Novel 5-Fluorouracil Carbonate-Loaded Liposome: Preparation, In Vitro, and In Vivo Evaluation as an Antitumor Agent.

5-Fluorouracil (5-FU) is a chemotherapeutic drug against many types of cancers, especially colorectal cancer. However, its short plasma half-life and serious adverse reactions limit its wide clinical applications. To overcome these shortcomings, a novel lipophilic 5-FU carbonate [XL-01, (5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl) methyl tetradecyl carbonate] was designed, synthesized, and encapsulated into liposome (LipoXL-01) by a thin-film dispersion method through formulation screening and optimization. LipoXL-01 was characterized by a particle size of around 100 nm, polydispersity index of 0.200, ζ-potential value of -41 mV, encapsulation efficiency of 93.9%, and drug-loading efficiency of 11.6%. The cellular uptake of LipoXL-01 was increased in a concentration-dependent manner on HCT15 cells. LipoXL-01 could enhance the induction of cell apoptosis and the inhibition of cell migration and arrest the ability of the cell cycle at the S-phase on HCT15 cells better than 5-FU. Additionally, LipoXL-01 exhibited a slow drug release profile with a cumulative release rate of 12% in 8 h. The results of pharmacokinetic and biodistribution studies revealed that LipoXL-01 had a long plasma half-life (7.21 h) and a high tumor accumulation (733 nmol/g at 8 h). The in vivo antitumor effect study also showed that LipoXL-01 had more potent efficacy than 5-FU (65 vs 48% of the tumor-inhibition rate). Simultaneously, negligible systemic toxicity was observed via analyzing the body weight as well as hematological and pathological parameters in the tested mice. The current study suggested that LipoXL-01 might be a promising nanocandidate for chemotherapy of colorectal cancer.

Evaluation of antitumor efficacy of folate-poly(2-ethyl-2-oxazoline)-distearoyl phosphatidyl ethanolamine based liposome.

This study aims to explore and evaluate the antitumor efficacy of doxorubicin (DOX)-loaded liposomes containing the novel tri-block polymer folate-poly (2-ethyl-2-oxazoline)-distearoyl phosphatidyl ethanolamine (F-PEOz-DSPE), compared with folate-polyethylene glycol-distearoyl phosphatidyl ethanolamine (F-PEG-DSPE) to offer an alternative for PEG decorated carriers. PEOz, a pH-sensitive polymer, exhibits similar solubility and segmental flexibility to PEG. In our previous study, PEOz was employed to an F-PEOz-DSPE which was segmentally similar to F-PEG-DSPE and exhibited selective targeting and pH-sensitivity in tumor cells. In this work, DOX-loaded liposomes containing F-PEOz-DSPE (F-PEOz liposome) or F-PEG-DSPE (F-PEG liposome) were prepared. In vivo/vitro antitumor efficacy and biodistribution were compared between the two liposomes. F-PEOz liposome showed higher in vitro antitumor activity and significantly stronger inhibition of tumor growth in HeLa tumor-bearing nude mice (tumor inhibition rate, 81.20 vs 52.99% with the treatment of 9 mg/kg DOX-loaded F-PEOz liposome/F-PEG liposome) and much less toxicity than free DOX. In vivo fluorescence imaging experiment confirmed that F-PEOz liposome accumulated much more than F-PEG liposome in tumor. Based on the above, F-PEOz liposome may be a promising carrier in tumor chemotherapy to achieve better therapeutic efficacy.

Small-sized polymeric micelles incorporating docetaxel suppress distant metastases in the clinically-relevant 4T1 mouse breast cancer model.

BACKGROUND: The small size of ultra-small nanoparticles makes them suitable for lymphatic delivery, and many recent studies have examined their role in anti-metastasis therapy. However, the anti-metastatic efficacy of small-sized nanocarriers loaded with taxanes such as docetaxel has not yet been investigated in malignant breast cancer. METHODS: We encapsulated docetaxel using poly(D,L-lactide)1300-b-(polyethylene glycol-methoxy)2000 (mPEG2000-b-PDLLA1300) to construct polymeric micelles with a mean diameter of 16.76 nm (SPM). Patient-like 4T1/4T1luc breast cancer models in Balb/c mice, with resected and unresected primary tumors, were used to compare the therapeutic efficacies of SPM and free docetaxel (Duopafei) against breast cancer metastasis using bioluminescent imaging, lung nodule examination, and histological examination. RESULT: SPM showed similar efficacy to Duopafei in terms of growth suppression of primary tumors, but greater chemotherapeutic efficacy against breast cancer metastasis. In addition, lung tissue inflammation was decreased in the SPM-treated group, while many tumor cells and neutrophils were found in the Duopafei-treated group. CONCLUSION: Small-sized mPEG2000-b-PDLLA1300 micelles could provide an enhanced method of docetaxel delivery in breast cancer metastasis, and may represent a valid chemotherapeutic strategy in breast cancer patients with resected primary tumors.

Lipid-like gemcitabine diester-loaded liposomes for improved chemotherapy of pancreatic cancer.

Gemcitabine (GEM) is a non-selective chemotherapeutic agent used in the treatment of pancreatic cancer. Its antitumor efficacy is limited by a short plasma half-life and severe adverse reactions. To overcome these shortcomings, four novel lipid-like GEM diesters were synthesized and encapsulated into liposomes. Through optimization, dimyristoyl GEM (dmGEM)-loaded liposomes (LipodmGEM) were successfully obtained with an almost complete encapsulation efficiency. Compared to free GEM, LipodmGEM showed enhanced cellular uptake and cell apoptosis, improved inhibition of cell migration on AsPC-1 cells and a greatly extended half-life (7.22 vs. 1.78 h). LipodmGEM succeeded in enriching the drug in the tumor (5.28 vs. 0.03 µmol/g at 8 h), overcoming a major shortcoming of GEM, showed excellent anticancer efficacy in vivo and negligible systemic toxicity, superior to GEM. Attractive as well, suspensions of LipodmGEM remained stable at 2-10 °C away from light for no <2 years. Our results suggest that LipodmGEM might become of high interest for treating pancreatic cancer while the simple strategy we reported might be explored as well for converting other antitumor drugs with high water-solubility and short plasma half-life into attractive nanomedicines.

Synthesis of a novel polymeric material folate-poly(2-ethyl-2-oxazoline)-distearoyl phosphatidyl ethanolamine tri-block polymer for dual receptor and pH-sensitive targeting liposome.

The in vivo distribution of antitumor drugs is usually lack of selectivity, and thus, leading to a low efficacy of chemotherapy on cancers and high toxicity to normal cells. Receptor-mediated targeting liposome with pH-sensitivity as a dual drug delivery system is one of the efficient approaches to overcome the disadvantages. The study was to synthesize a novel smart polymeric material (folate-poly(2-ethyl-2-oxazoline)-distearoyl phosphatidyl ethanolamine, F-PEOz-DSPE), which can combine with the folate-receptor (FR) over-expressed on cancer cells and respond to pH changes in endosome-lysosome system in cancer cells to rapidly release drug simultaneously. The F-PEOz-DSPE was synthesized by the method of asymmetric synthesis of organic polymer and characterized by IR, (1)H-NMR, electrospray ionization (ESI)-MS and gel permeation chromatography (GPC). To investigate the properties of targeting and pH-sensitivity of F-PEOz-DSPE, blank liposomes, blank fluorescently labeled liposomes and doxorubicin (DOX)-loaded liposomes containing F-PEOz-DSPE or PEOz-DSPE or DSPE were prepared. The cytotoxicity, cellular uptake and drug cumulative release in vitro were investigated. Blank liposomes modified with PEOz block had little cytotoxicity in vitro. The liposomes containing F-PEOz-DSPE showed a higher affinity to human ovarian cancer cell SKOV3, a FR(+) cancer cells, than those with PEOz-DSPE. A higher drug cumulative release from DOX-loaded liposomes containing F-PEOz-DSPE or PEOz-DSPE in vitro was found in phosphate buffered saline at pH 5.0 medium than at pH 7.4. These results indicate that F-PEOz-DSPE exhibits selective targeting, pH-sensitivity and little cytotoxicity, and may be a promising polymeric material for dual receptor and pH-sensitive targeting liposome.

Antisense microRNA185 loaded liposome for efficient inhibition of the hepatic endogenous microRNA185 level.

MicroRNA185 (miR185), an endogenous noncoding RNA with 23 nucleotides, is one of key posttranscriptional modulators of cholesterol metabolism in hepatic cells. The antisense inhibitor of miR185 (miR185i) could decrease cholesterol level in vivo, providing a promising agent for anti-atherosclerosis strategy. In this work, a novel LipomiR185i was constructed by thin film hydration method and post-PEGylation as DOPE: DOTAP: Chol: DSPE-PEG2000 at the molar ratio of 1:1:1:0.1 with a nitrogen-to-phosphate ratio of 3, through the optimization of three cationic lipids (DOTAP, DODMA and DLin-MC3-DMA), six helper lipids (PC-98T, HSPC, DOPE, DMPC, DPPC and DSPC), different amounts and incorporation approaches of DSPE-PEG2000 and nitrogen-to-phosphate ratio. LipomiR185i was characterized with a particle size of 174 ± 11 nm, a zeta potential of 7.0 ± 3.3 mV, high encapsulation efficiency and transfection activity. It could protect miR185i from the rapid degradation by nucleases in serum, enhance cellular uptake and promote lysosomal escape in HepG2 cells. LipomiR185i could accumulate in the liver and remain for at least two weeks. More importantly, LipomiR185i significantly down-regulated the hepatic endogenous miR185 level in vitro and in vivo without significant tissue damage at 14 mg⋅kg-1. The construction of LipomiR185i provides a potential anti-atherosclerotic nanodrug as well as a platform for delivering small RNAs to the liver efficiently and safely.

Effect of a poloxamer 407-based thermosensitive gel on minimization of thermal injury to diaphragm during microwave ablation of the liver.

AIM: To assess the insulating effect of a poloxamer 407 (P407)-based gel during microwave ablation of liver adjacent to the diaphragm. METHODS: We prepared serial dilutions of P407, and 22.5% (w/w) concentration was identified as suitable for ablation procedures. Subsequently, microwave ablations were performed on the livers of 24 rabbits (gel, saline, control groups, n = 8 in each). The P407 solution and 0.9% normal saline were injected into the potential space between the diaphragm and liver in experimental groups. No barriers were applied to the controls. After microwave ablations, the frequency, size and degree of thermal injury were compared histologically among the three groups. Subsequently, another 8 rabbits were injected with the P407 solution and microwave ablation was performed. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and creatinine (Cr) in serum were tested at 1 d before microwave ablation and 3 and 7 d after operation. RESULTS: In vivo ablation thermal injury to the adjacent diaphragm was evaluated in the control, saline and 22.5% P407 gel groups (P = 0.001-0.040). However, there was no significant difference in the volume of ablation zone among the three groups (P > 0.05). Moreover, there were no statistical differences among the preoperative and postoperative gel groups according to the levels of ALT, AST, BUN and Cr in serum (all P > 0.05). CONCLUSION: Twenty-two point five percent P407 gel could be a more effective choice during microwave ablation of hepatic tumors adjacent to the diaphragm. Further studies for clinical translation are warranted.

Preparation, Characterization, and Antitumor Activities of Miriplatin-Loaded Liposomes.

Because of the insolubility of miriplatin in water, miriplatin and lipiodol suspension is the sole formulation of miriplatin approved in Japan to treat hepatocellular carcinoma by transcatheter arterial chemoembolization. Until now, there have been no reports of other pharmaceutical formulations of miriplatin except miriplatin/lipiodol suspension. In this study, we aimed not only to develop miriplatin-loaded liposomes (lipomiriplatins) which could be administrated systematically for tumors besides hepatocellular carcinoma but also to ascertain whether miriplatin, like its analog of NDDP, was a liposome-dependent antitumor agent. We found that miriplatin could be successfully incorporated into liposomes, and both the stability and antitumor activity of lipomiriplatins were independent of the liposomal compositions. Especially, HPLC was successfully established as the quantitative method for lipomiriplatins, which completely eliminated the interference of cholesterol. Lipomiriplatins possessed favorable colloidal properties (99.71 ± 0.56 nm, -50 mV), high drug-loading capacity (about 2.2 mg/mL), excellent entrapment efficiency (>95%), and robust stability. The remarkable antitumor activities of lipomiriplatin were proved to be mediated by inducing cell apoptosis and were comparable to that of the commercial cisplatin and oxaliplatin injections, indicating that lipomiriplatins showed great promise for future potential clinical application via systematic administration.

Inhibition of murine breast cancer growth and metastasis by survivin-targeted siRNA using disulfide cross-linked linear PEI.

Biodegradable disulfide-containing polyethyleneimine (PEI) derivatives showed great potential as siRNA vectors for the treatment of cancer due to the reduction-sensitive property. In this study, we developed and characterized a hyperbranched disulfide cross-linked PEI (lPEI-SS) based on linear PEI (lPEI) by ring-opening reaction of propylene sulfide. We evaluated the efficiency of lPEI-SS as a siRNA vector in vitro with luciferase reporter gene system, and investigated the anti-tumor efficacy of survivin-targeted siRNA (siRNA(sur)) on 4T1 murine breast cancer model using lPEI-SS synthesized here. Results from cytotoxicity and hemolysis assay proved that lPEI-SS showed favorable cell and blood compatibility. lPEI-SS/siRNA polyplexes prepared under the optimized condition were compact spherical particles with the average size of 229.0nm and zeta potential of 42.67mV. Cellular uptake of lPEI-SS/siRNA polyplexes was significantly improved due to the higher branching degree of lPEI-SS over the parent lPEI. lPEI-SS/siRNA(sur) exhibited great anti-proliferation effect on 4T1 cell line, which was found to be caused by the induction of apoptosis. Most importantly, results of tumor volume, tumor weight and histological observation demonstrated that lPEI-SS/siRNA(sur) polyplexes effectively inhibited the tumor growth and metastasis of 4T1 murine breast cancer model.

Local targeted therapy of liver metastasis from colon cancer by galactosylated liposome encapsulated with doxorubicin.

Since regional drug administration enables to maintain a high drug concentration within tumors, we compared the plasma concentration and biodistribution of doxorubicin (Dox) from drug-loaded conventional liposomes by local or systemic administration. The results demonstrated that drug concentration was substantially improved in liver as well as a decrease in blood and other organs by spleen injection mimicking portal vein perfusion (regional administration). To further investigate the targeted therapeutic effect of galactosylated liposome encapsulated doxorubicin (Dox) by regional administration, liver targeting liposomes were prepared by incorporating galactosylated-DPPE to conventional liposomes. Liposome uptake and targeting were verified in vitro and in vivo by fluorescence microscopy and xenogen IVIS imaging system, respectively. The results showed that galactose targeted liposomes presented a stronger specific cell uptake by human hepatocellular carcinoma HepG2 cells compared to the non-targeted liposomes. In vivo fluorescence imaging showed that the intra-hepatic deposition of conventional and galactosylated liposomes via spleen injection was more than that via tail vein administration, and galactosylated liposomes had higher fluorescent intensity over conventional liposomes in the liver post spleen administration. The anti-tumor effect of various drug administration routes for both liposomal formulations was evaluated using a murine liver metastasis model of colon cancer. The results indicated that tumor progression in the liver and mesenteric lymph nodes was significantly suppressed by Dox-loaded galactosylated liposomes via spleen injection, while no significance was observed in non-targeted formulations. Our data indicated that local perfusion of galactosylated liposomal doxorubicin had a great promise for the treatment of liver metastasis from colon cancer.