Liposomes are closed vesicles that are formed when phospholipids (constituents of biological membranes) are dispersed in water at relatively low concentrations. Since Bangham discovered liposomes in 1965, these liposomes have been studied on both basic and applied levels. Liposomes have contributed significantly to drug delivery, as well as analysis of cellular function, due to their mimicry of biological membranes and closed properties. Hybrid liposomes, which have been developed by Ueoka can be prepared by ultrasonication of vesicular and micellar molecules in a buffer solution (1). The physical properties of these liposomes such as shape, size, membrane fluidity, and the temperature of their phase transition can be controlled by changing the constituents and compositional ratios. Significantly prolonged survival in rats was obtained by using hybrid liposomes as drug carriers in the treatment of brain tumors (2). Hybrid liposomes without drugs were also demonstrated to inhibit the proliferation of various tumor cells in vitro and in vivo (3-7).

In this study, we examined inhibitory effects of hybrid liposomes composed of phosphatidylcholine and polyoxyethylenealkyl ether on the growth of lymphoma cells in vitro, in vivo and clinical applications.

We examined morphology of hybrid liposomes composed of dimyristoyl- phosphatidylcholine (DMPC) and 5 mol % polyoxyethylene(23)dodecyl ether (C₁₂(EO)₂₃) on the basis of electron microscopy. A clear solution of hybrid liposomes having hydrodynamic diameter of 80-100 nm could be kept over one month on the basis of dynamic light scattering measurements. It is noteworthy that hybrid liposomes of DMPC/5mol% C₁₂(EO)₂₃ having diameter of 100 nm could avoid the clearance by reticular endothelial system (RES) in vivo.

We next examined the fifty percent inhibitory concentration (IC₅₀) of hybrid liposomes on the growth of human B lymphoma cells (RAJI) in vitro. The IC₅₀ values were 0.16mM for hybrid liposomes of DMPC/5mol%C₁₂(EO)₂₃ and 0.34mM for liposomes on the basis of the DMPC concentration, respectively. These results indicate that the inhibitory effects of hybrid liposomes of DMPC/5mol% C₁₂(EO)₂₃ should be advantage as compared with single-component DMPC liposomes.

How do hybrid liposomes suppress the growth of tumor cells? Then, we tried to examine a plausible mechanism for inhibition of hybrid liposomes on the growth of RAJI cells. Firstly, the DNA content of RAJI cells treated with DMPC/5mol%C₁₂(EO)₂₃ hybrid liposomes was measured using flow cytometer. A marked increase in the apoptotic DNA was observed in RAJI cells treated with hybrid liposomes. Next, we examined the nuclear DNA fragmentation with hybrid liposomes using agarose gel electrophoresis. It is noteworthy that exposure of RAJI cells to the hybrid liposomes caused DNA fragmentation characteristic of apoptosis. In addition, we examined induction of apoptosis by hybrid liposomes on the basis of confocal microscopy using staining method with YO-PRO-1 and PI. The green color was observed in the cells after adding hybrid liposomes, although green color in the cells was not observed in the case of DMPC liposomes and C₁₂(EO)₂₃ micelles. These results indicate that hybrid liposomes should induce apoptosis for RAJI cells. The green color was also observed in fluorescence micrograph of RAJI cells treated with hybrid liposomes on the basis of TUNEL method.

Furthermore, we examined therapeutic effects of DMPC/10mol%C₁₂(EO)₂₃ hybrid liposomes on mice model of carcinoma in vivo. The prolonged survival of mice was obtained by the treatment with hybrid liposomes of DMPC/5mol%C₁₂(EO)₂₃. It is of interest that significantly prolonged survival rate (150%) was obtained after the treatment with hybrid liposomes in a dose of 136 and 203 mg/kg for DMPC.

Assessment of chronic toxicity of hybrid liposomes of DMPC/5mol%C₁₂(EO)₂₃ was carried out using normal rats after the injections of hybrid liposomes for six months. The number of red and white blood cells of rats treated with hybrid liposomes were within normal limits. In addition, all of the other biochemical parameters, such as ALP, GOT and GPT activities, as well as levels of albumin, urea nitrogen, creatinin, glucose, total protein, calcium, inorganic phosphorus, sodium, potassium and chloride, were the same as those observed in the controls. Furthermore, no weight loss was observed in the rats. These results indicate that hybrid liposomes should have no side effect in vivo.

Clinical applications of hybrid liposomes of DMPC/5mol%C₁₂(EO)₂₃ without any drug for patients with lymphoma were examined after passing the committee of bioethics. The clinical diagnosis of one severe-stage patient at that time was as follows : (a) No effects of all chemotherapeutics were observed. (b) It was suggested that the future lifetime would be a few months. So, we tried to examine the treatment with hybrid liposomes for the severe-stage patient. The hybrid liposomes were administered once every day in a daily dose of 11.0-16.5 mg/kg for DMPC. There were not abnormal findings on routine blood test and hematochemistry. The high g-GTP values recovered to the stage of normal ones after the administration of hybrid liposomes having a diameter of 100 nm. This result indicates that hybrid liposomes could avoid the reticular endothelial system. The prolonged survival more than one year was attained in the severe-stage patient with lymphoma after the intravenous injection of hybrid liposomes without any side effect. It is also worthy to note that the remarkable reduction of lymph node neoplasm (solid tumor) was obtained after the local administration of hybrid liposomes.

Moreover, we examined induction of apoptosis for lymph node neoplasm (solid tumor) using TUNEL method. Tissue sections were prepared from reduced lymph nodes and stained by a TUNEL-based method. The green color was obtained in the lymph node neoplasm (solid tumor) after the injection of hybrid liposomes, indicating that hybrid liposomes should induce apoptosis for lymph node. During the period of treatment, no abnormal findings were obtained on the basis of various hematological and biochemical tests. These results demonstrate that hybrid liposomes should be safe in clinical applications.

In conclusion, we clearly demonstrated that hybrid liposomes of DMPC/5mol%C₁₂(EO)₂₃ had highly inhibitory effects on the growth of tumor cells along with apoptosis. The noteworthy aspects of this study are as follows : (a) Hybrid liposomes of DMPC/5mol%C₁₂(EO)₂₃ having diameter of 100 nm could avoid the clearance by the reticular endothelial system (RES). (b) The fifty percent inhibitory concentration (IC₅₀) of hybrid liposomes on the growth of RAJI cells was one half of that of DMPC liposomes. (c) The induction of apoptosis by hybrid liposomes was verified for RAJI cells on the basis of flow cytometry, gel electrophoresis and staining method. (d) The significantly prolonged survival rate was obtained for mice model of carcinoma after the treatment with hybrid liposomes in vivo. (e) Hybrid liposomes should have no side effects using normal rats in vivo. (f) The remarkable reduction of lymph node neoplasm (solid tumor) and the prolonged survival for a severe-stage patient with lymphoma was attained after the treatment with hybrid liposomes in clinical applications.

It is very important that chemotherapy with drug-free hybrid liposomes could be established without any side effects. Such successful clinical applications of hybrid liposomes without drugs were for the first time.

References

1. J. Am. Chem. Soc., 110: 1588-1595, 1988.
2. Cancer Res., 56: 3986-3992, 1996.
3. Bioorg. Med. Chem. Lett., 9: 1937-1940, 1999.
4. Bioorg. Med. Chem. Lett., 12: 3251-3254, 2002.
5. Drug Delivery System, 14: 37-42, 1999.
6. Int. J. Cancer, 115: 377-382, 2005.
7. Bioorg. Med. Chem. Lett., 16: 782-785, 2006.