorbance of the cells incubated with different treatments, OD control is the absorbance of the cells incubated with the culture medium only, and OD blank is the absorbance of the medium in plate wells without cell seeded.

Cellular uptake assay
The cellular uptake of the DOX and DOX-ANPs was determined by fluorescence microscopy using intrinsic fluorescence of DOX. The UMR-106 cells were seeded onto cover slips placed in six-well plates with a density of 5 £ 10 5 cells per well. After 24 h of incubation, the culture medium was removed and replaced with fresh culture medium, including DOX and DOX-ANPs with an equivalent concentration of DOX at 0.5 mg/ml for 0.5, 5 and 24 h at 37 C. Thirty minutes before the incubation ended, Lyso-ID green dye was added into the culture medium to label the endosomes and lysosomes. Subsequently, the cells were washed and fixed with 4% paraformaldehyde for 30 min. The fixed cells were rinsed with PBS and mounted on microscope slides. The fluorescent distribution was visualised under a fluorescent microscope (Nikon ECLIPSE Ti S, Japan).

Cell apoptosis analysis
Cell apoptosis was evaluated using a FITC Annexin V apoptosis detection kit (BD Pharmingen, USA). Briefly, following each treatment, the floating and adherent cells were harvested by centrifugation. The cells were suspended with 100 ml of 1£ binding buffer. Next, 5 ml of Annexin V-FITC and 5 ml of PI solution were added and incubated for 30 min on ice in the dark. Finally, 400 ml of 1£ binding buffer was added. All the samples were analysed with flow cytometer (BD Bioscience, USA).

Morphological examination
UMR-106 cells were treated with DOX-ANPs and DOX for 48 h. The cellular morphology and characteristics of apoptosis were directly observed and the images were captured using an inverted light microscope (Olympus, Japan). For TEM samples preparation, after incubation for 48 h, cells were fixed with 4% glutaraldehyde for 24 h at 4 C and postfixed with 1% osmium tetraoxide at 4 C for 2 h. After each fixing, cells were washed three times with 0.1 M sodium cacodylate buffer. Dehydration of the cells was carried out with increasing concentrations of acetone. The cells were then infiltrated with resin and embedded with 100% resin in beam capsule. Fine sections were prepared and stained with uranyl acetate. The cell ultra-structure was observed using TEM (Hitachi H-7100, Japan). For SEM samples preparation, after the cells dehydration with acetone, cells were then critical point-dried from CO 2 by the critical point drier (CPD 030, Bal-TEC, Switzerland) for 30 min. The dried cells were coated in gold using an SEM coating unit (E5100 Polaron, UK). The samples were observed using SEM (JOEL 64000, Japan).

Statistical analyses
All statistical analyses were performed using SPSS software (IBM SPSS Statistics version 23, USA). All experiments were performed at least three times. The results were expressed as mean § standard deviation. Comparisons between groups were determined using oneway analysis of variance (ANOVA) with post hoc group comparison of Duncan's multiple range test. A value of p < 0.05 was regarded to be significant unless indicated otherwise.

Morphology and surface characterisation
The morphologies of the ANPs before and after DOX loading were observed by TEM and FESEM (Figures 1 and 2). As shown in Figure 1, the blank ANPs exhibited a uniform spherical shape, with a distributed size of 20-60 nm ( Table 1). The images also indicated a porous nature for the synthesised ANPs (Figure 1(A)), which was suitable for drug loaded onto the nanoparticles by physical adsorption. After the DOX encapsulation, the morphological structure of the nanoparticles did not show obvious changes; only the size distribution slightly increased ( Figure 2).

Zeta potential measurements
Zeta potential, as one of the surface properties, is generally used to predict the stability of colloidal dispersions. The value of zeta potential for good stability is above §30 mV [26]. High zeta potential prevents aggregation of the particles due to electric repulsion. As shown in Table 1, the pure ANPs showed a negative zeta potential of ¡46.17 § 3.82 mV, while a lower negative value of ¡40.57 § 3.80 was recorded for DOX-ANPs ( Figure 3). The difference between the blank ANPs and DOX-ANPs can be attributed to the additional positive charge of DOX, which indicated that DOX had been loaded onto the ANPs    successfully. Due to the electrostatic interaction, positively charged DOX can be loaded onto the ANPs easily. Therefore, ANPs synthesised in this study can be a suitable drug delivery system for DOX.

DOX loading content and loading efficiency
The drug loading content and loading efficiency are important factors for evaluating the usability of ANPs as a drug delivery system. The loading of DOX on ANPs was achieved at different ANPs:DOX ratios. As shown in Table 2, with the increase in the feeding concentration of DOX (2, 4 and 6 mg), high loading content was observed (3.98 § 0.02%, 7.93 § 0.02% and 11.09 § 0.72%), while the loading efficiencies of the three formulations were all above 99%. This demonstrated minimal loss of the drug during the loading process. The successful loading of positively charged DOX on ANPs can be ascribed to the negative surface charge of ANPs, which promoted an electrostatic interaction. DOX-ANPs [3] got the highest loading content (11.09 § 0.72) and selected for subsequent works in this study.

FTIR analysis
The FTIR spectra of ANPs, DOX and DOX-ANPs were presented in Figure 4. The characteristic aragonite spectrum was shown at 1478, 1082, 860 and 712 cm ¡1 , respectively [27] ( Figure 4(a)). The spectra of ANPs are typically descriptive of the aragonite polymorph of CaCO 3 . The FTIR spectrum of DOX is shown in Figure 4(c). DOX bears functional groups, such as hydroxide (OH ¡ ) and amino (-NH 2 ). The broad vibrational band centred around 3430 cm ¡1 in the spectrum of DOX-ANPs (Figure 4(b)) is strong OH ¡ stretching vibration of DOX and/or absorbed water. The absorption peak at 2931 cm ¡1 related to the -NH 2 group of DOX was found overlapped with the broad band of OH ¡ stretching vibration [28]. The existence of DOX-typical peaks indicated the effective loading of the drug into the ANPs. Lack of any important shifting in the characteristic bands of aragonite in the spectrum of DOX-ANPs evidenced that the aragonite phase did not change during the drug loading process.

XRD analysis
The XRD patterns of DOX, ANPs and DOX-ANPs are shown in Figure 5. The XRD pattern of ANPs ( Figure 5    DOX-ANPs; the presence of these elements were undoubtedly from the DOX (C 27 H 29 NO 11 ), and thus the XRD analysis was a qualitative determination that DOX was loaded onto the ANPs.

3.6.
In vitro DOX release profile pH 4.8 and 7.4 were selected to simulate the weakly acidic microenvironment of tumour and normal physiological environment, respectively. A small amount (36%) of DOX released in PBS solution (pH 7.4) at the first 120 min. With time increasing, approximately 80% of DOX was released slowly and sustainedly within 1740 min. When DOX-ANPs were exposed to an acidic PBS buffer (pH 4.8), approximately 72% of DOX was released rapidly within 60 min, and almost 90% was released within 540 min ( Figure 6). This result was similar to those reported by other researchers [19,29]. The result indicated that DOX was hardly released from ANPs in the blood or normal tissues, which could minimise the side effects of anticancer drug to normal tissues, while DOX release could be triggered responsively in the acidic environment of tumour tissues. This feature makes ANPs an excellent pH-responsive carrier which has the potential to precisely release drugs into tumour tissues and reduce the side effects to normal tissues.

Biocompatibility assay of blank ANPs
A normal osteoblast cell line and an OS cancer cell line were chosen for this biocompatibility study. As shown in Figure 7, the viability percentage was higher than 80%, even at high concentration of 500 mg/ml, which indicated the blank ANPs did not have apparent cytotoxicity. Because of the gravity sedimentation [30], we deduced that ANPs precipitated and covered the cell surface, which would block the cells to get enough nutrients and oxygen from the culture medium. Consequently, when the cells were exposed to extremely high concentrations (500-1000 mg/ml), the cell viability was decreased; even so, the viability percentages were still above 70%. These results suggest ANPs can be considered as a potential material for biomedical applications due to the good biocompatibility.

Cytotoxicity evaluation of DOX and DOX-ANPs
Cytotoxicity studies were evaluated on the UMR-106 cells using an MTT assay. As shown in Figure 8, at the low and middle concentrations (0.125-1 mg/ml), the cells were more sensitive to free DOX compared to DOX-ANPs (p < 0.05), because free DOX immediately contacted with the cells, which may initially induce toxicity to the cells. While in the high concentration (2 mg/ml), DOX-ANPs showed the same cell viability compared to

Cellular uptake of DOX-ANPs and free DOX
The inherent fluorescent property of DOX enables the use of fluorescence microscopy to visualise the distribution of the drug [31]. The cells treated with DOX showed a strong red fluorescence in the nucleus with less in the cytoplasm (Figure 9). The cells co-cultured with DOX-ANPs showed obviously red fluorescence within the cytoplasm at both 0.5 and 5 h, indicating the nanoparticles accumulation in the cytoplasm. Strong red fluorescence was observed in the nucleus after 24 h co-cultured with DOX-ANPs, which indicated the DOX released from DOX-ANPs, and diffused from the cytoplasm to the nucleus. After labelling endosomes and lysosomes, the result indicated obvious co-localisation of DOX-ANPs with endosomes and lysosomes ( Figure 10). The subcellular co-localisation assay suggested endosomes and lysosomes were involved in the internalisation of nanoparticles. The explanation of this trend may lie with the different cellular uptake mechanisms [32]. Free DOX is transported into cells via a passive diffusion mechanism, while drug-loaded nanoparticles enter the cells by endocytosis-mediated cellular uptake [33]. These results revealed the ability of ANPs to deliver DOX into the cells and also offered evidence of cellular uptake of DOX-ANPs through endocytosis.

Morphological examinations
UMR-106 cells were treated with blank ANPs, free DOX and DOX-ANPs for a period of 48 h, and the morphological observations ( Figure 11) revealed that both control cells and cells treated with blank ANPs showed adherent growth and a regular polygon cell shape, while the number of the cells treated with DOX or DOX-ANPs decreased obviously compared to control group. In addition, cell shrinkage, detachment and floating in the culture medium could be observed in these two groups ( Figure 11(C-E)). Many small dark dots (the aggregated nanoparticles) could be observed on the surface or inside the cells (Figure 11(F)). The marked cell number decrease and morphological changes qualitatively demonstrated the occurrence of apoptosis. Characteristic ultrastructural changes of UMR-106 cells were observed by SEM as displayed in Figures 12. The control cells showed a typical morphological feature of cancer cells, such as numerous microvilli and lamellipodia on the surface (Figure 12(A,B)). Morphological changes are witnessed in the treated groups ( Figure 12(C-F)). Cells treated with free DOX exhibited a decrease in the number of microvilli, while the cells treated with DOX-ANPs showed a relatively smooth surface with microvilli disappearance. Additionally, cell shrinkage, irregular plasma membrane blabbing and separated apoptotic bodies could be observed in the treatment groups. Cell shrinkage is the most characteristic feature of the early process of apoptosis. Membrane blabbing is one of a series of distinctive morphological events during apoptosis. Apoptosis produces cell fragments called apoptotic bodies, which suggests that it is in a late stage of apoptosis. The treatments resulted in distinct morphological alterations, which are typical characteristics of apoptosis [34,35].
TEM micrographs of ultrastructure of UMR-106 cells are displayed in Figure 13. The micrographs of control group showed complete integrity cell organelles, clear nuclear membrane and well-distributed chromatin (Figure 13(A)). The ultrastructural characteristics were notably changed after treatment, showing typical apoptotic phenomena ( Figure 12(B-F)). The major signs of early apoptosis including cell shrinkage, chromatin condensation and margination can be observed in both treatment groups. A large number of vesicles formed in the cytoplasm, and a few vacuoles contained DOX-ANPs coacervates (Figure 12(E)). This indicated DOX-ANPs can be internalised into the cells. The typical apoptosis phenomena, including membrane blabbing and apoptotic bodies, were also observed in Figure 12, which indicated that DOX-ANPs can induce apoptosis of OS cancer cells.

Apoptotic induction by DOX-ANPs
The induction of apoptosis of DOX-ANPs was further corroborated using Annexin V/PI apoptosis flow cytometry analysis. Since externalisation of phosphatidyl serine (PS) occurs in the earlier stage of apoptosis, and PS has a high binding affinity towards Annexin V. Annexin V-FITC staining can identify apoptosis at an early stage, while PI can detect necrotic cells due to its permeability through damaged cell membranes. As shown in   .53 § 0.75%, respectively. Treatment with DOX-ANPs significantly increased the percentage of apoptotic cells, compared to the control (p < 0.05). A similar trend was observed in DOX treatment group. However, there was no significant difference between blank ANPs group and the control group (p > 0.05). Overall, both DOX and DOX-ANPs can induce apoptosis of UMR-106 cells in a time-dependent manner.

Induction of cell cycle arrest by DOX-ANPs
Based on DNA content, cell cycle consists of four phases: G0/G1 (normal DNA content, 2 N), S (DNA synthesis), G2/M (double DNA content, 4N) [36]. Apoptotic cells contain fractional DNA content, defined as sub G0/G1. The effect of DOX-ANPs on cell cycle was evaluated using flow cytometric analysis. The DNA content histograms of UMR-106 cells treated with DOX and DOX-ANPs are shown in Figure 15. The cells in control group incubated 24 h showed a normal predominant distribution in G0/G1 phase (55.37 § 1.19%) and a relatively minor G2/M phase (19.92 § 1.01%) and sub G0/G1 (0.63 § 0.04%), while an increased amount of the cell population in the G2/M phase (45.07 § 1.43%) and sub G0/G1 phase (19.27 § 0.48%) can be observed in DOX-ANPs Figure 14. Annexin V assay of UMR-106 cells for 24, 48 and 72 h treatment in different groups. In all panels, LL represents viable cells, LR represents early apoptosis, UR represents late apoptosis and UL represents necrosis.
group. The same trend was observed in DOX-positive control group: the G2/M phase was 39.73 § 1.78% and sub G0/G1 phase was 27.94 § 0.12%. Cells treated with DOX or DOX-ANPs exhibited a large significant increase of the G2/M phase and sub G0/G1 phase, compared with the control group (p < 0.01). However, there was no significant difference between blank ANPs group and the control group in sub G0/G1 and G2/M phases (p > 0.05). These results indicated that DOX-ANPs induced cell cycle arrest in the G2/M phase and apoptosis in UMR-106 cells. These results are consistent with the DOX antitumour mechanism [37]. The cytotoxicity of DOX is attributed to its intercalation in DNA and inhibition the function of the topoisomerase (Topo) II. Topo II is a nuclear enzyme that participates in the topological rearrangement of DNA at mitosis. As a result, DOX can induce the accumulation of double-stranded DNA and cause cell cycle arrest in the