Ex vivo nonviral gene delivery of µ-opioid receptor to attenuate cancer-induced pain.

Virus-mediated gene delivery shows promise for the treatment of chronic pain. However, viral vectors have cytotoxicity. To avoid toxicities and limitations of virus-mediated gene delivery, we developed a novel nonviral hybrid vector: HIV-1 Tat peptide sequence modified with histidine and cysteine residues combined with a cationic lipid. The vector has high transfection efficiency with little cytotoxicity in cancer cell lines including HSC-3 (human tongue squamous cell carcinoma) and exhibits differential expression in HSC-3 (∼45-fold) relative to HGF-1 (human gingival fibroblasts) cells. We used the nonviral vector to transfect cancer with OPRM1, the µ-opioid receptor gene, as a novel method for treating cancer-induced pain. After HSC-3 cells were transfected with OPRM1, a cancer mouse model was created by inoculating the transfected HSC-3 cells into the hind paw or tongue of athymic mice to determine the analgesic potential of OPRM1 transfection. Mice with HSC-3 tumors expressing OPRM1 demonstrated significant antinociception compared with control mice. The effect was reversible with local naloxone administration. We quantified ß-endorphin secretion from HSC-3 cells and showed that HSC-3 cells transfected with OPRM1 secreted significantly more ß-endorphin than control HSC-3 cells. These findings indicate that nonviral delivery of the OPRM1 gene targeted to the cancer microenvironment has an analgesic effect in a preclinical cancer model, and nonviral gene delivery is a potential treatment for cancer pain.

Efficient in vivo gene delivery using modified Tat peptide with cationic lipids.

A combination of modified HIV-1 Tat (mTat) peptide and cationic lipids, FuGENE HD (FH), dramatically enhanced transfection efficiency across a range of cell lines when compared to mTat or FH alone (Biomaterials 35:1705-1715 2014). The efficiency of this Tat peptide combination was significantly higher than many commercial non-viral vectors. In this present study, we tested the feasibility of this non-viral vector, mTat/FH, in vivo using plasmid DNA encoding a luciferase gene. The results of the in vivo studies showed that animals administered mTat/FH/DNA intramuscularly had significantly higher and longer luciferase expression (≈7 months) than those with mTat/DNA, FH/DNA, or DNA alone. Histological evaluation showed little immune response in the muscles, livers, and kidneys of mice administered with the mTat/FH. The combination of mTat with FH could significantly improve transfection efficiency, expanding the potential use of non-viral gene vectors in vivo.

The effect of a bioactive collagen membrane releasing PDGF or GDF-5 on bone regeneration.

Regenerative procedures using barrier membrane technology are presently well established in periodontal/endodontic surgery. The objective of this study was to compare the subsequent effects of the released platelet-derived growth factor (PDGF) and growth/differentiation factor 5 (GDF-5) from collagen membranes (CMs) on bone regeneration in vitro and in vivo. In vitro studies were conducted using MC3T3-E1 mouse preosteoblasts cultured with or without factors. Cell viability, cell proliferation, alkaline phosphatase (ALP) activity and bone marker gene expression were then measured. In vivo studies were conducted by placing CMs with low or high dose PDGF or GDF-5 in rat mandibular defects. At 4 weeks after surgery new bone formation was measured using µCT and histological analysis. The results of in vitro studies showed that CM/GDF-5 significantly increased ALP and cell proliferation activities without cytotoxicity in MC3T3-E1 cells when compared to CM/PDGF or CM alone. Gene expression analysis revealed that Runx2 and Osteocalcin were significantly increased in CM/GDF-5 compared to CM/PDGF or control. Quantitative and qualitative µCT and histological analysis for new bone formation revealed that although CM/PDGF significantly enhanced bone regeneration compared to CM alone or control, CM/GDF-5 significantly accelerated bone regeneration to an even greater extent than CM/PDGF. The results also showed that GDF-5 induced new bone formation in a dose-dependent manner. These results suggest that this strategy, using a CM carrying GDF-5, might lead to an improvement in the current clinical treatment of bone defects for periodontal and implant therapy.

Long-term efficient gene delivery using polyethylenimine with modified Tat peptide.

Polyethylenimine (PEI), a cationic polymer, has been widely studied and shown great promise as an efficient gene delivery vehicle. Likewise, the HIV-1 Tat peptide, a cell-permeable peptide, has been successfully used for intracellular gene delivery. To improve the favorable properties of these two vectors, we combine PEI with the modified Tat peptide sequence bearing histidine and cysteine residues (mTat). In vitro mTat/PEI-mediated transfection was evaluated by luciferase expression plasmid in two cell types. mTat/PEI produced significant improvement (≈5-fold) in transfection efficiency of both cell lines with little cytotoxicity when compared to mTat alone, PEI alone, or four commercial reagents. The particle size of mTat/PEI/DNA complex was significantly smaller than mTat or PEI alone, and it was correlated with higher transfection efficiency. Filipin III, an inhibitor of caveolae-mediated endocytosis, significantly inhibited mTat/PEI transfection. In contrast, chlorpromazine, an inhibitor of clathrin-mediated endocytosis, did not. This suggested caveolae-mediated endocytosis as the transfection mechanism. Furthermore, the results of in vivo studies showed that animals administered mTat/PEI/DNA intramuscularly had significantly higher and longer luciferase expression (≈7 months) than those with mTat/DNA, PEI/DNA, or DNA alone, without any associated toxicity. The combination of mTat with PEI could significantly improve transfection efficiency, expanding the potential use as a non-viral gene vector both in vitro and in vivo.

In vitro wear assessment of titanium alloy teeth.

PURPOSE: Wear of commercially pure (CP) titanium prosthetic teeth has frequently been observed. The greatest wear has been found when the same grades of CP titanium are used for both maxillary and mandibular teeth. This study examined the wear behavior of teeth made with cast titanium alloy and compared these results with those for CP titanium and gold alloy teeth. MATERIALS AND METHODS: All tooth specimens were cast with grade 3 alpha titanium, 3 metastable beta alloys [Ti-15Mo-2.8Nb-0.2Si (Timetal 21 SRx), Ti-13Nb-13Zr, and Ti-15V-3Cr-3Sn-3Al], and 2 alpha+beta alloys (Ti-6Al-7Nb and Ti-6Al-4V). As a control, Type IV gold alloy was also cast conventionally. All teeth (both maxillary and mandibular) were secured in an in vitro 2-body wear testing apparatus that simulated chewing function (60 strokes/min; grinding distance, 2 mm under flowing water). Wear resistance was assessed as volume loss (mm(3)) at 5 kgf (grinding force) after 50,000 strokes. The results (n = 5) were analyzed using analysis of variance or Fisher's exact test (alpha = 0.05). RESULTS: Of the titanium teeth, the wear of 2 of the metastable beta alloys (Timetal 21 SRx and Ti-15V-3Cr-3Sn-3Al) was found to be significantly (p <0.05) higher than that of CP titanium and the 2 alpha+beta alloys. The Type IV gold alloy exhibited better wear resistance than all of the titanium teeth tested. No correlation was found between wear loss and hardness among all the metals tested. CONCLUSIONS: Among the titanium teeth examined, the alpha+beta alloys exhibited significantly less wear than the other types of titanium. The dental casting gold alloy tested exhibited the best wear resistance among all of the metals tested.