Hwan-Seok Jeong

The effect of mannosylation of liposome-encapsulated indocyanine green on imaging of sentinel lymph node

Abstract The imaging of sentinel lymph nodes (SLN) has been researched for its role in assessing cancer progression and postsurgical lymphedema. Indocyanine green (ICG) is a near-infrared (NIR) optical dye that has been approved by the Food and Drug Administration. It is known that liposome-encapsulated ICG (LP-ICG) has improved stability and fluorescence signal compared with ICG. We designed mannosylated liposome-encapsulated ICG (M-LP-ICG) as an optical contrast agent for SLN. M-LP-ICG has a higher UV absorbance spectrum and fluorescence intensity than LP-ICG. The stability of M-LP-ICG measured in 50% fetal bovine serum solution by a dialysis method was better than that of LP-ICG. M-LP-ICG demonstrated a high uptake in RAW 264.7 macrophage cell because the density of mannose is high. There were differences between M-LP-ICG and glucosylated liposome-encapsulated ICG (G-LP-ICG), which are geometrical isomers. The result of an inhibition study of M-LP-ICG showed a statistically significant decrease in uptake in RAW 264.7 cells after either co-treatment or pre-treatment with d-(+)-mannose as an inhibitor. Results from an in vitro experiment demonstrated that M-LP-ICG was specifically taken up by macrophage cells through the mannose receptor on its surface. The time-series images acquired from a normal mouse model after subcutaneous injection showed that the signal from M-LP-ICG in SLN and other organs appeared early and disappeared quickly in comparison with signals from LP-ICG. Not only the sentinel but also the draining lymph nodes were observed partly in M-LP-ICG. M-LP-ICG appears to increase the specificity of uptake and retention in macrophages, making it a good candidate contrast agent for an optic imaging system for SLN and the lymphatic system.

Effect of blue light emitting diodes on melanoma cells: Involvement of apoptotic signaling

The present study was undertaken to examine whether blue LED irradiation induces cellular apoptosis in B16-F10 cells and whether it blocks the early growth of melanoma cells in mice. Irradiation with blue LED was observed to reduce cell viability and to induce apoptotic cell death, as accompanied by exposure of phosphatidylserine on the plasma outside membrane and an accumulation of a sub-G1 population. Furthermore, the mitochondrial membrane potential increased, and mitochondria-related apoptotic proteins (cytochrome c, caspase 3, and PARP) were observed. In addition, the level of intracellular superoxide anion (O2(-)) gradually increased. Interestingly the phosphorylation of p53 increased at earlier times under blue LED irradiation, but reduced after exposure for a longer time. Additionally, the thickness of the mice footpad injected with B16-F10 cells decreased significantly until the 9th day of blue LED irradiation, indicating the inhibition of the early growth rate of the melanoma cells. Our data demonstrate that blue LED irradiation induces apoptotic cell death by activating the mitochondria-mediated pathway and reduces the early growth rate of melanoma cells. Further studies are needed to elucidate the precise mechanism of blue LED in melanoma cells.

Peptide-loaded Nanoparticles and Radionuclide Imaging for Individualized Treatment of Myocardial Ischemia

PURPOSE To determine whether chitosan hydrogel nanoparticles loaded with vascular endothelial growth factor (VEGF) peptides (81-91 fragments) capable of targeting the ischemic myocardium enhance angiogenesis and promote therapeutic effects and whether radionuclide image-guided dosage control is feasible. MATERIALS AND METHODS Experimental procedures and protocols were approved by the Institutional Animal Care and Use Committee. Rats (n = 32, eight per group) were subjected to myocardial ischemia (control group) and received chitosan hydrogel nanoparticles with VEGF165 proteins (chitosan VEGF) or VEGF81-91 peptides (chitosan peptides) via apical puncture. Ischemic hearts receiving chitosan without angiogenic factors served as the chitosan control. Myocardial perfusion was examined 7 days after surgery by using technetium 99m ((99m)Tc) tetrofosmin (37 MBq) autoradiography, and changes in vascular density with immunohistochemical staining were reviewed. Kruskal-Wallis test and Bonferroni corrected Mann-Whitney U test were used for multiple comparisons. Wilcoxon signed rank test was used to compare myocardial retention of (99m)Tc chitosan. RESULTS Thirty minutes of myocardial ischemia resulted in perfusion defects (median, 54%; interquartile range [IQR], 41%-62%). Chitosan VEGF decreased perfusion defect extent (median, 68%; IQR, 63%-73%; P = .006 vs control) and increased vascular density (median, 81 vessels per high-power field; IQR, 72-100; P = .009 vs control). Administration of chitosan peptides reduced the degree of perfusion defects (median, 66%; IQR, 62%-73%; P = .006 vs control) and increased vascular density (median, 82 vessels; IQR, 78-92; P = .006 vs control). The effects of chitosan peptides on perfusion and vascular density were comparable to those seen with chitosan VEGF proteins (P = .713 and P = .833, respectively). Chitosan radiolabeled with (99m)Tc was administered twice at reperfusion with a 1-hour interval to determine whether image-guided dosage control is feasible. The hearts initially retained 4.6% (IQR, 4.1%-5.0%) of (99m)Tc chitosan administered and 9.2% (IQR, 6.6%-12.7%; P = .068) with subsequent injection. CONCLUSION VEGF peptides have angiogenic potential and resulted in therapeutic effectiveness. Adjunct use of single photon emission computed tomography was also demonstrated for individualized treatment of myocardial ischemia by further tailoring the therapeutic dosing. Online supplemental material is available for this article.

Blue light emitting diode induces apoptosis in lymphoid cells by stimulating autophagy.

The present study was performed to examine the induction of apoptotic cell death and autophagy by blue LED irradiation, and the contribution of autophagy to apoptosis in B cell lymphoma A20 and RAMOS cells exposed to blue LED. Irradiation with blue LED reduced cell viability and induced apoptotic cell death, as indicated by exposure of phosphatidylserine on the plasma outside membrane and fragmentation of DNA. Furthermore, the mitochondrial membrane potential increased, and apoptotic proteins (PARP, caspase 3, Bax, and bcl-2) were observed. In addition, the level of intracellular superoxide anion (O2(-)) gradually increased. Interestingly the formation of autophagosomes and level of LC3-II were increased in blue LED-irradiated A20 and RAMOS cells, but inhibited after pretreatment with 3-methyladenine (3-MA), widely used as an autophagy inhibitor. Inhibition of the autophagic process by pretreatment with 3-MA blocked blue LED irradiation-induced caspase-3 activation. Moreover, a significant reduction of both the early and late phases of apoptosis after transfection with ATG5 and beclin 1 siRNAs was shown by the annexin V/PI staining, indicating a crucial role of autophagy in blue LED-induced apoptosis in cells. Additionally, the survival rate of mice irradiated with blue LED after injection with A20 cells increased compared to the control group. Our data demonstrate that blue LED irradiation induces apoptosis via the mitochondrial-mediated pathway, in conjunction with autophagy. Further studies are needed to elucidate the precise mechanism of blue LED-induced immune cell death.

Improving Cerebral Blood Flow through Liposomal Delivery of Angiogenic Peptides: Potential of 18F-FDG PET Imaging in Ischemic Stroke Treatment

Strategies to promote angiogenesis can benefit cerebral ischemia. We determined whether liposomal delivery of angiogenic peptides with a known biologic activity of vascular endothelial growth factor benefitted cerebral ischemia. Also, the study examined the potential of 18F-FDG PET imaging in ischemic stroke treatment. Methods: Male Sprague–Dawley rats (n = 40) underwent 40 min of middle cerebral artery occlusion. After 15 min of reperfusion, the rats (n = 10) received angiogenic peptides incorporated into liposomes. Animals receiving phosphate-buffered solution or liposomes without peptides served as controls. One week later, 18F-FDG PET imaging was performed to examine regional changes in glucose utilization in response to the angiogenic therapy. The following day, 99mTc-hexamethylpropyleneamine oxime autoradiography was performed to determine changes in cerebral perfusion after angiogenic therapy. Corresponding changes in angiogenic markers, including von Willebrand factor and angiopoietin-1 and -2, were determined by immunostaining and polymerase chain reaction analysis, respectively. Results: A 40-min period of middle cerebral artery occlusion decreased blood perfusion in the ipsilateral ischemic cortex of the brain, compared with that in the contralateral cortex, as measured by 99mTc-hexamethylpropyleneamine oxime autoradiography. Liposomal delivery of angiogenic peptides to the ischemic hemisphere of the brain attenuated the cerebral perfusion defect compared with controls. Similarly, vascular density evidenced by von Willebrand factor–positive staining was increased in response to angiogenic therapy, compared with that of controls. This increase was accompanied by an early increase in angiopoietin-2 expression, a gene participating in angiogenesis. 18F-FDG PET imaging measured at 7 d after treatment revealed that liposomal delivery of angiogenic peptides facilitated glucose utilization in the ipsilateral ischemic cortex of the brain, compared with that in the controls. Furthermore, the change in regional glucose utilization was correlated with the extent of improvement in cerebral perfusion (r = 0.742, P = 0.035). Conclusion: Liposomal delivery of angiogenic peptides benefits cerebral ischemia. 18F-FDG PET imaging holds promise as an indicator of the effectiveness of angiogenic therapy in cerebral ischemia.

Effect of space length of mannose ligand on uptake of mannosylated liposome in RAW 264.7 cells: In vitro and in vivo studies

The most widely used method for increasing uptake on macrophage is specific targeting for mannose receptor (MR) presented on macrophages. Efficiency of the uptake for MR is influenced by the space length and flexibility of mannose ligand in liposome (LP). We prepared mannosylated liposomes (M-EGn-LP-ICG) encapsulated indocyanine green (ICG) with mannose ligand of various ethylene glycol units (EG), LP-ICG, and mannosylated liposome (M-LP-ICG) incorporated with p-aminophenyl-α-d-mannopyranoside. We studied the effect of space length of the mannose ligand in vitro and in vivo with prepared liposomes. A space length of two ethylene glycol units at least was needed for uptake by macrophages and the uptake was increased as the space length increased up to EG4. We measured near-infrared (NIR) fluorescence intensity by ICG and the fluorescence value of cell-associated N-(4-nitrobenzo-2-oxa-1,3-diazole) (NBD) in liposome after cellular uptake. M-EG4-LP-ICG showed lower NIR fluorescence intensity but higher NBD fluorescence value than M-LP-ICG. The result of pre-treatment with d(+)-mannose as an inhibitor showed significant decreasing in uptake of mannosylated LP-ICG but no difference in LP-ICG. These were explained that mannosylated LP-ICG was taken up by macrophages through the MR and M-EG4-LP-ICG showed more specific uptake than M-LP-ICG. We obtained images as time passed in the NIR range after intravenous administration using a Balb/c mouse with inflammatory model. The results showed high uptake in liver at early time and rapid degradation of mannosylated LP-ICG. M-EG4-LP-ICG was more selectively taken up by macrophages than M-LP-ICG.

Effect of Angiogenesis Induced by Consecutive Intramuscular Injections of Vascular Endothelial Growth Factor in a Hindlimb Ischemic Mouse Model

PurposeAngiogenesis plays a major role in various physiological and pathological situations. Thus, an angiogenic therapy with vascular endothelial growth factor (VEGF) has been commonly recommended as a representative therapeutic solution to recover the insufficient blood supply of collateral vessels in an ischemic lesion. In this study, the injection method and injection time point of VEGF proteins were focused to discover how to enhance the angiogenic effect with VEGF.MethodsMouse models (n = 15) were divided into control, VEGF treatment by intra-venous injection (VEGF-IV) and VEGF treatment by intra-muscular injection (VEGF-IM). Right proximal femoral arteries of mice were firmly sutured to obstruct arterial blood-flow. In the VEGF-IV treatment group, VEGF proteins were injected into the tail vein and, in the VEGF-IM treatment group, VEGF proteins were directly injected into the ischemic site of the right thigh after postoperative day 5, 10, 15, 20 and 25 follow-ups. Blood-flow images were acquired by 99mTc Gamma Image Acquisition System to compare the ischemic-to-non-ischemic bloodstream ratio at postoperative days 5, 15, and 30.ResultsVEGF-IM treatment significantly induced higher an angiogenic effect rather than both the control group (P = 0.008) and VEGF-IV treatment group (P = 0.039) at the 30th day.ConclusionDuring all experiments, angiogenesis of VEGF-IM treatment represented the most evident effect compared with control and VEGF-IV group in a mouse model of hindlimb ischemia.

131I-labeled chitosan hydrogels for radioembolization: A preclinical study in small animals

INTRODUCTION The purpose of the study was to examine potential of 131I-labeled chitosan hydrogels (Chi) for treatment of liver cancer. METHODS Orthotopic hepatoma was induced by McA-RH7777-fLuc cells (1×107) that were injected into the left hepatic lobe of rats. Ten days later, tumor-bearing rats evidenced by bioluminescence received 125I-labeled Chi with left hepatic artery access. Pharmacokinetics and excretion (n=8) and biodistribution (n=6/time point) were studied after injection. To examine therapeutic potential, animals (n=8/group) were also treated with Chi labeled with or without 131I. Changes in tumor volume by magnetic resonance (MR) imaging were studied. RESULTS The rate of tumor induction assessed by bioluminescence imaging was 72% (68/95). Gamma counter and scintigraphy imaging analyses showed accumulation of 125I-labeled Chi dominantly in the liver. A small fraction of 125I-labeled Chi was detected in the stomach (2.02±3.07%ID) and muscle (1.37±1.48%ID) at 2 d post-treatment. Blood sample analysis showed the maximum blood concentration of 0.09±0.03%ID/mL, which peaked at 0.60±0.45 d. Over a 4-week period, 31.22±8.16%ID were excreted in the urine and 3.5±1.3% in the feces. Treatment of Chi (median, 876mm3; IQR, 496mm3-1413mm3) markedly reduced the extent of tumor growth, compared to controls (median, 12,085mm3; IQR, 7786mm3-25,832mm3; P<0.05 vs control). 131I Chi (median, 80mm3; IQR, 35mm3-172mm3; P<0.05 vs control) induced a greater tumor-suppressing effect, compared to Chi alone. CONCLUSIONS In this study, we have characterized a new radioembolization device, 131I Chi, in vivo and provided evidence for its therapeutic potential. ADVANCES IN KNOWLEDGE Transarterial embolization is a conceivable treatment option for patients with inoperable liver cancer to mitigate the disease progression. Recently, we have developed chitosan-based hydrogel microparticles. In the present study, the hydrogel microparticles were radiolabeled with 131I for treatment of liver cancer. Our results demonstrated that a hepatic arterial injection of 125I-labeled Chi resulted in substantial liver accumulation, which was accompanied by virtually no extrahepatic deposition. The results of the present study also showed that administration of 131I Chi markedly suppressed tumor growth, compared to controls and to animals receiving unlabeled Chi. 131I-labeled chitosan hydrogel microparticles represent a new therapeutic approach for treatment of liver cancer.

PEGylated nanoliposomes encapsulating angiogenic peptides improve perfusion defects: Radionuclide imaging-based study

INTRODUCTION Although liposomes hold promise for cancer therapy, the effectiveness of treating myocardial ischemia by promoting angiogenesis has yet to be proved. Nanoliposomes loaded with therapeutic agents can effectively target ischemic myocardium via enhanced permeability and retention. Surface polyethylene glycol (PEG) modification can further facilitate effective targeting by prolonging liposomal circulation. This study aimed to determine whether PEGylated nanoliposomes are effective in facilitating targeted drug delivery and treating myocardial ischemia. METHODS Rats subjected to 30min of myocardial ischemia were given (99m)Tc-hexamethylpropyleneamine oxime- or (99m)Tc-diethylenetriamine pentaacetate-labeled liposomes with mean diameters of ~100nm or ~600nm with or without PEG modifications to determine the extent of myocardial uptake in the different conditions. Therapeutic effectiveness was assessed by studying changes in myocardial perfusion defects with (99m)Tc-tetrofosmin autoradiography and vascular density with immunohistochemistry at 7days post-treatment. RESULTS The liver and spleen showed the largest capacity for liposome uptake. Uptake by the liver and spleen was more pronounced when the liposomes were larger. Conversely, myocardial liposome uptake was significantly greater when the liposomes were ~100nm rather than ~600nm in diameter. Surface modification with PEG significantly augmented myocardial uptake of ~100nm liposomes. PEG modification did not affect the size dependence. To investigate therapeutic efficacy, hearts subjected to ischemia received PEGylated nanoliposomes encapsulated with angiogenic peptides. Our data demonstrated that PEGylated nanoliposomes loaded with angiogenic peptides improved myocardial perfusion defects and increased vascular density. A 10-fold increase in liposomal concentration did not further benefit myocardial ischemia. CONCLUSIONS Liposomal angiogenic formulation with size control and PEG modification may be effective treatment strategy for myocardial ischemia. Increasing the concentration of liposomes does not necessarily benefit myocardial ischemia.

Liposomal angiogenic peptides for ischemic limb perfusion: comparative study between different administration methods.

Abstract Background: We investigated the therapeutic effectiveness of PEGylated liposomes loaded with angiogenic peptides for treating hindlimb ischemia. Methods: Rats received a femoral artery occlusion. Red blood cells collected from the animals were labeled with technetium-99m. Limb perfusion gamma imaging was performed. PEGylated liposomes loaded with angiogenic peptides were administered intra-arterially. Technetium-99m red blood cell imaging was repeated 1 week later. The animals were sacrificed the next day. The expression of angiogenic proteins was studied. Later, changes in limb perfusion after intra-arterial infusion versus intra-muscular injection were also compared to determine the therapeutic effectiveness of different administration methods. Results: Femoral artery occlusion dramatically reduced ischemic limb perfusion (by an average of 69%, compared to contralateral limb). This was not different among groups (p > 0.05). Liposomes loaded with angiogenic peptides significantly improved ischemic limb perfusion, compared to controls (210% of baseline, versus 100% of baseline in control; p < 0.05 versus controls). The enhanced ischemic limb perfusion was accompanied by an increased expression of CD 31 (an average of 1.6-fold increase of controls; p < 0.05). The liposomes or peptides treatment alone did not affect ischemic perfusion (liposomes alone: 100% of baseline; peptides alone: 120% of baseline; p > 0.05 versus controls, respectively) or the angiogenic response (1.1-fold of controls in liposomes alone; 1.0-fold of controls in peptides alone; p > 0.05 versus controls, respectively). Intra-muscular injection induced similar liposomal treatment effects on ischemic limb perfusion (230% of baseline) as those by intra-arterial infusion (210% of baseline; p < 0.05 versus intra-muscular). Conclusions: PEGylated liposomes loaded with angiogenic peptides improved ischemic limb perfusion and promoted angiogenic responses. Liposomal angiogenic treatment via intra-arterial infusion resulted in an equally effective therapeutic efficacy compared to that of intra-muscular injection. These results show the therapeutic potential of our liposomal strategy for treating peripheral limb ischemia.