Concomitant delivery of doxorubicin and cisplatin through liposome-based thermosensitive nanoparticles: perspective in the treatment of cancer in animal models.

The temperature sensitive liposomal formulations are a promising tool to improve the therapeutic index of the drugs with minimal toxicity. The aim of this study was to investigate the potential of concomitant delivery of cisplatin (Cis) and doxorubicin (Dox) containing thermosensitive liposomes (TSLs) with mild hyperthermia against cancer in vitro and in vivo. The polyethylene glycol coated DPPC/DSPC, thermosensitive and DSPC, non-thermosensitive liposomes incorporating Cis and Dox were prepared and characterized. A conventional Differential Scanning Calorimetry (DSC) technique and Fourier Transform Infrared Spectroscopy (FT-IR) were applied to study drug-phospholipid interaction and compatibility. The chemotherapeutic efficacy of these formulations was evaluated in benzo[a]pyrene (BaP) induced fibrosarcoma under hyperthermic condition. The size diameter of prepared thermosensitive liposomes was measured to be 120 ± 10 nm. The DSC data exhibited the changes in the curves of DSPC + Dox and DSPC + Cis while comparing the pure DSPC and drugs. However, the FITR showed same spectrum of phospholipids and drugs individually and in the mixture as well. The data showed higher efficacy of Cis-Dox-TSL as 84% inhibition in tumor growth was recorded in this group of animals in hyperthermic condition. The Kaplan-Meir curve revealed, 100% and 80% survival of the animals in the groups treated with Cis-Dox-TSL under hyperthermia and Cis-Dox-NTSL without hyperthermia, respectively. However, Cis-TSL as well as Dox-TSL exhibited 50% survival, while only 20% survival was recorded in the groups of animals treated with Dox-NTSL and Cis-NTSL. The flow cytometry analysis revealed that Cis-Dox-NTSL augments the induction of apoptosis in the tumor cells which was recorded as 18%. As expected, Cis-Dox-TSL showed great potential as 39% of cells were measured as apoptotic cells, significantly very high in comparison to Cis-Dox-NTSL, Dox-TSL and Cis-TSL as well. The apoptotic analysis of the cells by flow cytometry clearly indicated the effect of hyperthermia during the treatment while Cis-Dox-TSL formulation was administered. Finally, the immunohistochemical analysis of the tumor tissues by confocal microscopy exhibited several fold increases in the expression of pAkt in the animals treated with vehicles in Sham-NTSL as well as Sham-TSL. However, Cis-Dox-TSL showed great reduction in the expression of Akt, as it declined by 11-fold. The results of the present study directed the role of concomitant delivery doxorubicin and cisplatin containing thermosensitive liposomes under hyperthermic conditions for the development of a novel therapeutic strategy for the treatment of cancer.

In vitro and in vivo activity of thermosensitive liposomes loaded with doxorubicin and cisplatin.

Thermosensitive liposomes loaded with cisplatin and doxorubicin composed of DPPC, DSPC, and DPPE-PEG5000 with different ratios were prepared by thin film hydration method. The Differential Scanning Calorimetry (DSC) curves showed that the liposomes composed of DPPC-DSPC-DPPE-PEG5000 with phospholipid ratio 95:5:0.05 w/w were a suitable formulation as thermosensitive liposomes with a DSC peak at 42.1 °C. The effect of doxorubicin and cisplatin encapsulated non-thermosensitive and thermosensitive liposomes on cellular proliferation and IC50 in SKBR3 & MDA-MB-231 breast cancer and PC-3 & LNcaP prostate cancer cell lines was investigated. The results showed that doxorubicin loaded into thermosensitive liposomes showed 20-fold decrease in the IC50 at 42 °C while comparing it with the same at 37 °C. Also, the results showed a more than 35-fold and 12-fold decrease in the IC50 of cisplatin thermosensitive liposomes at 42 °C, while compared with free cisplatin and cisplatin thermosensitive liposomes at any temperature. The in vivo results showed that the effect of doxorubicin encapsulated thermosensitive liposomes at hyperthermic conditions during the treatment as the tumor growth inhibition was measured 1.5-fold higher than any of the liposomal formulations of doxorubicin. It was also noticed that the tumor volume reduced to 150 mm3 in doxorubicin thermosensitive liposomes (G8) after 3 weeks during the treatment, but increased to 196 mm3 after 4 weeks. The Kaplan-Meir curve showed the 100% survival of the animals from G8 (thermosensitive liposomes containing doxorubicin plus hyperthermia) after 12 weeks. The flow cytometry data revealed more than 25% apoptotic cells and 6.25% necrotic cells in the tumor cells from the tissues of the G8 group of the animals. The results clearly indicate the superior efficacy of doxorubicin and cisplatin containing thermosensitive liposomes treatment during hyperthermia.

Liposomal formulation of glycosphingolipids from Sphingomonas paucimobilis induces antitumour immunity in mice.

Natural Killer T (NKT) cells play an important role in host's anti-tumour immune response. Glycosphingolipids (GSLs) isolated from Sphingomonas paucimobilis have the ability to stimulate NKT cells. In this study, the activity of free GSLs or GSLs-incorporated liposomes (glycosphingosomes) was investigated against dimethyl-α-benzanthracene (DMBA)-induced tumours in mice. The anti-tumour immunity of GSLs- or glycosphingosomes-loaded bone marrow-derived dendritic cells (BMDCs) was investigated in tumour-bearing mice. The Immunotherapeutic potential of co-administration of liposomal doxorubicin (Lip-Dox) and GSLs or glycosphingosomes was assessed by measuring cytokine levels and VEGF in the tumour tissues. Pretreatment with glycosphingosomes significantly delayed the frequency of tumour formation. Immunotherapy with glycosphingosomes-loaded BMDCs increased serum IFN-γ level and survival rate in mice. The effect of immunotherapy was dependent on effector functions of NK cells because the depletion of NK cells abolished the effects of immunotherapy. There was reduced tumour growth with low expression of VEGF in the group of mice treated with glycosphingosomes and Lip-Dox combination. Moreover, the splenocytes secreted higher levels of IFN-γ, IL-12 and lower TGF-ß level. The results of this study indicate that glycosphingosomes can induce better antitumour immunity and may be considered a novel formulation in antitumour therapy.

Combination of glycosphingosomes and liposomal doxorubicin shows increased activity against dimethyl-α-benzanthracene-induced fibrosarcoma in mice.

The present study aimed to assess the antitumor effect of glycosphingolipid-incorporated liposomes (glycosphingosomes) in combination with liposomal doxorubicin (Lip-Dox) in a mouse model of fibrosarcoma. Glycosphingosomes were prepared by incorporating glycosphingolipids isolated from Sphingomonas paucimobilis into the liposomes of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, cholesterol, and cardiolipin. Tumors were induced by administering dimethyl-α-benzanthracene, and tumor-bearing mice were treated with various formulations of Dox, including free Dox, Lip-Dox, or glycosphingosomes + Lip-Dox. Mice were observed for 90 days to monitor their survival and tumor size. Free Dox, but not Lip-Dox or a combination of glycosphingosomes and Lip-Dox, caused the substantial depletion of leukocytes and significantly increased the levels of lactate dehydrogenase and creatinine kinase in mice. Tumor-bearing mice treated with a combination of glycosphingosomes and Lip-Dox showed restricted tumor growth and increased survival when compared to those treated with free Dox or Lip-Dox. The results of the present study suggest that a combination of glycosphingosomes and Lip-Dox may prove to be very effective in the treatment of tumors.

Coadministration of doxorubicin and etoposide loaded in camel milk phospholipids liposomes showed increased antitumor activity in a murine model.

Small unilamellar vesicles from camel milk phospholipids (CML) mixture or from 1,2 dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) were prepared, and anticancer drugs doxorubicin (Dox) or etoposide (ETP) were loaded. Liposomal formulations were used against fibrosarcoma in a murine model. Results showed a very high percentage of Dox encapsulation (~98%) in liposomes (Lip) prepared from CML-Lip or DPPC-Lip, whereas the percentage of encapsulations of ETP was on the lower side, 22% of CML-Lip and 18% for DPPC-Lip. Differential scanning calorimetry curves show that Dox enhances the lamellar formation in CML-Lip, whereas ETP enhances the nonlamellar formation. Differential scanning calorimetry curves also showed that the presence of Dox and ETP together into DPPC-Lip produced the interdigitation effect. The in vivo anticancer activity of liposomal formulations of Dox or ETP or a combination of both was assessed against benzopyrene (BAP)-induced fibrosarcoma in a murine model. Tumor-bearing mice treated with a combination of Dox and ETP loaded into CML-Lip showed increased survival and reduced tumor growth compared to other groups, including the combination of Dox and ETP in DPPC-Lip. Fibrosarcoma-bearing mice treated with a combination of free (Dox + ETP) showed much higher tumor growth compared to those groups treated with CML-Lip-(Dox + ETP) or DPPC-Lip-(Dox + ETP). Immunohistochemical study was also performed to show the expression of tumor-suppressor PTEN, and it was found that the tumor tissues from the group of mice treated with a combination of free (Dox + ETP) showed greater loss of cytoplasmic PTEN than tumor tissues obtained from the groups of mice treated with CML-Lip-(Dox + ETP) or DPPC-Lip-(Dox + ETP).

Etoposide incorporated into camel milk phospholipids liposomes shows increased activity against fibrosarcoma in a mouse model.

Phospholipids were isolated from camel milk and identified by using high performance liquid chromatography and gas chromatography-mass spectrometry (GC/MS). Anticancer drug etoposide (ETP) was entrapped in liposomes, prepared from camel milk phospholipids, to determine its activity against fibrosarcoma in a murine model. Fibrosarcoma was induced in mice by injecting benzopyrene (BAP) and tumor-bearing mice were treated with various formulations of etoposide, including etoposide entrapped camel milk phospholipids liposomes (ETP-Cam-liposomes) and etoposide-loaded DPPC-liposomes (ETP-DPPC-liposomes). The tumor-bearing mice treated with ETP-Cam-liposomes showed slow progression of tumors and increased survival compared to free ETP or ETP-DPPC-liposomes. These results suggest that ETP-Cam-liposomes may prove to be a better drug delivery system for anticancer drugs.