Safe and efficient pH sensitive tumor targeting modified liposomes with minimal cytotoxicity
Introduction
Numerous active anti-tumor drugs [1] have been developed in recent decades. However, quite a number of active anti-tumor drugs present equal cytotoxicity to normal cells while inhibiting tumor cells. Research of tumor targeting drug delivery systems hence aimed at increasing the efficiency of tumor targeting or reducing cytotoxicity to normal cells [2]. Nanoparticles have become a popular direction of tumor targeting development because of the “enhanced permeability and retention (EPR) effect” [3]. Various targeting mechanisms, including the EPR effect, receptor-mediated active targeting [4] and pH sensitive drug controlled release [5], have been widely applied in a rational design of targeting delivery systems.
Designed nanoparticles, based on the above 3 targeting mechanisms have been obtained through different materials such as surfactants [6], poly (amidoamines) [7] and carbon nanotubes [8]. Each kind of nanoparticle has higher efficiency than the homologous drug. Among the above-mentioned mechanisms, pH sensitive systems continuously attract a lot of attention in recent years since it is widely known that the pH level in tumor tissues is lower than in normal tissues. Dozens of synthesized pH-sensitive materials such as the polymers based on poly acrylic acid have been developed but cytotoxicity and non-degradability remain their drawbacks [9].
A liposome is a kind of biofilm analog consisting of phospholipids with minimal cytotoxicity. They can be self-assembly in an aqueous phase, so that their polar head groups are exposed to water and their hydrophobic tails are sandwiched in the middle. It is found that liposomes can target the reticulo-endothelial system and enhance the in vivo stability of drugs [10]. The main shortcomings of “conventional” liposomes (LPs) as tumor targeting drug carriers are their fast elimination from the blood circulation and their poor efficiency of targeting tumor cells. In previous research, polyethylene glycol was initially applied in modifying liposomes to extend the blood circulation times [11], [12], and different materials have been used as modifier to make the liposomes sensitive toward tumor tissue, including iron oxide [13], carbon nanotubes [8] and polyacrylic acid [14]. Although modified substances turn the LPs into functional liposomes, they will at the same time introduce cytotoxicity. Although some biocompatible molecules such as chitin have been utilized in preparing pH sensitive microspheres to act as controlled drug delivery system [15], the possibility of microspheres to be tumor targeting drug carriers or the cytotoxicity of the microsphere have not been previously examined [15].
As opposed to many polymerized materials. The degradation products of PASP is easily excreted or utilized by physiological processes in the body. PASP has free carboxylic acid groups and its conformation is diverse due to the ionization of carboxylate in different acidity conditions [2]. Carboxylate groups in the PASP chain are protonated at pH value below 5.0, whilst ionized at pH above 5.0, since the pKa of PASP is 4.88 [16]. With its fully biodegradable, water-soluble properties and pH-sensitivity, PASP has become an attractive candidate for drug delivery [17]. Dissociated negatively charged PASP can adhere to the positively charged head of phospholipids (choline group) through electrostatic forces and PASP modified liposomes (PLPs) can thus be obtained without any crosslinker or toxic monomer [18]. However, these electrostatic forces are too weak to maintain the stability of PLPs, which will weaken the targeting effect. One method to overcome this shortcoming is to graft a hydrophobic chain onto the PASP backbone which could anchor into the hydrophobic segment of the bilayer of the liposomes [2]. In our research, PASP-g-C8 was prepared based on the above discussion. Compared with PLPs, OPLPs present a better stability than PLPs due to the existing octyl group as “anchor”.
In view of these aspects and to develop a “high efficiency and minimal cytotoxicity” tumor targeting drug carrier, OPLPs containing both biocompatible PASP-g-C8 as acidic triggered molecules, and liposomes as the matrix, were investigated. In the present article, the broad spectrum of the anti-tumor drug Cytarabine (CYT) was chosen as the model drug. The pH sensitivity, the tumor targeting and in vitro cytotoxicity of OPLPs were assessed. Transmission electron microscope (TEM) was used to observe the morphology of OPLPs. Dynamic Light Scattering (DLS) and zeta potential measurements analyzer were used to characterize their surface property.
Section snippets
Materials
Polysuccinimide (PSI) was purchased from Luoyang Mining Run Environmental Protection Material Co. Ltd. (China). PASP was provided by Kang’en Chemical Co. Ltd. (China). Octylamine was purchased from Beijing Kehua Jingwei Tech. Co. Ltd. (China). Lecithin from soybean was purchased from Sinopharm Chemical Research Co. Ltd. (China) (phosphatidylcholine accounts for 95%, pH = 5.0–7.0). Cholesterol was purchased from Beijing Aoboxing Biotechnology Co. Ltd. (China). D2O was purchased from Sinopharm
Preparation and mechanism of PLPs
Considering that tumor targeting drug delivery systems should present environmental response and very low toxicity, the biocompatible molecule PASP was hence selected. Liposomes consist of phospholipid with positive charged choline as its polar head. The preparation process and operating mode of PLPs have been shown in Fig. 1. PASP ionized abundantly in dilute NaOH solution: 
COO− was largely produced and PLPs could be prepared via electrostatic adsorption (Fig. 1b). As seen in Fig. 1c, since
Conclusions and perspective
OPLPs, a pH-sensitive drug carrier, composed of liposomes and PASP-g-C8 were successfully prepared. The “anchor-chain” molecule (PASP-g-C8) was synthesized and successfully implanted into liposomes to obtain OPLPs. The moderate DS of the anchor chains helped PASP-g-C8 implant into liposomes, and the in vitro drug release profile corresponded to the variation of DS. The in vitro drug release studies showed that OPLPs with DS of 7.8% possessed optimum pH sensitivity and achieved no more than 40
Acknowledgements
The authors want to express their thanks for the supports from National Basic Research Program (973 Program) of China (2014CB745103), the (863) High Technology Project (2012AA021402), the Project sponsored by SRF for ROCS, SEM (LXJJ2012-001) and the Chinese Universities Scientific Fund (JD1417).
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