Optimization of a cationic liposome-based gene delivery system for the application of miR-145 in anticancer therapeutics.

In order to improve the delivery efficiency of microRNA (miRNA or miR)-145, the present study examined several factors which may affect cationic liposome (CL)-based transfection, including the hydration medium used for the preparation of liposomes, the quantity of the plasmid, the molar ratio of N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP)/cholesterol (chol), or DOTAP/chol, and the weight ratio of DOTAP/DNA. In order to enhance the transfection efficiency, protamine was selected as a DNA-condensing agent to form liposome­protamine­DNA (LPD) ternary complexes. An agarose gel retardation assay was used to examine the DNA binding affinity of the CLs. Following transfection, GFP fluorescence images were captured and flow cytometry was performed to determine the transfection efficiency. Furthermore, an MTT assay was performed to determine the cytotoxicity of the liposome complexes. The final optimal conditions were as follows: 5% glucose as the hydration medium, a molar ratio of DOTAP/chol at 3:1 for the preparation of CLs, a weight ratio of DOTAP/protamine/DNA of 3:0.5:1, with 8 µg plasmid added for the preparation of the LPD complexes. In vitro, the LPD complexes exhibited an enhanced transfection efficiency and low cytotoxicity, which indicated that the presented LPD vector enhanced the transfection efficiency of the CLs. The HepG2 cells were found to have the lowest expression levels of miR­145 out of the cell lines tested (A549, BGC-823, HepG2, HeLa, LoVo and MCF-7). Following the transient transfection of the HepG2 cells with miR­145, the results revealed that the overexpression of miR­145 inhibited the proliferation of the HepG2 cells and downregulated the expression of cyclin-dependent kinase 6 (CDK6), cyclinD1, c-myc, and Sp1 transcription factor (Sp1). In conclusion, in this study, we optimized a liposome­based delivery system for the efficient delivery of miR­145 into cancer cells. This may provide a foundation for further research into the use of miR­145 in anticancer therapeutics.

[Inhibitive effect of local perfusion of tanshinone II A nanoparticles on MMP-2 secretion].

OBJECTIVE: To test the effect of Tanshinone II A nanoparticles on the expression of MMP-2. METHODS: Thirty five male New Zealand white rabbits were randomly divided into three groups. In group A (15 rabbits), the carotid arteries of the rabbits were stripped. In group B (5 rabbits) and C (15 rabbits), nanoparticles and Tanshinone II A nanoparticles were perfused respectively in the denudated arteries of the rabbits. The neointimal areas and the IOD values of MMP-2 secretion were measured. RESULTS: A significant reduction of neointimal hyperplasia in group C was found compared to the other two groups in terms of the intimal area and the intima-media ratio. Group C had significant lower IOD values than group A. CONCLUSION: Local administration of nanoparticles with incorporated Tanshinone I A not only inhibits neointimal hyperplasia but also inhibits the expression of MMP-2 in stripped arteries.

Solid lipid nanoparticles of mitoxantrone for local injection against breast cancer and its lymph node metastases.

In an attempt to improve the therapeutic effect of mitoxantrone (MTO) against breast cancer and its lymph node metastases, solid lipid nanoparticles (SLN) of MTO were prepared, characterized and evaluated on mice. Film dispersion-ultrasonication method was used to prepare MTO-SLN, optimized by central composite design. MTO-SLN were prepared with a mean size of 61 nm, drug content (DC) of 4.18+/-0.10% and encapsulation yield (EY) of 87.23+/-2.16%. MTO-SLN were lyophilized and their mean size became 79 nm without significant change in DC and EY. The in vitro release study revealed a profile of sustained release of MTO from MTO-SLN without burst effect: the cumulative release rate Q24 h = 25.86 +/- 0.82%, t50 = (5.25 +/- 1.10)d and t90 = (28.38 +/- 4.50)d. The drug concentration of MTO-SLN in local lymph nodes was much higher and the drug concentrations in other tissues lower than that of MTO solution (MTO-Soln). Human MCF-7 breast cancer in nude mice and animal model of P388 lymph node metastases in Kunming mice were applied to investigate the therapeutic effects. There was no observed toxicity to the main tissues after local injection of MTO-SLN, but, for MTO-Soln, medium to serious toxicity to liver and lung was produced. The percent inhibition of MTO-SLN against breast cancer was 81.81 +/- 14.03%, while that of MTO-Soln with a double dose was 82.86 +/- 11.13%. The tests for lymph node metastases showed that MTO-SLN gave a mean size of lymph node of 41.85 +/- 27.42 mm3, while that of the MTO-Soln was 119.32 +/- 57.30 mm3 and that of the placebo was 186.83 +/- 77.71 mm3. This study opens a new perspective of active delivery of antitumor drug against breast cancer and its lymph node metastases with inspiring therapeutic effect and little side effects.

Mitoxantrone-loaded BSA nanospheres and chitosan nanospheres for local injection against breast cancer and its lymph node metastases. II: Tissue distribution and pharmacodynamics.

Bovine serum albumin (BSA) and chitosan (CS) nanospheres of mitoxantrone (MTO) were comparatively evaluated in terms of tissue distribution, acute toxicity and therapeutic efficiency against breast cancer and its lymph node metastases. After local injection in rats, MTO nanospheres showed a slower elimination rate and a much higher drug concentration in lymph nodes compared with MTO solution, and a lower drug concentration in other tissues. There was no observed acute toxicity to the main tissues of Kunming mice after local injection of MTO-BSA-NS. Mild toxicity to liver and lung was observed for MTO-CS-NS, but, for MTO solution, severe toxicity to liver and lung and much lower number of white blood cells were observed. Human MCF-7 breast cancer in nude mice and animal model of P388 lymph node metastases in Kunming mice were applied to investigate the therapeutic efficiency. The inhibition rate of the nanospheres against breast cancer was much higher than that of MTO solution, and lymph node metastases were efficiently inhibited by the nanospheres, especially MTO-BSA-NS.

Mitoxantrone-loaded BSA nanospheres and chitosan nanospheres for local injection against breast cancer and its lymph node metastases. I: Formulation and in vitro characterization.

Positively charged mitoxantrone (MTO) was absorbed by negatively charged blank bovine serum albumin (BSA) and chitosan (CS) nanospheres to form MTO-BSA-NS and MTO-CS-NS, respectively. In addition to other conditions, values of pH of every step were optimized. On optimized conditions, MTO-BSA-NS of a mean size of 77 nm with an encapsulation yield (EY) of (98.86+/-1.43)% [drug loading rate (DL) (19.82+/-0.29)%] and MTO-CS-NS of a mean size of 75 nm with an EY of (97.57+/-1.00)% [DL (9.78+/-0.10)%] were obtained. After lyophilization and sterilization by (60)Co, the mean size increased about 10% but no significant change was observed in EY and DL. Tests for in vitro release in physiological saline or physiological saline containing 0.5% (w/v) ascorbic acid by a dialysis bag showed sustained release and little burst effect.