Nanocapsules: lipid-coated aggregates of cisplatin with high cytotoxicity

Abstract

Cisplatin is one of the most widely used agents in the treatment of solid tumors, but its clinical utility is limited by toxicity. The development of less toxic, liposomal formulations of cisplatin has been hampered by the low water solubility and low lipophilicity of cisplatin, resulting in very low encapsulation efficiencies. We describe a novel method allowing the efficient encapsulation of cisplatin in a lipid formulation; it is based on repeated freezing and thawing of a concentrated solution of cisplatin in the presence of negatively charged phospholipids. The method is unique in that it generates nanocapsules, which are small aggregates of cisplatin covered by a single lipid bilayer. The nanocapsules have an unprecedented drug-to-lipid ratio and an in vitro cytotoxicity up to 1000-fold higher than the free drug. Analysis of the mechanism of nanocapsule formation suggests that the method may be generalized to other drugs showing low water solubility and lipophilicity.The clinical use of cis-diamminedichloroplatinum(II) (cisplatin) and many of its analogs faces three major problems: 1) serious dose-limiting toxicities in particular nephrotoxicity and neurotoxicity; 2) rapid inactivation of the drug as a result of complexation to plasma and tissue proteins; and 3) the frequent occurrence of platinum resistance [1][2][3][4][5] . In general, these problems can be reduced by shielding of a drug from the extracellular environment by means of a lipid coating. However, in many cases this approach fails because of inefficient encapsulation of the drug in lipid formulations resulting in low drug uptake by the tumor 6 . This is particularly true for cisplatin: the low water solubility and low lipophilicity of cisplatin result in lipid formulations with a very low drug-to-lipid ratio 7-9 . One approach is to synthesize lipophilic derivatives of cisplatin that can be efficiently encapsulated in large multilamellar liposomes 10 . Here, we describe a new method to efficiently encapsulate native, non-derivitized cisplatin in a lipid formulation. Nanocapsules of cisplatinOur method involves hydration of a dry lipid film composed of equimolar amounts of dioleoyl-phosphatidylserine (PS) and dioleoyl-phosphatidylcholine (PC), with a buffered solution (pH 7.4) of 5 mM cisplatin followed by 10 freeze-thaw (FT) cycles, and removal of free (extravesicular) cisplatin by centrifugation. The cisplatincontaining lipid suspension (cisPt-PS/PC) was extremely cytotoxic (Fig. 1a) with a typical IC 50 (the drug concentration at which cell growth is inhibited by 50%) of approximately 2 nM as compared with 0.5 µM for the free drug (conventional cisplatin). A lipid suspension not loaded with cisplatin (blank) was not cytotoxic, and mixing conventional cisplatin with the blank lipid suspension did not increase the cytotoxicity of cisplatin.Omitting the freeze-thaw step, or leaving out the negatively charged PS in the lipid mixture, resulted in a dramatic decrease in cytotoxicity (Fig. 1b). This decrease in cytotoxicity was paralleled by a sim...

Micronuclei to detect in vivo chemotherapy damage in a p53 mutated solid tumour

“…In vivo induction of apoptosis in established 9L tumours was assessed after local tumour irradiation (20 Gy) or systemic injection of a nontoxic dose of cisplatin (1 mg kg À1 ) (Burger et al, 2002). Data from TUNEL assays (Figure 2A -C) and from antiactive caspase-3 immunostaining (data not shown) revealed the presence of apoptotic cells only after irradiation but not after cisplatin treatment.…”

New clues for platinum antitumor chemistry: Kinetically controlled metal binding to DNA

“…A different approach toward prodrugs has been the formation of membraneencapsulated cisplatin, which can be formed only by repeated freeze-thaw cycles (75). The resulting products are highly soluble and appear to result in slow releases and high activity (76).…”

Nanocapsules: A Novel Formulation Technology For Platinum-Based Anticancer Drugs

“…Key factors are likely the low water solubility (8 mM at 37°C) and low lipophilicity of cisplatin [51], which lead to liposomal formulations with relatively low drug-to-lipid molar ratios (in the order of 0.05-0.15). Burger et al recently discovered an alternative method to encapsulate cisplatin in a lipid formulation with superior efficiency [52]. The method, which takes advantage of the limited solubility of the drug in water, produces cisplatin nanocapsules, nanoprecipitates of cisplatin that are surrounded by a lipid layer.…”

“…Nanocapsules have a characteristic bean shape with a typical width of 50 nm and a length between 50 and 250 nm (Figure 2). Importantly, the cisplatin nanocapsules have an unprecedented cisplatin-to-phospholipid molar ratio of 11:1, and an in vitro cytotoxicity up to 100-fold higher than the free drug (Figure 2) [52].…”

“…Instead of DOPS, the negatively charged phospholipids dioleoyl-phosphatidylglycerol (DOPG) and dioleoyl-phosphatidic acid (DOPA) can be used to prepare cisplatin nanocapsules [52,53]. The formation of nanocapsules is incompatible with high chloride concentration and alkaline pH [52], conditions that prevent the formation of the aqua species of cisplatin.…”