Arsenic trioxide (ATO) is the main active ingredient of traditional Chinese medicine (TCM) Arsenic. In the 1970s, it was first applied to acute promyelocytic leukemia (APL) with significant efficacy [1
] and was approved by the National Medical Products Administration (NMPA) and Food and Drug Administration (FDA) as a first-line treatment for APL in 1999 and 2000, respectively [2
]. ATO can induce cell differentiation, inhibit apoptosis, and exert anti-tumor effect [4
]. In recent years, research studies have confirmed the significant growth inhibition and apoptosis induction effect of ATO in solid tumors, such as liver cancer, breast cancer, stomach cancer, glioma and lung cancer [5
]. At present, ATO injection has been employed clinically in the treatment of APL and advanced primary liver cancer. However, the unique physicochemical properties of ATO allow it to be rapidly cleared from blood, and it requires daily administration during clinical treatment. At the same time, the uptake of the reticuloendothelial system (RES) makes only a slight amount of ATO reach the tumor site. Nevertheless, considering the potent toxicity of ATO, increasing the dose of ATO will increase the systemic toxicity and cause damage to the liver, kidney, heart, and peripheral nerve [12
Based on the size advantage, nanoparticles (NPs) can exude through the tumor vasculature and effectively delivery the drugs to cells through enhanced permeability and retention (EPR) effects [15
]. Therefore, it is considered to be a kind of formulation with low toxicity and high stability. Different ATO delivery systems (DDS) have been developed, including magnetic nanoparticles [16
], chitosan nanoparticles [17
], microspheres [18
], liposomes [19
], and mesoporous silica nanoparticles [10
]. These formulations can achieve sustained release of ATO, which can reduce the transient plasma concentration and toxicity of drugs to a certain extent. However, they are still deficient in biocompatibility, and the safety of these systems needs to be verified [21
Sodium alginate (SA) is a sort of polyanionic polysaccharide alginic acid salt found in brown algae that is water-soluble and has the advantages of anti-tumor effect, immune regulation, non-toxic, biodegradability, and excellent biocompatibility [22
]. It has been approved by FDA for the pharmaceutical industry as an excipient [24
]. For the past few years, NPs prepared from SA as drug carrier systems have also attracted more attention [26
]. Red blood cells membrane (RBCM) will be formed into vesicles (RVs) using extrusion or sonication methods [28
]. As a drug carrier, it can be attached to the surface of NPs to sustain the release of drugs, avoid elimination by the immune system, increase drug stability, improve biocompatibility and thus prolong drug circulation in vivo [29
Moreover, RBCM coating nanotechnology already has excellent precedents. Che-Ming J et.al [31
] demonstrated the synthesis of an RBCM coated polymeric nanoparticle for long-circulating cargo delivery, Jinghan Su et al. [32
] extensively studied the effectiveness of RBCM-camouflaged NPs for treating metastatic breast cancer. In addition, if the non-toxic SA nanoparticles can be encapsulated by natural RBCM and combine the superiorities of sustained release and prolonging residence time, the nano-system can achieve the purpose of maintaining efficacy and reducing toxicity. In addition, this can provide a new possibility for safe application of ATO.
In this study, RBCM-camouflaged ATO-loaded sodium alginate nanoparticles (RBCM-SA-ATO-NPs, RSANs) were prepared as shown in Figure 1
. ATO-loaded sodium alginate nanoparticles (SA-ATO-NPs, SANs) were prepared by the ion crosslinking method, followed by coating of RBCM to obtain RSANs. It was then systematically characterized and evaluated for its efficacy and toxicity. The results indicated that the system might become a promising delivery system for the safe, effective, and sustained release of ATO.
2. Materials and Methods
SA (200–500 Pa·s) was purchased from Shanghai Taitan Technology Co., Ltd., Shanghai, China, anhydrous calcium chloride (CaCl2) was purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd., Shanghai, China and Polosham 188 (F-188) was purchased from Shaanxi Zhengyi Pharmaceutical Accessories Co., Ltd. Carbon support copper mesh (230 mesh) and phosphotungstic acid were obtained from Beijing Zhongjing Keyi Technology Co., Ltd., Beijing, China. 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (Dil, cell membrane green fluorescent probe), Hoechst 33342, 4% paraformaldehyde fix solution, antifade mounting medium and Cell Counting Kit-8 (CCK-8), were all purchased from Biyuntian Biotechnology Co., Ltd., (sShanghai, China) 5(6)-aminofluorescein was bought from Nanjing Xinfan Biotechnology Co., Ltd., Nanjing, China. Polycarbonate film was bought from Whatman Company, City, UK. Dialysis bag (Cut-off molecular weight = 3500Da) was obtained from United States for carbonization. Fetal bovine serum (FBS), RPMI 1640 medium, and DMEM medium were ordered from the Shanghai Chenyi Biotechnology Company, Shanghai, China. Trypsin and penicillin-streptomycin were purchased Yingjie Jieji (Shanghai) Trading Co., Ltd., Shanghai, China.
2.2. Cells and Animals
RAW264.7 cells (mice macrophages) and HEK-293 cells (normal human embryonic kidney cells) were bought from the Shanghai Cell Bank of the Chinese Academy of Sciences, Shanghai, China, and cultured with DMEM complete medium. SMMC-7721 cells (human liver cancer cells) and NB4 cells (APL cells) were obtained from Shanghai Jihe Biotechnology Co., Ltd., Shanghai, China and cultured with RPMI 1640 complete medium. Culture of the cells was performed in an incubator kept at 5% CO2 and 37 °C. The male BALB/c nude mice (SCXK 2017-0005) were obtained from Shanghai Slack Laboratory Animals Co., Ltd., Shanghai, China and kept in the Specific Pathogen Free (SPF) animal room of School of Pharmacy, Shanghai Jiao Tong University. Guidelines for care and use of laboratory animals of Shanghai Jiao Tong University were used to perform animal studies and these studies were duly approved by the animal ethics committee of Shanghai Jiao Tong University (No: A2019046, Date: 5 July 2019).
2.3. Preparation of SANs
SANs were prepared by the ion crosslinking method. In brief, 1.5 mL of 2 mg/mL CaCl2 was slowly added into 10 mL of 0.3 mg/mL SA (pH = 5) under stirring. After sonication for 5 min at 250 W, 0.1 mL of 10 mg/mL F-188 was added and then stirred for 30 min. To obtain SANs, 0.2 mL of 8 mg/mL ATO was added and stirred for other 30 min.
2.4. Preparation of RBCM
RBCM was extracted by hypotonic rupture method. In brief, whole blood of SD rats (bought from Shanghai Jiesijie Experimental Animal Co., Ltd., Shanghai, China) was collected through abdominal aorta. The blood was centrifuged (2000 rpm, 5 min, 4 °C), to obtain red blood cells (RBCs), and then washed with 1× phosphate buffer saline (1× PBS) for 3 times. To collect RBCM, 900 μL EDTA (0.2 mM) was added to disrupt the RBCs, followed by centrifugation (13,200 rpm, 10 min, 4 °C), and the above steps were repeated until the supernatant turned colorless. The obtained RBCM was resuspended in EDTA, and then stored in −80 °C refrigerator.
2.5. Preparation of RSANs
The prepared RBCM was sonicated at 250 W for 3 min to obtain RVs, and then sequentially extruded through polycarbonate films of 800 nm, 400 nm, and 200 nm by LF-50 extruder (Avestin Inc, agented by Shanghai Narujie Biotechnology Co., LTD, Shanghai, China) for at least 15 times respectively. The solutions of RVs and SANs were mixed at a ratio of 1:8(v/v). The prepared mixture was then extruded through polycarbonate films of 400 nm and 200 nm at least ten times, respectively, to obtain RSANs.
2.6. Characterization and Stability Test
The particle size and polydispersity index (PDI) of SANs and RSANs were determined by Malvern Zetasizer (He-Ne, 4.0 Mw, λo = 633 nm, Marvin instruments Ltd., Marvin, United Kingdom. The stability test was investigated simultaneously. The particle size and PDI of SANs and RSANs were measured for 15 days consecutively at both 4 °C and 37 °C.
2.7. Morphological Observation
Transmission electron microscopy (TEM, Thermo Fisher Scientific, Shanghai, China) was performed to evaluate the morphology of RSANs. 10 μL sample solutions were dropped on carbon-supported copper, air-dried and then rinses with 10 μL ultrapure water. Drip 10 μL 2% phosphotungstic acid to stain the samples, and the excess dye was removed with filter paper from the edge. After baking 30 min under an infrared baking lamp, the morphology was observed by TEM.
2.8. Drug Loading Capacity and In Vitro Release Study
The release study of ATO encapsulated in nanoparticles was performed in 1×PBS (pH 7.4). 2 mL ATO solution (final concentration of 135.6 μg/ mL), SANs, and RSANs solution were placed into dialysis bags, respectively (Mw
= 3500). The dialysis bags were fastened at both ends and maintained under sink conditions at 37 °C using 50 mL PBS, then magnetically stirred at 100 rpm. At time point of 3 h, encapsulation efficiency (EE) and drug loading capacity (DL) of the nanoparticles were analyzed by extracting 1 mL release solution. The optimal DL and EE were investigated by adding different concentrations of ATO. For the determination of in vitro release, 1 mL release sample was taken at 0.5, 1, 2, 4, 8, 12, 24, 36, 48, 72, and 84 h, and then replaced with 1 mL of fresh PBS. The aliquots were filtered through 0.22 μm microfiltration membrane and diluted up to 50 times. Inductively coupled plasma-atomic emission spectroscopy (ICP-AES) was used to detect the concentration of ATO. In addition, the following formulas were used to calculated DL and EE.
Encapsulation Efficiency (%) = (M1 − M2)/M1 × 100%
Drug Loading Capacity (%) = (M1 − M2)/(M1 − M2 + M3) × 100%
means the total amount of used ATO, M2
is the amount of ATO in the dialysis solution, and M3
is the amount of used SA.
2.9. Hemolysis Test
Since it is administered by intravenous injection (iv), it is necessary to ensure that the nano-preparation will not cause hemolysis or cell aggregation. RBCs from the blood of SD rats were collected and normal saline was added to obtain a 2% (v
) RBC suspension. The water, normal saline, RBC suspension, and RSANs were mixed into 12 tubes according to the ratio shown in Table 1
. Tube 1 (RSANs) replaced with ultrapure water served as a positive control, while tube 2 (RSANs) replaced with saline as negative control. The results after incubation of 3 h and 24 h at 37 °C were recorded. Meanwhile, the absorbances of supernatant were determined at 3 h and 24 h through an ultraviolet spectrophotometer at the wavelength of 540 nm, and then the percentage of hemolysis was calculated according to formula:
Percentage of Hemolysis (%) = (Asample − Atube 2)/(Atube 1 − Atube 2)
2.10. Macrophage Uptake Study
The immune escape ability of nanoparticles was investigated through RAW 264.7 cells. SA was labeled with 5(6)-aminofluorescein, and SANs and RSANs were prepared with labeled SA. A 12-well plate (2 × 105 cells per well) was used for 24 h incubation. Serum-free medium was used to starve cells for 1 h and then replaced by complete medium containing SANs or RSANs (SA concentration was 50 μg/mL). A complete medium without nanoparticles was used as blank group. The cells were observed with laser scanning confocal microscope (LSCM) after incubation for 2 h. Before being photographed, the cells were fixed with 4% paraformaldehyde. The excitation wavelength of 5(6)-aminofluorescein was 493 nm. Also, three parallel groups were set up to detect the uptake quantitatively. After incubation with the drugs, each group was first digested with trypsin and then washed with PBS for 3 times, then resuspended in 500 μL PBS. The detection was performed through the FITC channel of a flow cytometer.
2.11. In Vitro Cellular Uptake
To investigate the uptake of nano-formulations by tumor cells and the structural integrity of NPs during this process, the test was implemented on NB4 and 7721 cells. The determination method was described in Section 2.10
. For the qualitative detection, RSANs were prepared with labeled SANs and RVs (the RVs was labeled with Dil). The density of 7721 cells was 2 × 105
cells per well, and the cells were cultured for 24 h. The cells were then treated in the same way as Section 2.10
. After an incubation of 2 h, Hoechst 33342 was used to stain the nuclei for 15 min, and then the uptakes were observed with LSCM. The cells were fixed with 4% paraformaldehyde before being photographed. Since NB4 cells is a kind of suspension cells, the density of inoculation was doubled to avoid loss during the experiment, and finally immobilized on a glass slide coated with polylysine. The other steps were the same as the 7721 cells. The excitation wavelengths of 5(6)-aminofluorescein, Dil and Hoechst 33342 were 493 nm, 549 nm, and 405 nm, respectively.
2.12. Cytotoxicity Test
To investigate the toxicity of blank carrier material, the prepared SA nanoparticles (SNs) and RBCM-SA nanoparticles (RSNs) without ATO were diluted with DMEM complete medium to obtain SA concentrations of 8, 12, 20, 30, 40, 50, 60 μg/mL respectively. To investigate the toxicity of the nanoparticles after drug loading, free ATO solution, SANs, and RSANs were diluted to obtain different concentrations of ATO (1, 2, 4, 6, 8, 10, 12, 20 μg/mL). The above groups were administration groups (AG). 100 μL 293 cells (5 × 104
cells/mL) were placed into a 96-well culture plate for 24 h. The nutrient medium was then replaced with 100 μL AG. At 24 h time interval, all the groups were cultured with 100 μL CCK-8 for 2 h. Only DMEM medium was set as a blank group (BG) and only 293 cells were set as a control group (CG). The optical density (OD) of samples were determined at 450 nm by a microplate reader and the following formula was used to calculate the cell viability.
Cell viability (%) = (ODAG − ODBG)/(ODCG − ODBG) × 100%
2.13. In Vitro Efficacy Study
NB4 and 7721 cells were selected to evaluate efficacy. 100 μL NB4 and 7721 cells (5 × 104
cells/mL) were inoculated in a 96-well culture plate overnight, respectively. Then free ATO solution, SANs, and RSANs were administered to the cells, respectively. The concentrations of ATO were diluted to 1, 2, 4, 6, 8, 10, 12, 20 μg/mL. After 24 h, 100 μL CCK-8 were administered to evaluate the cell viability. To further investigate the inhibitory effect, the concentration of the ATO of each group was fixed at 1 μg/mL. The OD was measured after incubation of 4, 8, 12, 24, 36, 48, and 60 h, and the cell viability was determined. The blank and control groups were same as mentioned in Section 2.12
2.14. In Vivo Toxicity and Safety Test
Due to the potent toxicity of ATO, the administration concentration at a safe level should be determined at first. For 2 weeks, ATO with high (40 μg/mL), medium (20 μg/mL) and low concentration (10 μg/mL) was administered through the tail vein respectively once a week at a dose volume of 0.2 mL per mouse. The mental state and death of nude mice were registered during the period.
The aim of the safety trial was to investigate whether the continuous intravenous injection of RSANs would cause lesions on systemic, hematological, and major organs. Afterward, the healthy nude mice were divided into the saline group, ATO group, SANs group, and RSANs group. The drugs were administered once every 2 days at dose volume of 0.2 mL per mouse and repeated seven times. The weight of the mice was recorded at 1, 3, 5, 7, 9, 11, 13 days after administration. Meanwhile, the mental state and death of mice were observed during the process. 2 days after the last administration, orbital blood was collected into the tubes, pre-mixed with heparin sodium, and white blood cells (WBC), glutamate pyruvic transaminase (ALT), aspartate aminotransferase (AST) were analyzed. In addition, after the mice were sacrificed by CO2
asphyxiation, the principal organs were excised. The viscera coefficients were calculated after weighing. Furthermore, the tissues were fixed with paraffin solution for immunohistochemical analysis (H & E) to examine its structure and morphology.
Visceral coefficient = weight of organ/body weight
2.15. In Vivo Anti-Tumor Studies
Male nude mice 6–8 weeks old with 7721 cells were used to investigate the anti-tumor effects of our nanoparticles. First we established xenograft tumor model. For the establishment of tumor model, 7721 cell suspension (2 × 107 cells / mL) in a volume of 0.2 mL was injected subcutaneously in the armpit of the upper limb. In addition, then the formula width2 × length/2 was used to calculate the tumor volume every other day. At tumor volume of 100–250 mm3, then mice were grouped into four treatment groups (n = 5): (1) saline, (2) ATO, (3) SANs, and (4) RSANs. The drugs were administered intravenously at a dose of 1.3 μg/g every two days and repeated seven times. The CG was given normal saline for seven times. At the same time, the body weight and the tumor volume were recorded to evaluate for tumor inhibition efficacy as well as systemic toxicity. Two days after the last administration, the mice were sacrificed by CO2 asphyxiation, and the tumors were excised from each animal. The tumors were rinsed with normal saline, dried, and photographed, and the average tumor weight of each group was determined.
2.16. Statistics and Data Analysis
Data expression was shown as ± SD of the mean. Significant differences between SANs and RSANs were analyzed by Tukey Kramer multiple comparison tests, using GraphPad Prism Software, v.6.01 (GraphPad Software, Inc.). Results with p < 0.05 were considered significant and very significant with p < 0.01.
The objective of this project was to realize sustained release of ATO and ensure the safety and efficacy of the DDS. SANs were prepared by coating ATO with SA, and then RSANs with a shell-core bilayer structure was obtained by wrapping the RBCM on the surface of the SANs. The nanoparticles were homogeneous with spherical structures and stable for 15 days with an average EE and DL of 14.31% and 4.98%, respectively. Compared to free ATO, the SANs and RSANs showed excellent sustained release in vitro for 3 days. Generally, biological characteristics of RBCM could be used to avoid the recognition of macrophages. Even when ingested by NB4 cells and 7721 cells, RSANs still could maintain its nuclear-shell structure. At the same time, in vitro safety test showed the safety of carrier materials and without observable toxicity to 293 cells or hemolysis and aggregation of red blood cells. Free drug can kill nearly half of cells at a low concentration, while RSANs can significantly reduce the toxicity of ATO. After continuous administration of ATO formulations through IV tail injection, the results revealed that the SANs and RSANs had no significant systemic, blood, and organ toxicity. Moreover, the RSANs could maintain inhibition of NB4 cells and 7721 cells. After in vivo administration, RSANs can avoid rapid clearance and exert their sustained release properties, thereby enough drugs can be transported to the treatment site and achieve the effect. This may allow RSANs greater advantages in the treatment of tumors. Therefore, it could be concluded that the Nano-drug delivery system can decrease the toxicity of ATO with high safety and the potential for treating APL as well as anti-hepatocarcinoma. Meanwhile, RSANs can prolong the duration of ATO in vivo, thus reducing administration times and enhancing patient compliance. In consequence, the DDS can be developed into a safe and sustained release delivery system for ATO.