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Objective@#To evaluate the effectiveness of reactive oxygen species (ROS)-responsive nanomedicine in suppressing corneal neovascularization (CNV) in vivo.@*Methods@#ROS-responsive nanomedicine (ROS-TK-5/siVEGF), which consists of vascular endothelial growth factor (VEGF) small interfering RNA (siRNA) and thioketal linkage was synthesized by the Michael addition.The cumulative release of siVEGF from nanomedicine under oxidant conditions was assessed by agarose gel electrophoresis.Thirty-nine VEGFR2-luc-KI transgenic mice were used in this study, of which 30 mice were randomly divided into a normal control group, a PBS control group, an ROS-TK-5/NC group, an ROS-TK-5/siVEGF group, and a ranibizumab group, with 6 mice in each group.The ROS levels in the corneal tissue after alkali burning were tested by dihydroethidium (DHE) staining in the other 9 mice.In each group, alkali-burned mice were subconjunctivally injected with 10 μl of a different formula every two days.The effectiveness of nanomedicine in attenuating CNV was evaluated by slit-lamp microscopy and an in vivo imaging system (IVIS) at 7, 14, and 21 days after alkali burning. The use and care of the animals complied with the Statement of the Association for Research in Vision and Ophthalmology (ARVO) and the Guidelines of the Animal Experimental Committee of Liberation Army General Hospital.The study protocol was approved by the Ethics Committee of Liberation Army General Hospital (No.2018-X14-82).@*Results@#After treathrent with an aqueous solution without ROS, only 5%-10% of the siVEGF was released from the nanoparticles within 10 hours.In contrast, about 70% of the siVEGF was released from the nanoparticles after treatment with 10 mmol/L H2O2 within 10 hours.The relative fluorescent intensities in the corneal stromal layer at 7 days and 14 days after alkali burning were 5.403±0.306 and 2.930±0.255, respectively, which was significantly greater than those in the normal control group (1.003±0.015) (both at P<0.05). The CNV areas were statistically different among the four groups at various time points (Fgroup=49.855, P<0.01; Ftime=65.556, P<0.01). The CNV area was significantly reduced in the ROS-TK-5/siVEGF and ranibizumab groups compared with the PBS control and ROS-TK-5/NC groups at 7 days and 14 days after modeling, and the CNV area was more effectively reduced in the ROS-TK-5/siVEGF group than the ranibizumab group at 7 days and 14 days after modeling (all at P<0.05). At day 21 after modeling, the CNV area was significantly reduced in the ROS-TK-5/siVEGF and ranibizumab groups compared to the PBS control and ROS-TK-5/NC groups (all at P<0.05). IVIS showed that the corneal fluorescent intensity was statistically different among the four groups at various times (Fgroup=27.193, P=0.003; Ftime=51.062, P<0.01). The corneal fluorescent intensities were significantly reduced in the ROS-TK-5/siVEGF and ranibizumab groups compared to the PBS control and ROS-TK-5/NC groups at 7 days and 14 days after modeling; in addition, the corneal fluorescent intensity was more effectively reduced in the ROS-TK-5/siVEGF group in comparison with the ranibizumab group at 7 days and 14 days after modeling (all at P<0.05). At 21 days after modeling, the corneal fluorescent intensity was significantly reduced in the ROS-TK-5/siVEGF and ranibizumab groups compared to the PBS control and ROS-TK-5/NC groups (all at P<0.05).@*Conclusions@#ROS-TK-5/siVEGF nanomedicine effectively attenuates alkali burn-induced CNV formation and appears to have a better effect in comparison with ranibizumab at an early stage.
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Objective To evaluate the effectiveness of reactive oxygen species (ROS)-responsive nanomedicine in suppressing corneal neovascularization (CNV) in vivo.Methods ROS-responsive nanomedicine (ROS-TK-5/siVEGF),which consists of vascular endothelial growth factor (VEGF) small interfering RNA (siRNA) and thioketal linkage was synthesized by the Michael addition.The cumulative release of siVEGF from nanomedicine under oxidant conditions was assessed by agarose gel electrophoresis.Thirty-nine VEGFR2-1uc-KI transgenic mice were used in this study,of which 30 mice were randomly divided into a normal control group,a PBS control group,an ROS-TK-5/NC group,an ROS-TK-5/siVEGF group,and a ranibizumab group,with 6 mice in each group.The ROS levels in the corneal tissue after alkali burning were tested by dihydroethidium (DHE) staining in the other 9 mice.In each group,alkali-burned mice were subconjunctivally injected with 10 μl of a different formula every two days.The effectiveness of nanomedicine in attenuating CNV was evaluated by slit-lamp microscopy and an in vivo imaging system (IVIS) at 7,14,and 21 days after alkali burning.The use and care of the animals complied with the Statement of the Association for Research in Vision and Ophthalmology (ARVO) and the Guidelines of the Animal Experimental Committee of Liberation Army General Hospital.The study protocol was approved by the Ethics Committee of Liberation Army General Hospital (No.2018-X14-82).Results After treathrent with an aqueous solution without ROS,only 5％-10％ of the siVEGF was released from the nanoparticles within 10 hours.In contrast,about 70％ of the siVEGF was released from the nanoparticles after treatment with 10 mmol/L H2O2 within 10 hours.The relative fluorescent intensities in the corneal stromal layer at 7 days and 14 days after alkali burning were 5.403±0.306 and 2.930±0.255,respectively,which was significantly greater than those in the normal control group (1.003±0.015) (both at P＜0.05).The CNV areas were statistically different among the four groups at various time points (Fgroup =49.855,P＜0.01;Ftime =65.556,P＜0.01).The CNV area was significantly reduced in the ROS-TK-5/siVEGF and ranibizumab groups compared with the PBS control and ROS-TK-5/NC groups at 7 days and 14 days after modeling,and the CNV area was more effectively reduced in the ROS-TK-5/siVEGF group than the ranibizumab group at 7 days and 14 days after modeling (all at P＜0.05).At day 21 after modeling,the CNV area was significantly reduced in the ROS-TK-5/siVEGF and ranibizumab groups compared to the PBS control and ROS-TK-5/NC groups (all at P＜ 0.05).IVIS showed that the corneal fluorescent intensity was statistically different among the four groups at various times (Fgroup =27.193,P =0.003;Ftime =51.062,P ＜ 0.01).The corneal fluorescent intensities were significantly reduced in the ROS-TK-5/siVEGF and ranibizumab groups compared to the PBS control and ROS-TK-5/NC groups at 7 days and 14 days after modeling;in addition,the corneal fluorescent intensity was more effectively reduced in the ROS-TK-5/siVEGF group in comparison with the ranibizumab group at 7 days and 14 days after modeling (all at P＜ 0.05).At 21 days after modeling,the corneal fluorescent intensity was significantly reduced in the ROS-TK-5/ siVEGF and ranibizumab groups compared to the PBS control and ROS-TK-5/NC groups (all at P ＜ 0.05).Conclusions ROS-TK-5/siVEGF nanomedicine effectively attenuates alkali burn-induced CNV formation and appears to have a better effect in comparison with ranibizumab at an early stage.
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PURPOSE: Poloxamer 407 (P407) thermo-sensitive hydrogel formulations were developed to enhance the retention time in the urinary bladder after intravesical instillation. MATERIALS AND METHODS: P407 hydrogels (P407Gels) containing 0.2 w/w% fluorescein isothiocyanate dextran (FD, MW 4 kDa) as a fluorescent probe were prepared by the cold method with different concentrations of the polymer (20, 25, and 30 w/w%). The gel-forming capacities were characterized in terms of gelation temperature (G-Temp), gelation time (G-Time), and gel duration (G-Dur). Homogenous dispersion of the probe throughout the hydrogel was observed by using fluorescence microscopy. The in vitro bladder simulation model was established to evaluate the retention and drug release properties. P407Gels in the solution state were administered to nude mice via urinary instillation, and the in vivo retention behavior of P407Gels was visualized by using an in vivo imaging system (IVIS). RESULTS: P407Gels showed a thermo-reversible phase transition at 4℃ (refrigerated; sol) and 37℃ (body temperature; gel). The G-Temp, G-Time, and G-Dur of FD-free P407Gels were approximately 10℃–20℃, 12–30 seconds, and 12–35 hours, respectively, and were not altered by the addition of FD. Fluorescence imaging showed that FD was spread homogenously in the gelled P407 solution. In a bladder simulation model, even after repeated periodic filling-emptying cycles, the hydrogel formulation displayed excellent retention with continuous release of the probe over 8 hours. The FD release from P407Gels and the erosion of the gel, both of which followed zero-order kinetics, had a linear relationship (r²=0.988). IVIS demonstrated that the intravesical retention time of P407Gels was over 4 hours, which was longer than that of the FD solution ( < 1 hour), even though periodic urination occurred in the mice. CONCLUSIONS: FD release from P407Gels was erosion-controlled. P407Gels represent a promising system to enhance intravesical retention with extended drug delivery.
Subject(s)
Animals , Mice , Administration, Intravesical , Dextrans , Drug Liberation , Fluorescein , Hydrogels , Hydrogels , In Vitro Techniques , Kinetics , Methods , Mice, Nude , Microscopy, Fluorescence , Optical Imaging , Phase Transition , Poloxamer , Polymers , Urinary Bladder , UrinationABSTRACT
Objective: To evaluate the pulmonary retention, safety, and local drug concentration of Ranuncoli Ternati Radix extracts- ion sensitive in situ gels (RTRE-ISG). Methods: Sodium alginate as excipient, RTRE (70% ethanol) as solvent, it was swollen at room temperature using magnetic stirring to prepare RTRE-ISG which was administered via endotracheal tube to rats, The levels of inflammatory cytokines and fibrosis factors were detected in BALF to screen the relative safe priscription. Then using CY5.5 as fluorescent dye, the pulmonary retention of RTRE-ISG was evaluated with the aid of the small animal in vivo imaging system. In the end, the lung lavage was performed by using the improved alveolar lavage technique and the local drug concentration was determined. Results: The safety of RTRE-ISG prepared by 1% sodium alginate was better, the retention time in lung was 120-144 h revealed by fluorescence in vivo imaging system. The results showed that the release of drug reached to a peak of 5.24 μg/mL in 5 h after administration and 0.08 μg/mL in 144 h after administration. Conclusion: RTRE-ISG was good safety with the retention time in lung more than 120 h, which is a nevol sustained preparation of Chinese materia medica for the treatment of pulmonary tuberculosis.
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Aim Todiscusstheapplicationoftissue spontaneous fluorescence in myocardial ischemia injury of rats based on specific fluorescence detection technol-ogy.Methods Thechangeofspontaneousfluores-cence was compared between the myocardial tissue of normal rats and those of rats with myocardial ischemia injury and an quantitative analysis was then made.Re-sult Theresultsshowedthatspontaneousfluorescence of myocardial tissue for myocardial ischemia injury changed significantly.Spontaneous fluorescence signal of injury considerably was enhanced.The fluorescence signal which was quantified by FX Pro had statistical significance compared with normal myocardial tissue,P<0.01orP<0.05.Conclusion Ourresearchshows that spontaneous fluorescence of myocardial tissue can be enhanced obviously after myocardial ischemia inju-ry.Our research provides a method for the research and evaluation of myocardial ischemia injury model in rats which can be used in positioning,qualitative and quan-titative comparative study and in pathological,physio-logical,pharmacological and mechanism study.
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Aim To discuss the application of tissue spontane-ous fluorescence for draw focal cerebral ischemia reperfusion in-jury in rats based on specific fluorescence detection technology. Methods The change of spontaneous fluorescence WAS COM-PARED between the brains of normal rats and those of rats with draw focal cerebral ischemia-reperfusion injury and an quantita-tive analysis was then made. Result The results showed that spontaneous fluorescence of brain tissue for focal cerebral ische-mia reperfusion injury changed significantly. Spontaneous fluo-rescence signal of injury considerably enhanced. The fluores-cence signal which was quantified by IVIS had significant statisti-cal significance compared with normal brain tissue, P <0. 05. Conclusion Our research shows that spontaneous fluorescence of brain tissue enhances obviously after focal cerebral ischemia-reperfusion injury. Our research provides a method for the re-search and evaluation of focal cerebral ischemia-reperfusion inju-ry model in rats.
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In vivo imaging system(IVIS)is a new and rapidly expanding technology,which has a wide range of applications in life science such as cell tracing.By counting the number of photons emitted from a specimen,IVIS can quantify biological events such as tumor growth.We used B16F10-luc-G5 tumor cells and 20 Babl/C mice injected subcutaneously with B16F10-luc-G5 tumor cells(1×106 in 100 μL)to develop a method to quantitatively analyze cells traced by IVIS in vitro and in vivo,respectively.The results showed a strong correlation between the number of tumor cells and the intensity of bioluminescence signal(R2=0.99)under different exposure conditions in in vitro assay.The results derived from the in vivo experiments showed that tumor luminescence was observed in all mice by IVIS at all days,and there was significant difference(P<0.01)between every two days from day 3 to day 14.Moreover,tumor dynamic morphology could be monitored by IVIS when it was invisible.There was a strong correlation between tumor volume and bioluminescence signal(R2=0.97)by IVIS.In summary,we demonstrated a way to accurately carry out the quantitative analysis of cells using IVIS both in vitro and in vivo.The data indicate that IVIS can be used as an effective and quantitative method for cell tracing both in vitro and in vivo.