Sonodynamic effects of protoporphyrin IX disodium salt on isolated sarcoma 180 cells

The cytotoxic effect of PPIX on isolated sarcoma 180 cells induced by ultrasound was investigated. Tumor cells suspended in air-saturated PBS (pH 7.2) were exposed to ultrasound at 2.2 MHz for up to 60 s in the presence and absence of protoporphyrin IX disodium salt (PPIX). The viability of cells was determined by a trypan blue exclusion test. The rate of ultrasonically induced cell damage was increased with 40–160 μM PPIX, while no cell damage was observed with 160 μM PPIX alone. This enhancement of cell damage with PPIX was inhibited by histidine. The participation of lipid peroxidation products in the cell damage process was also investigated. Scanning electron microscope (SEM) observation of the surface of cells was performed to evaluate the morphological changes induced by ultrasonic irradiation. The results indicate the involvement of a sonochemical mechanism.     

Study of cell killing effect on S180 by ultrasound activating protoporphyrin IX

The present study was initiated to investigate the potential biological mechanism of cell killing effect on isolate sarcoma 180 (S180) cells induced by ultrasound activating protoporphyrin IX (PPIX). S180 cells were exposed to ultrasound for 30 s duration, at a frequency of 2.2 MHz and an acoustic power of 3 W/cm² in the presence of 120 μM PPIX. The viability of cells was evaluated using trypan blue staining. The generation of oxygen free radicals in cell suspensions was detected immediately after treatment using a reactive oxygen detection kit. A copper reagent colorimetry method was used to measure the level of FFAs released into cell suspensions by the process of cell damage induced by ultrasound and PPIX treatment. Oxidative stress was assessed by measuring the activities of key antioxidant enzymes (i.e., SOD, CAT, GSH-PX) in S180 tumor cells. Treatment with ultrasound and PPIX together increased the cell damage rate to 50.91%, while treatment with ultrasound alone gave a cell damage rate to 24.24%, and PPIX alone kept this rate unchanged. Colorimetry and enzymatic chemical methods showed that the level of FFAs in cell suspension increased significantly after the treatment, while the activity of all the above enzymes decreased in tumor cells at different levels, and were associated with the generation of oxygen free radicals in cell suspension after treatment. The results indicate that oxygen free radicals may play an important role in improving the membrane lipid peroxidation, degrading membrane phospholipids to release FFAs, and decreasing the activities of the key antioxidant enzymes in cells. This biological mechanism might be involved in mediating the effects on S180 cells and resulting in the cell damage seen with SDT.     

Damage effects of protoporphyrin IX - sonodynamic therapy on the cytoskeletal F-actin of Ehrlich ascites carcinoma cells

In this study, we report evidence of the damage effects of sonodynamic therapy (SDT) on a novel intracellular target, cytoskeletal F-actin, that has great importance for cancer treatment. Ehrlich ascites carcinoma (EAC) cells suspended in PBS were exposed to ultrasound at 1.34MHz for up to 60s in the presence and absence of protoporphyrin IX (PPIX). To evaluate the polymeric state and distribution of actin filaments (AF) we employed FITC-Phalloidin staining. The percentage of cells with intact AF was decreased with 10-80muMu PPIX after ultrasonic exposure, while only few cells with disturbed F-actin were observed with 80muMu PPIX alone. The fluorescence intensity of FITC-Phalloidin labeled cells was detected by flow cytometry. The morphological changes of EAC cells were observed by scanning electron microscope (SEM). The nuclei were stained with Hoechst 33258 to determine apoptosis. Cytoskeletal F-actin and cell morphological changes were dependent on the time after SDT. Some cells suffered deformations of plasma membrane as blebs that reacted positively to FITC-Phalloidin at 2h after SDT treatment. Many of the cells showed the typically apoptotic chromatin fragmentation. The alterations were more significant 4h later. Our results showed that cytoskeletal F-actin might represent an important target for the SDT treatment and the observed effect on F-actin and the subsequent bleb formation mainly due to apoptosis formation due to the treatment.     

Preparation and in vitro evaluation of an ultrasound-triggered drug delivery system: 10-hydroxycamptothecin loaded PLA microbubbles

10-Hydroxycamptothecin (HCPT) loaded PLA microbubbles, used as an ultrasound-triggered drug delivery system, were fabricated by a double emulsion-solvent evaporation method. The obtained microbubbles were characterized by scanning electron microscope (SEM), transmission electron microscope (TEM), X-ray diffraction (XRD), differential scanning calorimetry (DSC) and confocal laser scanning microscope (CLSM). In addition, the effect of diagnostic ultrasound exposure on BEL-7402 cells combined with HCPT-loaded PLA microbubbles was evaluated using cytotoxicity assay, CLSM and flow cytometry (FCM). It was found that the HCPT-loaded PLA microbubbles showed smooth surface and spherical shape, and the drug was amorphously dispersed within the shell and the drug loading content reached up to 1.69%. Nearly 20% of HCPT was released upon exposure to diagnostic ultrasound at frequency of 3.5 MHz for 10 min. Moreover, HCPT fluorescence in the cells treated only with the HCPT-loaded PLA microbubbles was discernible, but less intense, while those treated with the microbubbles in conjunction with ultrasound exposure was evident and intense, indicating an increased cellular uptake of HCPT by ultrasound exposure. Cytotoxicity test on BEL-7402 cells indicated that the HCPT-loaded PLA microbubbles combined with ultrasound exposure were more cytotoxic than the microbubbles alone. The results suggest that the combination of drug loaded PLA microbubbles and diagnostic ultrasound exposure exhibit an effective intracellular drug uptake by tumor cells, indicating their great potential for antitumor therapy.     

Ultrasound induces cellular destruction of nasopharyngeal carcinoma cells in the presence of curcumin

Objectives

Curcumin, a natural pigment from the traditional Chinese herb, has shown promise as an efficient enhancer of ultrasound. The present study aims to investigate ultrasound-induced cellular destruction of nasopharyngeal carcinoma cells in the presence of curcumin in vitro.

Methods

Nasopharyngeal carcinoma cell line CNE2 cells were incubated by 10 μm curcumin and then were treated by ultrasound for 8 s at the intensity of 0.46 W/cm². Cytotoxicity was evaluated using MTT assay and light microscopy. Mitochondrial damage was analyzed using a confocal laser scanning microcopy with Rhodamine 123 and ultrastructural changes were observed using a transmission electron microscopy (TEM).

Results

MTT assay showed that cytotoxicity induced by ultrasound treatment alone and curcumin treatment alone was 18.16 ± 2.37% and 24.93 ± 8.30%, respectively. The cytotoxicity induced by the combined treatment of ultrasound and curcumin significantly increased up to 86.67 ± 7.78%. TEM showed that microvillin disappearance, membrane blebbing, chromatin condensation, swollen mitochondria, and mitochondrial myelin-like body were observed in the cells treated by ultrasound and curcumin together. The significant collapse of mitochondrial membrane potential (MMP) was markedly observed in the CNE2 cells after the combined treatment of curcumin and ultrasound.

Conclusions

Our findings demonstrated that ultrasound sonication in the presence of curcumin significantly killed the CNE2 cells and induced ultrastructural damage and the dysfunction of mitochondria, suggesting that ultrasound treatment remarkably induced cellular destruction of nasopharyngeal carcinoma cells in the presence of curcumin.     

Enhancement of ultrasonically induced cell damage by phthalocyanines in vitro

In this work, erythrocytes from carp were used as a nucleated cell model to test the hypothesis that the phthalocyanines (zinc - ZnPc and chloroaluminium -AlClPc) enhance ultrasonically induced damage in vitro. In order to confirm and complete our earlier investigation, the influence of ultrasound (US) and phthalocyanines (Pcs) on unresearched cellular components, was studied. Red blood cells were exposed to 1 MHz continuous ultrasound wave (0.61 and/or 2.44 W/cm²) in the presence or absence of phthalocyanines (3 μM). To identify target cell damage, we studied hemolysis, membrane fluidity and morphology of erythrocytes. To demonstrate the changes in the fluidity of plasma membrane we used the spectrofluorimetric methods using two fluorescence probes: 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5,-hexatriene (TMA-DPH) and 1,6-diphenyl-1,3,5-hexatriene (DPH). The effect of US and Pcs on nucleated erythrocytes morphology was estimated on the basis of microscopic observation.The enhancement of ultrasonically induced membrane damage by both phthalocyanines was observed in case of hemolysis, and membrane surface fluidity, in comparison to ultrasound. The authors also observed changes in the morphology of erythrocytes. The obtained results support the hypothesis that the Pcs enhance ultrasonically induced cell damage in vitro.Furthermore, the influence of ultrasound on phthalocyanines (Pcs) in medium and in cells was tested. The authors observed changes in the phthalocyanines absorption spectra in the medium and the increase in the intensity of phthalocyanines fluorescence in the cells. These data can suggest changes in the structure of phthalocyanines after ultrasound action.     

Sonodynamically induced anti-tumor effect with protoporphyrin IX on hepatoma-22 solid tumor

Objective

The purpose of this study was to evaluate sonodynamically induced anti-tumor effect of protoporphyrin IX (PPIX) in mice bearing hepatoma-22 (H-22) solid tumors, and the possible in vivo cell damage mechanism was also investigated.

Methods

The pharmacokinetics of PPIX was analyzed in plasma, skin, muscle and tumor of H-22 bearing mice. Tumors were irradiated with ultrasound (1.43 MHz, I_SATA 3 W/cm², 3 min) for three times at 8, 12 and 24 h after 5.0 mg/kg PPIX administration, respectively. The anti-tumor effects of sonodynamic therapy (SDT) were estimated by the tumor inhibition ratio (volume and weight). The bio-effects of SDT were evaluated by hematoxylin and eosin (H&E) staining, transmission electron microscope (TEM) observation, lipid peroxidation (LPO) measurement and anti-oxidative enzymes (glutathione peroxidase (GSH-PX), catalase (CAT) and superoxide dismutase (SOD)) assay.

Results

A significant anti-tumor effect was obtained by PPIX-mediated sonodynamic therapy (PPIX-SDT). At the fifteenth day after PPIX-SDT, the tumor growth and tumor weight inhibition ratios were 53.84% and 45.86%, respectively. In addition, the structure of tumor tissues and the anti-oxidative enzymes were obviously destroyed after SDT treatment.

Conclusions

A biochemical mechanism was involved in PPIX-SDT in vivo, and the free radicals produced by the synergistic treatment destroying the anti-oxidative system of tumor cells in vivo may play an important role in this action. Also, the thermal effect could not be excluded in inducing damage of cellular structures, like membrane disruption and chromatin condensation under current evaluation in this paper.     

三种肿瘤细胞对超声结合血卟啉敏感性的实验研究

实验采用频率为1．6MHz，强度为1W／cm^2、3W／cm^2、5W／cm^2、7W／cm^2的聚焦超声结合血卟啉分别对腹水型小鼠S180细胞（Sarcomal80，S180）、艾氏腹水瘤细胞（Ehrlich Ascites Tumor，EAT）及H-22肝癌细胞进行杀伤效应研究，利用台盼蓝拒染法检测处理后细胞存活率的变化。实验结果表明，不同类型的细胞对超声结合血卟啉的敏感性不同，声照条件相同时，三种细胞对1．6MHz频率超声的敏感性依次为：S18〉EAT〉H-22，且对超声结合血卟啉敏感性明显大于单纯超声。无论是单纯超声组还是超声结合血卟啉组，三种细胞的存活率均随超声强度的增加而下降。     

Ultrasonic irradiation enhanced the efficacy of antimicrobial photodynamic therapy against methicillin-resistant Staphylococcus aureus biofilm

Antimicrobial photodynamic therapy (aPDT) is a non-pharmacological antimicrobial regimen based on light, photosensitizer and oxygen. It has become a potential method to inactivate multidrug-resistant bacteria. However, limited by the delivery of photosensitizer (PS) in biofilm, eradicating biofilm-associated infections by aPDT remains challenging. This study aimed to explore the feasibility of combining ultrasonic irradiation with aPDT to enhance the efficacy of aPDT against methicillin-resistant staphylococcus aureus (MRSA) biofilm. A cationic benzylidene cyclopentanone photosensitizer with much higher selectivity to bacterial cells than mammalian cells were applied at the concentration of 10 μM. 532 nm laser (40 mW/cm², 10 min) and 1 MHz ultrasound (500 mW/cm², 10 min, simultaneously with aPDT) were employed against MRSA biofilms in vitro. In addition to combined with ultrasonic irradiation and aPDT, MRSA biofilms were treated with laser irradiation only, photosensitizer only, ultrasonic irradiation only, ultrasonic irradiation and photosensitizer, and aPDT respectively. The antibacterial efficacy was determined by XTT assay, and the penetration depth of PS in biofilm was observed using a photoluminescence spectrometer and a confocal laser scanning microscopy (CLSM). In addition, the viability of human dermal fibroblasts (WS-1 cells) after the same treatments mentioned above and the uptake of P3 by WS-1 cells after ultrasonic irradiation were detected by CCK-8 and CLSM in vitro. Results showed that the percent decrease in metabolic activity resulting from the US + aPDT group (75.76%) was higher than the sum of the aPDT group (44.14%) and the US group (9.88%), suggesting synergistic effects. Meanwhile, the diffusion of PS in the biofilm of MRSA was significantly increased by 1 MHz ultrasonic irradiation. Ultrasonic irradiation neither induced the PS uptake by WS-1 cells nor reduced the viability of WS-1 cells. These results suggested that 1 MHz ultrasonic irradiation significantly enhanced the efficacy of aPDT against MRSA biofilm by increasing the penetration depth of PS. In addition, the antibacterial efficacy of aPDT can be enhanced by ultrasonic irradiation, the US + aPDT treatment demonstrated encouraging in vivo antibacterial efficacy (1.73 log₁₀ CFU/mL reduction). In conclusion, the combination of aPDT and 1 MHz ultrasound is a potential and promising strategy to eradicate biofilm-associated infections of MRSA.     

Sonodynamically induced antitumor effect of hematoporphyrin on Hepatoma 22

The ultrasonically induced cytotoxic effects of hematoporphyrin (Hp) on Hepatoma 22 (H22) cells in vitro and vivo were investigated. Tumor cells were suspended in saline and exposed to ultrasound at 1.43 MHz for up to 60s in the presence and absence of Hp. The viability of cells was evaluated by trypan blue exclusion test. The ultra-structure changes of H22 cells induced by ultrasonic irradiation were evaluated by scanning electron microscope (SEM) and transmission electron microscope (TEM). Lipid peroxidation in cell was estimated by the thiobarbicturic acid (TBA) method. Our experiments indicated that the ultrasonic intensity of 2 W/cm(2), the Hp concentration of 100 microg/ml and the ultrasound exposure time of 60s were the best conditions for sonodynamic treatment in vitro. The tumor volume and weight after the combination of Hp with ultrasound were remarkably inhibited. SEM and TEM observation found the cell ultra-structure was significantly damaged, and lipid peroxidation level remarkably increased after sonodynamic treatment. This study suggested the ultra-structural changes may play a key role in cell destruction induced by sonodynamic treatment and the biological mechanism might be involved in mediating the killing effect on H22 cells in our experiment mode.     

Membrane fluidity altering and enzyme inactivating in sarcoma 180 cells post the exposure to sonoactivated hematoporphyrin in vitro

Sonodynamic therapy (SDT) is a novel tumor therapy method. We investigated membrane fluidity, activity of the enzymes and membrane morphology in vitro post hematoporphyrin-SDT treatment. Furthermore, the potential mechanisms behind the changes in membrane fluidity and enzymic activity were discussed. Tumor cells were exposed to ultrasound at 1.75 MHz for up to 3 min in the presence and absence of hematoporphyrin. Fluorescence polarization, contents of Malonaldehyde, and levels of free fatty acid were assessed. Activity of enzymes was checked by the plumbic nitrate detection method. For the morphologic study, a scanning electron microscope was used to observe the cellular surface. Ultrasonically induced cell damage increased in the presence of HPD (from 15% to 24%). Compared with ultrasound treatment alone, the fluidity decreased from 5.037 to 3.908, malonaldehyde content and free fatty acid level increased from 0.743 nmol/mL to 0.979 nmol/mL and from 237.180 μmol/L to 730.769 μmol/L, respectively, post ultrasound combined with HPD treatment. Inactivity of adenylate cyclase and guanylate cyclase and significant deformation of the cellular surface were also observed post SDT treatment. Our results suggested that alterations in membrane modality and lipid composition played important roles in SDT-mediated inhibition of tumor growth, even inducing tumor cell death, which might be attributed to a sono-chemical activation mechanism.     

Sonodynamic effect of erythrosin B on sarcoma 180 cells in vitro

The ultrasonically induced cytotoxic effect of erythrosin B (EB) on isolated sarcoma 180 cells was investigated. The tumor cells were suspended in an air-saturated phosphate buffered saline and exposed to ultrasound at 1.93 MHz in a standing-wave mode for up to 60 s in the presence and absence of EB. The rate of cell damage induction by ultrasound was enhanced by 4-5 times with 160-microM EB, while no cell damage was observed with EB alone. This enhancement was significantly inhibited by histidine. Sonochemical generation of active oxygen species in the presence of EB, measured by ESR spectroscopy, was also inhibited by histidine. These results indicate the involvement of a sonochemical mechanism.     

Sonodynamic action of pyropheophorbide-a methyl ester induces mitochondrial damage in liver cancer cells

Sonodynamic therapy with pyropheophorbide-a methyl ester (MPPa) presents a promising aspect in treating liver cancer. The present study aims to investigate the mitochondrial damage of liver cancer cells induced by MPPa-mediated sonodynamic action. Mouse hepatoma cell line H₂₂ cells were incubated with MPPa (2 μM) for 20 h and then exposed to ultrasound with an intensity of 0.97 W/cm² for 8 s. Cytotoxicity was investigated 24 h after sonodynamic action using MTT assay and light microscopy. Mitochondrial membrane potential (ΔΨm) was analyzed using flow cytometry with rhodamine 123 staining and ultrastructural changes were observed using transmission electron microscopy (TEM).The cytotoxicity of MPPa-mediated SDT on H₂₂ cell line was 73.00 ± 3.42%, greater than ultrasound treatment alone (28.12 ± 5.19%) significantly while MPPa treatment alone had no significant effect on H₂₂ cells. Moreover, after MPPa-mediated SDT cancer cells showed swollen mitochondria under TEM and a significant collapse of mitochondrial membrane potential. Our findings demonstrated that MPPa-mediated SDT could remarkably induce cell death of H₂₂ cells, and highlighted that mitochondrial damage might be an important cause of cell death induced by MPPa-mediated SDT.     

Enhancement of heat transfer in forced convection by using dual low-high frequency ultrasound

Combined sonication with dual-frequency ultrasound has been investigated to enhance heat transfer in forced convection. The test section used for this study consists of a channel with, on one hand, heating blocks normal to the water flow, equipped with thermocouples, and, on the other hand, two ultrasonic emitters. One is facing the heating blocks, thus the ultrasonic field is perpendicular, and the second ultrasonic field is collinear to the water flow. Two types of ultrasonic waves were used: low-frequency ultrasound (25 kHz) to generate mainly acoustic cavitation and high-frequency ultrasound (2 MHz) well-known to induce Eckart’s acoustic streaming. A thermal approach was conducted to investigate heat transfer enhancement in the presence of ultrasound. This approach was completed with PIV measurements to assess the hydrodynamic behavior modifications under ultrasound. Sonochemiluminescence experiments were performed to account for the presence and the location of acoustic cavitation within the water flow. The results have shown a synergetic effect using combined low-and-high-frequency sonication. Enhancement of heat transfer is related to greater induced turbulence within the water flow by comparison with single-frequency sonication. However, the ultrasonically-induced turbulence is not homogeneously distributed within the water flow and the synergy effect on heat transfer enhancement depends mainly on the generation of turbulence along the heating wall. For the optimal configuration of dual-frequency sonication used in this work, a local heat transfer enhancement factor up to 366% was observed and Turbulent Kinetic Energy was enhanced by up to 84% when compared to silent regime.     

Ultrasound exposure in the presence of hematoporphyrin induced loss of membrane integral proteins and inactivity of cell proliferation associated enzymes in sarcoma 180 cells in vitro

Ultrasonically induced effects of hematoporphyrin (HPD) on cell damage and membrane protein alteration of S180 isolated tumor cells in vitro were investigated, and the potential mechanisms of sonodynamic therapy (SDT) inhibiting tumor growth were discussed. Tumor cells suspended in air-saturated PBS (pH 7.2) were exposed to ultrasound at 1.8 MHz for up to 180 s in the presence and absence of HPD. The viability of cells was determined by a trypan blue exclusion test. To estimate the damage effects of SDT on plasma membrane of tumor cells primarily, membrane integral proteins (EGFR, Ras, Fas, FasL) and cell proliferation associated enzymes (adenylate cyclase and guanylate cyclase) were checked with immunochemical methods. The results indicated that the intensity threshold for ultrasonically induced cell damage at 1.8 MHz was 3 W/cm². At this condition, the expression of the integral proteins was obviously inhibited and the activity of the enzymes was decreased post ultrasound treatment in the presence of 20 μg/ml HPD. Loss of the membrane proteins and inactivity of AC and GC post SDT was time-dependent. This paper reveals SDT can cause the loss of tumor cell membrane integral proteins and inactivity of the enzymes associated with cell proliferation which might be attributed to a sonochemical activation mechanism. The mechanisms by that tumor growth is inhibited by SDT can be understood as that the growth signaling pathway is partially interdicted and the resistance of tumor cells to the specifically activated immune cells is weakened.     

Impact of combined ultrasound-microwave treatment on structural and functional properties of golden threadfin bream (Nemipterus virgatus) myofibrillar proteins and hydrolysates

The effects of microwave, ultrasound and combined ultrasound-microwave (UM) treatment with different intensities on structural and hydrolysis properties of myofibrillar protein (MP) were investigated. Free radical scavenging ability, angiotensin-I-converting enzyme (ACE) inhibitory activity, and cellular antioxidant and anti-inflammatory abilities of the related bioactive peptides were also evaluated. Raman spectroscopic analysis indicated that MP molecule tended to unfold and stretch with increasing in β-turn and random coil content under mild microwave (100 W), ultrasound (100–200 W) and combined UM treatments. Meanwhile, differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) revealed these treatments could also improve the thermal stability against heat-induced denaturation and degeneration. The 200 W ultrasound treatment clearly increased MP solubility by disrupting the highly-ordered aggregates into smaller filament and fragment structures. The 300 W ultrasound coupled with 100 W microwave treatment further enhanced these effects. The resulting partially denatured structure induced by suitable ultrasound and combined UM treatments increased the susceptibility of MP to exogenous enzymes, thereby accelerating hydrolytic process and yielding a high peptide concentration in MP hydrolysates. MP peptides could effectively inhibit free radical and ACE activity, which also improved the ability of antioxidant defence system, and suppressed the production of proinflammatory cytokines in RAW 264.7 cells stimulated by H₂O₂. The combination of 100 W microwave and 300 W ultrasound treatment was optimal method for generating bioactive MP peptides with the strongest multi-activity effects against H₂O₂-induced cell damage.     

Effects of ultrasound on apoptosis induced by anti-CD20 antibody in CD20-positive B lymphoma cells

AIM: The present study was conducted to examine the thermal and non-thermal effects of ultrasound on apoptosis induced by anti-CD20 monoclonal antibody (rituximab). MATERIALS AND METHODS: SU-DHL-4 cells, a CD20-positive cell line derived from B cell lymphomas with a BCL2 gene rearrangement, were exposed to continuous 1 MHz ultrasound for therapeutic use under an air- or CO(2)-saturated condition to control cavitation. Early apoptosis (EA) and secondary necrosis (SN) were examined by flow cytometry. Cavitation was determined by detecting the hydroxyl radicals derived from pyrolysis of water molecules using electron paramagnetic resonance-spin trapping. To assess thermal effects, cells were treated in a temperature-controlled water bath. RESULTS: There was a significant additive increase in EA and EA+SN observed in cells treated with rituximab combined with heat at 42 degrees C or non-thermal ultrasound at 0.5 W/cm(2) under an air-saturated condition, where heat or ultrasound induced some cell death. A significant synergistic increase in EA and EA+SN was observed in cells treated with rituximab and ultrasound at 2.5 W/cm(2) under CO(2)-saturated conditions, where inertial cavitations were completely suppressed. No enhancement was observed at a temperature less than 40 degrees C or ultrasound at 0.5 W/cm(2) under CO(2)-saturated conditions. CONCLUSION: These results suggest that the immuno-therapeutic application of ultrasound at relatively high-intensities combined with rituximab thus produces synergistic effects under conditions where the non-thermal and non-cavitational effects are predominant.     

人体血清中原卟啉Ⅸ自体荧光光谱研究

通过对人体血清样品和模拟人体生理条件下原卟啉Ⅸ和牛血清白蛋白混合溶液的荧光光谱测定和分析,表明人体血清中原卟啉Ⅸ的自体荧光光谱主要来自于原卟啉Ⅸ和血清白蛋白的结合形式。此外,还考察了血清白蛋白和原卟啉Ⅸ对原卟啉Ⅸ荧光发射峰的影响。和不含有白蛋白的原卟啉Ⅸ溶液相比较,白蛋白不但使原卟啉Ⅸ产生的荧光峰发生红移,而且具有很强的增敏效应。在白蛋白存在条件下,当原卟啉Ⅸ浓度值小于0.8×10-5 mol·L-1时,随着它的浓度增加,原卟啉Ⅸ的荧光发射峰稍有红移;而当原卟啉Ⅸ浓度值大于0.8×10-5 mol·L-1时,其荧光发射波长几乎不随原卟啉Ⅸ浓度而变化。     

Interaction between protoporphyrin IX and tryptophan silver nanoparticles

Silver nanoparticles (AgNPs) have been intensively studied for several purposes including therapeutic applications in cancer. When prepared with tryptophan and photoreduction, silver nanoparticles (TrpAgNPs) become an alternative to conventional anticancer drugs. In this study, the anticancer activity of synthesized TrpAgNPs against MCF-7 breast cancer cells was evaluated, and the inhibitory concentration (IC50) was found to be ~3.4 mg/mL. Since the protoporphyrin IX (PPIX) concentrations in tumor cells are elevate compared to normal cells, the PPIX-TrpAgNP interaction was studied to investigate if it could contribute for cell apoptosis. The investigation was performed using PPIX solution (0.9 μg/mL) with different TrpAgNP concentrations (from 0 to 13 mg/mL). PPIX was characterized by UV-Vis spectroscopy, steady-state and time-resolved fluorescence spectroscopy. The results have shown that the presence of spherical TrpAgNps with 16-nm diameter quench the PPIX fluorescence intensity. This quenching is strongly dependent on the concentration of the TrpAgNPs, and it is caused by a combination of a static and a dynamic process. The chemical binding leads to oxidation of tryptophan and formation of kynurenine, observed in the emission spectra around 470 nm. The strong reduction of the PPIX fluorescence decay lifetime with nanoparticle increasing concentration confirms the quenching processes due to charge transfer from the excited PPIX states to the resonant silver states. The present study confirms the anticancer activity of TrpAgNPs on the human breast cancer cell line (MCF-7) in vitro and indicates that PPIX-AgNP interaction could contribute with MCF-7 apoptosis.

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Graphical abstract Interaction between TrpAgNPs and PPIX

     

A novel approach to quantitative ultrasonic naked gene delivery and its non-invasive assessment

The purpose of this study was to investigate practical, safe, easy-to-use, non-cytotoxic, and reliable parameters to apply to an ultrasound (US) naked gene therapy system. The ultrasound pressure at the point of cell exposure was measured using a calibrated hydrophone and the intensity calculated. An acoustic power meter calibrated using a hydrophone was used to measure the power of the transducer. Four cell types were exposed to US with different exposure times and intensities. Fluorescent microscopy, spectrophotometry, scanning electron microscope, laser scanning confocal microscopy, flow cytometry and histogram analysis were used to evaluate the results of the study. The plasmid of green fluorescent protein (GFP) served as the reporter gene. The energy accumulation E in US gene delivery for 90% cell survival was defined as the optimal parameters (E=3.56+/-0.06), and at 80% cell survival was defined as the damage threshold (E=59.67+/-3.54). US safely delivered GFP into S180 cells (35.1 kHz) at these optimal parameters without obvious damage or cytotoxity in vitro. Exposed cell function was proved normal in vivo. The transfection rate was 35.83+/-2.53% (n=6) in viable cells, corresponding to 90.17+/-1.47% (n=6) cell viability. The intensity of GFP expression showed a higher fluorescent peak in the group of adeno-associated virus GFP vector (AVV-GFP) than in the control group (P<0.001). The effect of US gene delivery and cell viability correlated as a fifth order polynomial with US intensity and exposure time. With optimal parameters, US can safely deliver naked a gene into a cell without damage to cell function. Both optimal uptake and expression of gene depend on the energy E at 90% cell survival. E can be applied as a control factor for bioeffects when combined with other parameters. Stable caviation results in optimal parameters for gene delivery and the transient caviation may cause cell damage, which will bring about a sharp rise of permeabilization. The results may be applied to the development of a novel clinical gene therapeutic system.