Pharmaceutical Nanotechnology
Tumor-targeted PE38KDEL delivery via PEGylated anti-HER2 immunoliposomes

https://doi.org/10.1016/j.ijpharm.2009.03.018Get rights and content

Abstract

We previously reported the development of PE38KDEL-loaded anti-HER2 poly(lactic-co-glycolic acid) (PLGA) nanoparticles that bind and internalize in HER2-overexpressing breast cancer cells, enabling potent anti-tumor activity. To overcome the problems associated with the short half-lives of this drug delivery system, we have constructed PE38KDEL-loaded anti-HER2 PEGylated liposomes (PE-HER-liposomes). PE-HER-liposomes were constructed with Fab′ of recombinant humanized anti-HER2 monoclonal antibody (anti-HER2 Fab′) covalently linked to PEGylated liposomes containing PE38KDEL (PE-liposomes). We attached anti-HER2 Fab′ to the terminus of PEG (polyethylene glycol) on PEGylated liposomes. Incorporation of pyridylthiopropionoylamino-PEG-distearoylphosphatidylethanolamine (PDP-PEG-DSPE) into PEGylated liposomes followed by mild thiolysis of the PDP groups resulted in the formation of reactive thiol groups at the periphery of the liposomes. Efficient attachment of maleimide-derivatized anti-HER2 Fab′ took place under mild conditions. The characterization of PE-HER-liposomes, such as particle size, was evaluated by dynamic light-scattering detector. The Micro BCA method was used to determine the encapsulation efficiency of PE38KDEL and the quantity of conjugated Fab′. Flow cytometry and confocal microscopy showed that PE-HER-liposomes possessed receptor-specific binding and internalization for HER2-overexpressing SK-BR3 cells. Remarkably, PE-HER-liposomes were more cytotoxic than non-targeted PE-liposomes in HER2-overexpressing breast cancer cells. In conclusion, PE-HER-liposomes could serve as a promising therapeutic candidate for the treatment of HER2-overexpressing breast cancers.

Introduction

Immunotoxins are composed of antibodies or antibody fragments linked to a toxin, such as ricin or Pseudomonas exotoxin A (PE) (Pastan et al., 2006). Currently, PE-based immunotoxins have been widely applied to clinical trials. However, the clinical use of PE-based immunotoxins is limited by their severe non-specific toxicity (Pastan et al., 2007). PE38KDEL is a 38 kDa mutant form of PE and exhibited superior anti-tumor activity and less non-specific toxicity (Gao et al., 2008). We previously reported the development of PE38KDEL-loaded anti-HER2 poly(lactic-co-glycolic acid) (PLGA) nanoparticles (PE-NPs-HER) that bind and internalize in HER2-overexpressing breast cancer cells, enabling potent anti-tumor activity (Chen et al., 2008). The results showed that PE-NPs-HER were well tolerated in mice with a much higher MTD (maximally tolerated dose) than the control immunotoxins PE-HER constructed by chemically coupling PE38KDEL to rhuMAbHER2 (recombinant humanized anti-HER2 monoclonal antibody). Moreover, PE-NPs-HER was shown to exhibit a much better therapeutic efficacy in HER2-overexpressing breast cancer bearing mice.

However, the further development of PE-NPs-HER in clinical application in breast cancer treatment was still hampered by several problems. For example, the nanoparticles without PEG (polyethylene glycol) coating would be rapidly cleared from the circulation in a short time (Moghimi and Szebeni, 2003). Furthermore, the initial burst effect occurred frequently in PLGA nanoparticles, resulting in a rapid drug release. Although PLGA is a kind of biodegradable and biocompatible components that was previously approved by the Food and Drug Administration (FDA) for clinical use, there have not been any drug-loaded PLGA nanoparticles that are under clinical application. Liposomes (phospholipid bilayer vesicles) are the most advanced of the particulate drug carriers (Sapra and Allen, 2003). In 2007, FDA granted approval to doxorubicin HCl liposomes injection (Doxil, Alza Corporation) for use in combination therapy in patients with multiple myeloma. Many other liposomal anticancer drugs are also in clinical trials (Sapra and Allen, 2003). However, one of the major drawbacks of classical liposomes is their rapid clearance from the circulation (Allen and Cullis, 2004). Several reports showed that stealth liposomes (e.g. PEGylated vesicles) could avoid the uptake of RES (reticuloendothelial system), thus resulting in a much longer time in circulation (Moghimi and Szebeni, 2003, Allen and Cullis, 2004). Moreover, PEGylated liposomes are stable in the blood circulation and could even be stable in water solution for more than 1 week, indicating that PEGylated liposomes meets the requirement for an effective drug delivery system (Lv et al., 2005, Erjavec et al., 2006, Song et al., 2006). Hence, long-circulated PEGylated liposomes represent a novel strategy for cancer targeted therapy.

Immunoliposomes, in which monoclonal antibody (mAb) fragments are conjugated to liposomes, represent the next generation of molecularly targeted drug delivery systems (Sapra and Allen, 2003). By combining the tumor targeting properties of mAbs with the pharmacokinetics and drug delivery advantages of liposomes, immunoliposomes offer the promise of selective drug delivery to tumor cells, including internalization and intracellular drug release within targeted cells. In the present study, we developed a novel PE38KDEL-loaded PEGylated liposomes conjugated with anti-HER2 Fab′ (PE-HER-liposomes). The results showed that PE-HER-liposomes (less than 200 nm) have a high drug loading and antibody conjugation efficiency. Flow cytometry and confocal study demonstrated that PE-HER-liposomes could be efficiently bound to and were internalized into HER2-overexpressing tumor cells, resulting in potent cytotoxicity. Thus, the generated PE-HER-liposomes could represent a novel strategy for HER2-overexpressing breast cancer therapy.

Section snippets

Materials

Egg phosphatidylcholine (EPC) and methoxy (polyethylene glycol) (2000) distearoylphos-phatidylethanolamine (mPEG-DSPE) were purchased from Lipoid GmbH (Ludwigshafen, Germany). PDP-PEG-DSPE, pyridyldithiopropionoylamino poly(ethylene glycol) distearoylphosphatidylethanolamine were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Cholesterol (CHOL), dithiothreitol (DTT), N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES), N-succinimidyl-4-(p-maleimidophenyl)-butyrate (SMPB)

Particle size and zeta potential

Preparation of PEGylated immunoliposomes was carried out as summarized in Fig. 1. The resultant PE38KDEL-loaded PEGylated liposomes were sized at 181.40 ± 4.59 nm (mean ± SD; n = 3) (Table 1), which was not much larger than drug-free PEGylated liposomes (P > 0.05, Student's unpaired t-test). That PE38KDEL encapsulation did not increase the size was also observed in PEGylated immunoliposomes. However, compared with the size of PE-liposomes, there was an increase of 13 nm in the size of PE-HER-liposomes,

Conclusion

The clinical use of PE-based immunotoxins is severely hampered by the nonspecific toxicity of PE, which is usually characterized by hepatoxicity. Previously, we developed PE38KDEL-loaded anti-HER2 PLGA nanoparticles (PE-NPs-HER), which exhibited potent anti-tumor activity and less hepatoxicity. However, the short half-life, initial burst and biocompatibility restricted the clinical application of PE-NPs-HER. In the present study, we successfully constructed PE38KDEL-loaded PEGylated

Acknowledgements

We thank Ms. Yang Yang and Ms. Jing Xu for their technical assistance. This work was supported in part by the grants from National Natural Science Foundation of China, Shanghai Commission of Science & Technology, Ministry of Science and Technology of China (973&863 program projects), Pudong Commission of Science and Technology of Shanghai.

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