Type I fibrillar collagen is the most abundant protein in the human body, crucial for the formation and strength of bones, skin, and tendon. Proteolytic enzymes are essential for initiation of the assembly of collagen fibrils by cleaving off the propeptides. We report that Mep1a −/− and Mep1b −/− mice revealed lower amounts of mature collagen I compared with WT mice and exhibited significantly reduced collagen deposition in skin, along with markedly decreased tissue tensile strength. While exploring the mechanism of this phenotype, we found that cleavage of full-length human procollagen I heterotrimers by either meprin α or meprin β led to the generation of mature collagen molecules that spontaneously assembled into collagen fibrils. Thus, meprin α and meprin β are unique in their ability to process and release both C-and N-propeptides from type I procollagen in vitro and in vivo and contribute to the integrity of connective tissue in skin, with consequent implications for inherited connective tissue disorders.proteolysis | Ehlers-Danlos syndrome | proteomics | fibrosis | connective tissue
This study demonstrates the capability of multiwalled carbon nanotubes (MWNTs) coupled with laser irradiation to enhance treatment of cancer cells through enhanced and more controlled thermal deposition, increased tumor injury, and diminished heat shock protein (HSP) expression. We also explored the potential promise of MWNTs as drug delivery agents by observing the degree of intracellular uptake of these nanoparticles. To determine the heat generation capability of MWNTs, the absorption spectra and temperature rise during heating were measured. Higher optical absorption was observed for MWNTs in water compared with water alone. For identical laser parameters, MWNT-containing samples produced a significantly greater temperature elevation compared to samples treated with laser alone. Human prostate cancer (PC3) and murine renal carcinoma (RENCA) cells were irradiated with a 1,064-nm laser with an irradiance of 15.3 W/cm2 for 2 heating durations (1.5 and 5 minutes) alone or in combination with MWNT inclusion. Cytotoxicity and HSP expression following laser heating was used to determine the efficacy of laser treatment alone or in combination with MWNTs. No toxicity was observed for MWNTs alone. Inclusion of MWNTs dramatically decreased cell viability and HSP expression when combined with laser irradiation. MWNT cell internalization was measured using fluorescence and transmission electron microscopy following incubation of MWNTs with cells. With increasing incubation duration, a greater number of MWNTs were observed in cellular vacuoles and nuclei. These findings offer an initial proof of concept for the application of MWNTs in cancer therapy.
Inclusion of multi-walled carbon nanotubes (MWNTs) into tissue prior to laser therapy has the potential to enhance the selectivity and effectiveness of cancer therapy by providing greater and more controlled thermal deposition. The purpose of this study was to investigate the optical and thermal response of tissue representative phantoms containing MWNTs to optical radiation. Tissue representative phantoms 20 mm in diameter and 1 mm in thickness were created from sodium alginate. Following the inclusion of MWNTs (900 nm in length, 40-60 nm in diameter) in phantoms, the distribution of MWNTs was observed using transmission electron microscopy. A predominantly, evenly dispersed and randomly oriented distribution of MWNTs was observed with a rare presence of MWNT clustering or clumping. In order to characterize the response of MWNT inclusion on optical properties of phantoms, the transmittance and reflectance spectra of phantoms with and without MWNT inclusion were measured with a spectrophotometer over a wavelength range of 200-1400 nm. Inclusion of MWNTs in phantoms dramatically enhanced light absorption across the entire wavelength range as evidenced by a diminished transmittance and reflectance compared with phantoms without MWNTs. In order to evaluate the spatiotemporal temperature distribution associated with laser irradiation of phantoms with and without MWNTs, the temperature was measured at discrete radial distances from the center of the incident laser beam using thermocouples. The rate of temperature increase and peak temperature for phantoms containing MWNTs was much greater compared with phantoms without MWNTs at all measurement locations. In conclusion, MWNT inclusion in tissue phantoms increases the optical absorption and temperature elevation, which may enable more effective photothermal therapies of human disease utilizing lasers.
Laser therapies can provide a minimally invasive treatment alternative to surgical resection of tumors. However, the effectiveness of these therapies is limited due to nonspecific heating of target tissue which often leads to healthy tissue injury and extended treatment durations. These therapies can be further compromised due to heat shock protein (HSP) induction in tumor regions where non-lethal temperature elevation occurs, thereby imparting enhanced tumor cell viability and resistance to subsequent chemotherapy and radiation treatments. Introducing multi-walled nanotubes (MWNT) into target tissue prior to laser irradiation increases heating selectivity permitting more precise thermal energy delivery to the tumor region and enhances thermal deposition thereby increasing tumor injury and reducing HSP expression induction. This study investigated the impact of MWNT inclusion in untreated and laser irradiated monolayer cell culture and cell phantom model. Cell viability remained high for all samples with MWNT inclusion and cells integrated into alginate phantoms, demonstrating the non-toxic nature of both MWNTs and alginate phantom models. Following, laser irradiation samples with MWNT inclusion exhibited dramatic temperature elevations and decreased cell viability compared to samples without MWNT. In the cell monolayer studies, laser irradiation of samples with MWNT inclusion experienced up-regulated HSP27, 70 and 90 expression as compared to laser only or untreated samples due to greater temperature increases albeit below the threshold for cell death. Further tuning of laser parameters will permit effective cell killing and down-regulation of HSP. Due to optimal tuning of laser parameters and inclusion of MWNT in phantom models, extensive temperature elevations and cell death occurred, demonstrating MWNT-mediated laser therapy as a viable therapy option when parameters are optimized. In conclusion, MWNT-mediated laser therapies show great promise for effective tumor destruction, but require determination of appropriate MWNT characteristics and laser parameters for maximum tumor destruction.
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