A New Method Without Organic Solvent to Targeted Nanodrug for Enhanced Anticancer Efficacy
- Shichao Wu†1, 2, 3,
- Xiangrui Yang†1, 2, 3Email author,
- Mingyuan Zou1,
- Zhenqing Hou2Email author and
- Jianghua Yan1Email author
© The Author(s). 2017
Received: 12 April 2017
Accepted: 31 May 2017
Published: 15 June 2017
Since the hydrophobic group is always essential to the synthesis of the drug-loaded nanoparticles, a majority of the methods rely heavily on organic solvent, which may not be completely removed and might be a potential threat to the patients. In this study, we completely “green” synthesized 10-hydroxycamptothecine (HCPT) loaded, folate (FA)-modified nanoneedles (HFNDs) for highly efficient cancer therapy with high drug loading, targeting property, and imaging capability. It should be noted that no organic solvent was used in the preparation process. In vitro cell uptake study and the in vivo distribution study showed that the HFNDs, with FA on the surface, revealed an obviously targeting property and entered the HeLa cells easier than the chitosan-HCPT nanoneedles without FA modified (NDs). The cytotoxicity tests illustrated that the HFNDs possessed better killing ability to HeLa cells than the individual drug or the NDs in the same dose, indicating its good anticancer effect. The in vivo anticancer experiment further revealed the pronounced anticancer effects and the lower side effects of the HFNDs. This new method without organic solvent will lead to a promising sustained drug delivery system for cancer diagnosis and treatment.
Experiment conditions in Fig. 3
Ultrasonic power (W)
In recent years, the non-spherical particle shape has been attracting more and more attention for their potential effect on drug delivery [14–19]. There has been already evidence that shape has an influence on many properties of the particles, such as the biodistribution and the degradation [20–22]. Most of all, the cellular internalization was proved to be strongly shape dependent [23–25]. Because the particles must be able to enter cancer cells and act on their therapeutic targets to kill them. And many studies have found that the cancer cells preferred particles with high aspect ratio [10, 26].
However, most of these methods rely heavily on organic solvents, mainly due to the hydrophobic group needed in the nanoparticle preparation processes . These organic solvents may be residual within the particles and cannot be completely removed by conventional practices, such as reduced pressure distillation or freeze drying. As a result, trace amounts of organic solvents remain in the medicine, which are called residual solvents. Although residual solvents are very little and may meet the special directions published in pharmacopeias which have been strictly controlling the maximum allowable amounts of the residual solvents in pharmaceutical products, the residual solvents will be accumulating in the body and may accentuate the disease or cause other serious issues. Hence, the manufacturers have been aspiring to minimize the amount of the organic solvents used in the drug production process. Therefore, it will be a pretty major leap for the medicine, the human health, and the environment to use “green” chemistry into the pharmaceutical industry, although facing amounts of difficulties.
In this study, we developed HCPT-loaded, folate (FA)-modified nanoneedles (HFNDs) with a high aspect ratio and sharp ends via a completely green method without using any organic solvent. The pH-controlled precipitation of the HCPT and FA-modified chitosan (CS-FA) lead to nucleation of nanoneedles with nanocrystalline HCPT as the core wrapped with CS-FA as steric stabilizers. The HFNDs were found to possess good properties of targeting and imaging capability. In vitro and in vivo studies were then systematically investigated. These results highlight the great potential of FA-modified, imaging-functional nanoneedles for highly efficient chemotherapy, as well as for cancer diagnostic applications.
All chemicals are of analytical grade and used as received without further purification. Deionized (DI) water was used in all experiments. FA was purchased from Bio Basic Inc. 10-hydroxycamptothecine (HCPT; purity >99%) was purchased from Lishizhen Pharmaceutical Co., Ltd. Chitosan (Mw = 70 000, 90% degree of deacetylation) was obtained from Zhejiang Aoxing. N-Hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were purchased from Sigma-Aldrich.
Synthesis of the FA-Chitosan Conjugate
FA (10 mg), chitosan (20 mg), EDC (4 mg), and NHS (4 mg) were added into 2 mL PBS buffer solution (pH 5.5) and stirred at rt for 12 h to obtain the CS-FA suspension. Then, the suspension was dialyzed against a buffer solution (pH 10) to remove excess FA molecules. The remaining suspension was centrifuged (5000 rpm) and lyophilized for 24 h to obtain the dry CS-FA powder.
Preparation of HFNDs
HCPT (10 μg) was dissolved in 200 μL of NaOH aqueous solution (0.1 M) to obtain solution A, and CS-FA (10 μg) was dissolved in 200 μL HCl (0.1 M) to obtain solution B. Afterwards, solution A and solution B were added dropwise into pure water (1 mL) under vigorous stirring for 30 s, and the mixture was sonicated (200 W) in an ice bath for 6 min. The suspension was centrifuged (10,000 rpm, 5 min) and lyophilized for 24 h. For the preparation of NDs, the chitosan solution was used to replace the solution B.
In Vitro Drug Release Study
The in vitro drug release study of HFNDs was performed using the dialysis technique. The HFNDs were dispersed in a PBS buffer solution (10 mL) and placed in a pre-swelled dialysis bag (MWCO 3500 Da). The dialysis bag was then immersed in PBS (0.1 M, 200 mL, pH 7.4) and oscillated continuously in a shaker incubator (100 rpm) at 37 °C. All samples were assayed by fluorescence spectrophotometry.
Confocal Imaging of Cells
The confocal imaging of cells was performed using a Leica laser scanning confocal microscope. Imaging of HCPT was carried out under the 382-nm laser excitation, and the emission was collected in the range of 500–550 nm. HeLa cells were seeded and preincubated at 37 °C for 24 h (5% CO2) before incubated with the HFNDs for 8 h.
Cellular Uptake Measured by Fluorescence Measurement
HeLa cells were seeded in a 24-well plate (1 × 106 mL/well). The plate was then incubated at 37 °C for 24 h in a humidified atmosphere (5% CO2). The cells were then incubated with NDs and HFNDs at equivalent concentrations of HCPT. The drug-treated cells were incubated for 6 h at 37 °C, followed by being washed twice with PBS, and digested by trypsin (0.05%)/EDTA treatment. The suspensions were centrifuged (1000 rpm, 4 °C) for 4 min. The cell pellets were washed with PBS to remove the background fluorescence in the medium. After two cycles of washing and centrifugation, the cells were resuspended with 2 mL PBS and disrupted completely by vigorous sonication. The amount of HCPT in the sonicated mixture was analyzed by fluorescence spectroscopy (excitation at 382 nm). Blank cells in the absence of drug were measured to determine the cells auto-fluorescence level as the control.
The cytotoxicity of HFNDs was determined by MTT assay. Briefly, an adequate number of exponential phase HeLa cells were plated in quintuplicate in a 96-well flat bottomed microplate and incubated for 24 h in the presence of drug/particles. In this study, 20 μL 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) solution (5 mg/mL in PBS) was added in each well, and the plates were incubated at 37 °C for another 4 h. Afterwards, a volume of 150 μL dimethylsulfoxide (DMSO) was added, and the plate was agitated on a water bath chader at 37 °C for 30 min. The absorbance at 570 nm was measured using a Microplate Reader (model 680; Bio-Rad).
For in vivo fluorescence imaging, DiR was encapsulated into the NDs and HFNDs. DiR-NDs and DiR-HFNDs were intravenously administered into HeLa tumor-bearing nude mice via tail veins at an equivalent dose of DiR-HCPT per kilogram mouse body weight. At predetermined time intervals, the mice were anesthetized and imaged with the Maestro in vivo imaging system (Cambridge Research & Instrumentation, Woburn, MA, USA). After 24 h, the mice were sacrificed, and the tumor as well as the major organs (spleen, liver, kidney, lung, and heart) was excised, followed by washing the surface with 0.9% NaCl for the ex vivo imaging.
Tumor Inhibition In Vivo
When HeLa tumor volume of the HeLa tumor-bearing mice was approximately 60 mm3, the mice were randomly divided into four groups and treated by intravenous injection of 0.9% NaCl, free HCPT, NDs, and HFNDs every 3 days at a dose of 80 μg HCPT per mouse. The tumor volume and the body weight were monitored every 3 days. The tumor volume was calculated by the following formula: tumor volume = 0.5 × length × width2.
After 21 days, the mice were sacrificed, followed by the tumors excised and weighed. Then, the tumors were fixed in 4% paraformaldehyde overnight at 4 °C, embedded into paraffin, sectioned (4 μm), stained with hematoxylin and eosin (H&E), and observed using a digital microscopy system.
The statistical significance of treatment outcomes was assessed using Student’s t test (two-tailed); P < 0.05 was considered statistically significant in all analyses (95% confidence level).
Results and Discussion
Synthesis of the FA-Chitosan Conjugate
Preparation of HFNDs
Figure 3b, c shows the optimized needle-shaped morphology of the HFNDs with an average length of about 800 nm and the width of about 80 nm. The result of DLS measurement shows a size of 104.3 ± 5.7 nm (Fig. 4g) and a zeta potential of +16.3 ± 1.9 mv (Fig. 4h). What’s more, a 2 wt% HFNDs dispersion showed good stability for 2.5 days at least. Since there is no fluorescence signals from CS-FA, we can measure HCPT drug-loading content of the HFNDs by using the fluorescence characteristics of HCPT. The HCPT drug-loading content of the HFNDs was 70.2 ± 3.1%, and the encapsulation efficiency was 83.1%. And the FA content of the HFNDs was 7.0%, which was calculated via the percentage of FA in CS-FA.
In Vitro Drug Release Studies
To evaluate the tumor target ability of dual-drug nanoneedles, DiR was used as a near-infrared fluorescence probe to be encapsulated into free HCPT, NDs, and HFNDs at the equivalent DiR concentration. 0.9% NaCl, DiR-NDs, and DiR-HFNDs were injected intravenously into the mice-bearing tumors derived from human cervical carcinoma HeLa cells, and their in vivo biodistributions were investigated.
Tumor Inhibition In Vivo
The study herein presents a completely green approach to obtain FA-modified, HCPT-loaded nanoneedles for the highly efficient chemotherapy with high drug loading, targeting property, and imaging capability. The drug release profile revealed that the HFNDs showed a sustained and prolonged release. The CLSM demonstrated the more effective cellular internalization of HFNDs than NDs. The MTT experiment indicated that the HFNDs not only showed a much higher cytotoxicity than the individual drugs and NDs. This illustrated the good targeting property of the HFNDs. This work opens a door to design new dosages of nanoparticles with completely green method, which might have a powerful effect on environmental protection in the future.
This project was supported by the Natural Science Foundation of Xiamen city, Fujian Province, China (No. 3502Z20163007).
SW and XY conceived and carried out experiments, analyzed data, and wrote the paper. XY, ZH, and JY designed the study, supervised the project, analyzed data, and wrote the paper. MZ assisted in the synthesis and characterizations of the NPs. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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