Palmitoyl ascorbate and doxorubicin co-encapsulated liposome for synergistic anticancer therapy
Introduction
Nowadays, chemotherapy is still the ultimate therapy strategy for cancer treatment. However, the clinical application of chemotherapy is severely restricted by the toxic side effects (Corti et al., 2012, Wang et al., 2011). To address this dilemma, the major approach is the combination of two or more drugs with similar anticancer mechanism. The combination can reduce the toxicity and enhance the anticancer ability (Duan et al., 2013). Nevertheless, simple combination of free drugs is difficult to obtain expected therapeutic effect due to their different physicochemical properties and poor pharmacokinetic profiles (Lv et al., 2014).
Nanoparticle-based drug delivery systems provide the possibility of combining drugs with different properties into a single vehicle (Lv et al., 2014). Nanocarriers, such as liposomes, nanoparticles and micelles, can deliver multiple agents to the tumor sites simultaneously through the enhanced permeability and retention (EPR) effect (Maeda et al., 2000); and it can prolong blood circulation time to improve the pharmacokinetic profiles, thus raise the anti-tumor potency (Liu et al., 2014). One of the most widely used nanocarriers is liposome. The structure of liposome is mainly comprised of phospholipid and cholesterol. Compared with other delivery systems, the liposomes possess better biocompatibility (Zununi Vahed et al., 2017). Several liposomal agents, such as Doxil® (Barenholz, 2012), DaunoXome® (Fassas and Anagnostopoulos, 2005) and Visudyne® (Bressler and Bressler, 2000), have been released to the market and proved clinical therapeutic values.
As an anthracycline chemotherapeutic agent, doxorubicin (DOX) is widely used for treatment of many types of cancer, such as breast cancer, lung cancer, liver cancer, soft tissue sarcoma, and myeloma (Mohan and Rapoport, 2010). The anticancer mechanism of DOX mainly involves two ways: (1) intercalating into DNA strands and interfering with topoisomerase II mediated DNA repair (Nitiss, 2009); (2) generating reactive oxygen species (ROS) which leads to DNA damage (Sterba et al., 2013). Although DOX is a first-line anticancer agent, the serious side effects, such as cardiotoxicity, hepatotoxicity, and renal toxicity, have severely limited its wide utility in the clinic. The toxicity of DOX is mainly caused by DOX induced non-selective apoptosis in tissues and organs (Misra and Sahoo, 2010). Therefore, minimizing the toxicity of DOX is crucial.
Ascorbic acid (AA), as an antioxidant, has been recently used in cancer treatment (Sunil Kumar et al., 2015). At high concentrations, it can induce apoptosis in tumor cells by promoting ROS generation (Baek et al., 2017, Lim et al., 2016). It has been demonstrated that AA can enhance anti-tumor effect of numerous anticancer agents, such as DOX, paclitaxel, cisplatin, vincristine, and arsenic trioxide (Chiang et al., 1994, Huang et al., 2014, Kurbacher et al., 1996). However, the clinical anticancer application of AA is compromised by the need for extremely high dose and chemical instability (D'Souza et al., 2008). Palmitoyl ascorbate (PA), a lipophilic derivative of AA, is significantly more stable and more efficacious in suppressing the proliferation of cancer cells and DNA synthesis (Asada et al., 2012, Austria et al., 1997, Kato et al., 2011). With the similar mechanism of cell death as AA, PA can produce more ROS than AA (Miwa et al., 1988).The previous study showed that PA-encapsulated liposome at a lower dose is superior to free AA in suppressing tumor growth (Sawant et al., 2012). Furthermore, early investigations indicated that AA and its derivatives, especially PA, can effectively reduce the cardiac toxicity of DOX but not impede the anticancer activity of DOX (Shimpo et al., 1991). The cardiotoxicity of DOX is related to peroxidation of cardiac lipids (Myers et al., 1977), and PA can effectively alleviate the toxicity caused by DOX via sweeping the peroxidative products.
Since PA has a similar anticancer mechanism with DOX (produce ROS), and effectively reduces the toxicity of DOX, DOX and PA co-encapsulated liposome was designed in this study with the aid of nanocarriers. In addition to the characterization of the liposomes, the advantages of co-loaded liposome were studied by both in vitro and in vivo study. The cytotoxicity study and the in vivo tumor growth inhibition study were used to analyze synergistic anticancer effects, and the underlying mechanisms were studied by cellular uptake and immunohistochemical assay. The toxicity of DOX was assessed by H&E staining, and the intracellular distribution of DOX was evaluated by confocal laser scanning microscopy (CLSM). The research showed DOX and PA co-delivery liposome produced synergistic anticancer effects and exhibited superior pharmacokinetic profiles to overcome the clinical limitations of DOX caused by toxicity.
Section snippets
Materials
Doxorubicin hydrochloride (DOX·HCl) was obtained from Meilun Biology Technology Co., Ltd. (Dalian, China). PA was purchased from Sigma-Aldrich® (St. Louise, MO, USA). Phosphatidylcholine was purchased from Tywei Pharmaceutical Co., Ltd. (Shanghai, China). Cholesterol and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from J&K Scientific Ltd. (Shanghai, China). Amiloride, chlorpromazine and nystatin were purchased from Aladdin® (Shanghai, China). Sodium azide
Particle size, zeta potential and drug encapsulation
The sizes of the liposomes are in the range of 90–140 nm (Table 1), which was beneficial for the tumor accumulation by the EPR effect (Liao et al., 2014). In comparison with Blank-LPs, the sizes of drugs encapsulated liposomes were slightly increased, might indicate drugs were loaded into liposomes successfully. The narrow size distribution was evidenced by the PDI (lower than 0.3, shown in Table 1). The zeta potential of Blank-LPs was below − 20 mV (Table 1). When DOX was loaded into liposomes,
Conclusion
In summary, we have prepared DOX and PA co-encapsulated liposomes for improving cancer therapy of DOX. The co-loaded liposome (DOX:PA = 1:20) elevated the uptake of DOX and efficiently concentrated in the nucleus, therefore, it exhibited higher DOX accumulation in cancer cells, stronger anticancer effect but lower toxicity. Thus, co-delivery DOX and PA with liposome has significant potential in clinical combination treatment.
The following are the supplementary data related to this article. . The
Conflict of interest
The authors declare no competing financial interests.
Acknowledgement
This work was supported by the National Natural Science Foundation of China [no. 81473272, no. 81503148].
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