Evaluation of self-assembled HCPT-loaded PEG-b-PLA nanoparticles by comparing with HCPT-loaded PLA nanoparticles
- Xiangrui Yang†1,
- Shichao Wu†1, 2,
- Yange Wang1,
- Yang Li1, 2,
- Di Chang1,
- Yin Luo1,
- Shefang Ye1Email author and
- Zhenqing Hou1Email author
© Yang et al.; licensee Springer. 2014
Received: 11 November 2014
Accepted: 4 December 2014
Published: 19 December 2014
We present a dialysis technique to prepare the 10-hydroxycamptothecin (HCPT)-loaded nanoparticles (NPs) using methoxypolyethylene glycol-poly(d,l-lactide) (PEG-b-PLA) and PLA, respectively. Both HCPT-loaded PEG-b-PLA NPs and HCPT-loaded PLA NPs were characterized by differential scanning calorimetry (DSC), dynamic light scattering (DLS), transmission electron microscopy (TEM), scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). The results showed that the HCPT-loaded PEG-b-PLA NPs and HCPT-loaded PLA NPs presented a hydrodynamic particle size of 120.1 and 226.8 nm, with a polydispersity index of 0.057 and 0.207, a zeta potential of −31.2 and −45.7 mV, drug encapsulation efficiency of 44.52% and 44.94%, and drug-loaded content of 7.42% and 7.49%, respectively. The HCPT-loaded PEG-b-PLA NPs presented faster drug release rate compared to the HCPT-loaded PLA NPs. The HCPT-loaded PEG-b-PLA NPs presented higher cytotoxicity than the HCPT-loaded PLA NPs. These results suggested that the HCPT-loaded PEG-b-PLA NPs presented better characteristics for drug delivery compared to HCPT-loaded PLA NPs.
Nowadays, cancer is one of the most common causes of death all over the world. Chemotherapy is still a commonly used strategy for cancer treatment [1, 2], but their efficacy is largely limited by low stability, short circulating half-life, and the toxicity associated with the anticancer drugs to normal tissues [3–6]. The nanoparticles (NPs) have been proposed to overcome these inconveniencies, for NPs can encapsulate a series of poorly water-soluble anticancer drugs and reduce their toxicity by the way of releasing them in a sustained manner at their target site [1, 7–10]. In addition, the self-assembly technique is a simple and low-cost method for producing NPs in a controllable way [11, 12]. Hence, the self-assembled NPs have attracted considerable interest for their potential use in anticancer drug delivery system.
Although the NPs enhanced the stability and decreased the toxicity of drugs, they only solved part of the problems, since the particles could be still rapidly cleared by the reticuloendothelial system (RES) uptake [11, 13]. It was pointed that hydrophobic NPs were more prone to RES uptake as compared to the negatively charged NPs . So the NPs with surface modification have been devised to further improve the suboptimal pharmacokinetic properties [15–17]. Among all the strategies, PEGylation was proved to be the most effective, because of its unique physicochemical characteristics, such as good dispersibility and solubility [18–20]. With PEG chains on their surface, the NPs can entangle water molecules in the aqueous phase, which will render their surface hydrophilic and hinder protein binding and RES uptake . It can still enhance their solubility and diminish their aggregation and immunogenicity . More importantly, polyethylene glycol has been approved by FDA for human use. All these properties contribute to wide-spread use of PEG in many therapeutics [3, 16, 21].
10-Hydroxycamptothecin (HCPT) is a promising broad-spectrum antitumor agent which has shown to have a strong anti-tumor activity against gastric carcinoma, hepatoma, leukemia, and tumor of the head and neck in clinical practice . In spite of its remarkable success in early clinical trials, the application of HCPT is still largely limited because of its poor solubility and stability, which led to low therapeutic efficiency and a number of side effects owing to the conversion of HCPT from active lactone form to the inactive carboxylate form under physiologic conditions [5, 23]. For these reasons, we chose HCPT as the model chemotherapeutic agent, expecting to further improve the suboptimal pharmacokinetic properties and promote its clinical application.
Moreover, testing tools are also important for future clinic use, and fluorescence imaging technique is an effective research tool to investigate many in vivo processes in the life sciences, such as the locations and sizes of tumors and the distribution of chemotherapy agents [24, 25]. The intrinsically fluorescence properties of HCPT in the drug delivery system will provide a good signal readout for the detection, which will meet the requirements of cancer diagnosis and therapy simultaneously.
In this paper, we presented a dialysis technique to direct the self-assembly of the HCPT-loaded PEG-b-PLA NPs. The hydrophobic polymeric core of the platform readily encapsulated the water-insoluble drug for systemic drug delivery. The physicochemical properties of the HCPT-loaded PEG-b-PLA NPs were characterized by differential scanning calorimetry (DSC), dynamic light scattering (DLS), scanning electron microscope (SEM), transmission electron microscopy (TEM), and confocal laser scanning microscopy (CLSM). In vitro drug release profiles and cytotoxicity tests were also conducted. The HCPT-loaded PLA NPs were also prepared and characterized in the same way and used for comparison.
All chemicals were analytical grade and used as received without further purification. The ultrapure water (18 MΩ∙cm−1) was used throughout the work. The 10-HCPT (purity >99%) was purchased from Lishizhen Pharmaceutical Co, Ltd (Wuhan, Hubei, China). PLA (50 kDa) and PEG-b-PLA (10%) were provided by Daigang BIO Engineer Co., Ltd. (Shandong, China). A dialysis bag (Mw cutoff = 8,000 to 14,000 Da) was ordered from Greenbird Inc. (Shanghai, China).
Preparation of the HCPT-loaded PEG-b-PLA NPs and HCPT-loaded PLA NPs
The DSC analysis was performed on the solid samples using Netzsch model DSC-204 (Netzsch, Selb, Germany) with heating cycles of 0°C to 200°C. Samples (4 to 5 mg) were placed in a sealed aluminum pan and heated continuously at the rate of 10°C min−1 under a constant flow (40 mL min−1) of N2.
Particle size, polydispersity index, surface charge, morphology, and HCPT distribution
The average particle size, zeta potential, and polydispersity index (PDI) were determined DLS using Malvern Zetasizer Nano-ZS (Malvern Instruments, Worcestershire, UK). The morphology of the HCPT-loaded PEG-b-PLA NPs was examined by SEM (LEO 1530; ZWL, Pegnitz, Germany) and TEM (JEM-2100; JEOL Ltd., Tokyo, Japan) at 20 and 200 kV, respectively. One drop of the suspension was placed on a silicon wafer or a carbon-coated copper grid and dried in the air before observation. The distribution of HCPT within the HCPT-loaded PEG-b-PLA NPs was analyzed by CLSM (Olympus FV 1000; Olympus, Tokyo, Japan). The HCPT-loaded PLA NPs were used for comparison.
In vitro drug release behavior
The in vitro drug release studies of the HCPT-loaded PEG-b-PLA NPs were performed using the dialysis technique. The particles were dispersed in PBS (10 mL) and placed into a pre-swelled dialysis bag (MWCO 3,500 Da). The dialysis bag was then immersed in 0.1 M PBS at pH 7.4 and oscillated continuously in a shaker incubator (100 rpm) at 37°C. All samples were assayed by fluorescence spectrophotometry (HORIBA Fluoromax-4; HORIBA Ltd, Minami-ku, Kyoto, Japan). The HCPT-loaded PLA NPs were used for comparison.
In vitro cell viability assays
Human hepatocellular carcinoma cells (BEL-7402) were cultured in standard cell media recommended by the American Type Culture Collection. The cells seeded in 96-well plates were incubated with a series of increasing concentrations of the HCPT-loaded PEG-b-PLA NPs for 48 h. Subsequently, the relative cell viability was assessed by the standard MTT assay. Cells treated with free HCPT and cells treated with the HCPT-loaded PLA NPs were compared.
Results and discussion
Preparation of the HCPT-loaded PEG-b-PLA NPs
Particle size, PDI, surface charge, and morphology
In vitro drug release behavior
In vitro cell viability assays
In this study, we developed a simple but successful method to obtain both HCPT-loaded PEG-b-PLA NPs and HCPT-loaded PLA NPs with fine characteristics for drug delivery. Although both exhibiting a slow, prolonged release profile, the HCPT-loaded PEG-b-PLA NPs presented a smaller particle size, faster drug release, and higher cell cytotoxicity compared to the HCPT-loaded PLA NPs. The animal experiments are going on now. The results obtained in this study indicate that these NPs might become a promising drug delivery system for HCPT.
This work was funded by the National Natural Science Foundation of China (grant nos. 31271071 and 81472458).
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