Proceedings of the 2nd World Congress on New Technologies, 2016
Indocyanine green (ICG) is a FDA-approved near-infrared (NIR) fluorophore that is widely used in ... more Indocyanine green (ICG) is a FDA-approved near-infrared (NIR) fluorophore that is widely used in in vivo nearinfrared (NIR) fluorescence imaging of cancers . However, since ICG is "always on" agent, it has the potential problem of high background fluorescence when circulating in the blood stream, which can result in a low target-to-background ratio (TBR) in in vivo imaging. In addition, non-specific uptake by normal cells and tissues of ICG further reduced TBR. Due to these reasons, its applications for fluorescence imaging as well as phototherapy of cancers have been limited. Here we developed ICG-loaded hollow mesoporous silica nanoparticles (HMSNP) for selective NIR fluorescence imaging and phototherapy of cancers. We expected that NIR fluorescence and phototoxicity are turned off in normal cells or in the extracelluar regions but becomes highly fluorescent and phototoxic inside cancer cells, thereby enabling selective fluorescence imaging and therapy of cancers. The size of the prepared nanoparticles was about 180 nm in diameter. ICG was loaded into HMSNP by 12 w/w%. Comparison of the UV/Vis absorption spectra of ICG-loaded HMSNP and free ICG in aqueous solution confirmed aggregation of the loaded ICGs inside the nanoparticles. As expected, both NIR fluorescence and singlet oxygen generation of ICG-loaded HMSNP were significantly inhibited in comparison with free, ICG. In the in vitro cell study, strong fluorescence was observed in the cancer cells treated with ICG-loaded HMSNP indicating turn-on of its fluorescence inside cancer cells. Upon light irradiation, ICG-loaded HMSNP showed significant cytotoxicity higher than that of free ICG molecules. These features make HMSNP a promising platform for utilizing ICG in biomedical applications.
Sterilization procedure is one of the most important obstacles in the clinical applications of bi... more Sterilization procedure is one of the most important obstacles in the clinical applications of biodegradable microspheres. The microspheres prepared with poly( -hydroxy acid) were severely aggregated during ethylene oxide (EO) gas sterilization, and could not be used in clinical applications. In this study, the e!ects of EO gas sterilization on the poly(L-lactide) (PLLA) microspheres were analyzed by nuclear magnetic resonance spectroscopy (H-NMR), di!erential scanning calorimetry (DSC), gel permeation chromatography (GPC), scanning electron microscope (SEM) and size fractionation. The aggregation between the microspheres might be stimulated by high mobility of amorphous regions of PLLA on the microsphere surfaces since both water vapor and gas mixture can reduce glass transition temperature (¹ ) of PLLA below the sterilization temperature. During EO gas sterilization, there were no changes in the molecular structure and the molecular weight of PLLA in microspheres, but there were changes in the crystallinity of PLLA in microspheres. In this study, poly(L-lactide)-poly(ethylene glycol) diblock copolymers (PLE) were blended with PLLA homopolymers in various ratios to design the microsphere suitable for EO gas sterilization. Aggregation of PLLA microspheres was markedly prevented when more than 4 wt% of PLE was blended in the microspheres. This inhibition e!ect on aggregation may be due to the increased initial crystallinity of the microspheres, which help to maintain the microsphere morphology during EO gas sterilization.
All-trans-retinoic acid (atRA) has been proved to be effective against several malignancies in hu... more All-trans-retinoic acid (atRA) has been proved to be effective against several malignancies in human clinical trials. However, in many patients who were treated with atRA, the cancer relapsed after a brief remission. One reason for such relapse is that atRA is metabolized by specific P450s that are induced in the liver during prolonged atRA treatments. In order to overcome such a drawback of atRA, we prepared biodegradable microspheres to provide continuous release of atRA for a long period of time. These biodegradable microspheres were prepared by poly(L-lactide) (PLLA) and polyethylene glycol (PEG)-PLLA diblock copolymers (PLE) in various blending ratios to control the release rate of atRA. As the PLE content in microsphere was increased, the density of the hydrophilic PEG block of PLE on microsphere surfaces increased and the microspheres were dispersed well in PBS without any surfactants. Various release patterns of atRA were obtained according to PLE and atRA contents in the microspheres. Especially, the pseudo-zero-order release profiles were observed for 5 weeks when the contents of PLE and atRA in the microspheres were above 4 wt.%.
All-trans-retinoic acid (atRA) is a promising anticancer and antiwrinkle drug. However, its clini... more All-trans-retinoic acid (atRA) is a promising anticancer and antiwrinkle drug. However, its clinical application is limited because it is rapidly metabolized by the induced cytochrome P450 (P450). In this study, farnesol derivatives are proposed as new inhibitors to prevent P450-mediated metabolism. The farnesol derivatives were suc-farnesol and mal-farnesol, which were synthesized by the chemical conjugation of farnesol with succinic anhydride and maleic anhydride, respectively. The inhibition effects of farnesol, farnesoic acid, and farnesol derivatives on the atRA metabolism were evaluated in microsome and in AMC-HN-6 cells. In the microsome experiment, suc-farnesol and mal-farnesol strongly inhibited atRA metabolism at 10 -4 mol/L concentration by as much as 61% and 77%, respectively. In the cell experiment, the inhibition effects of farnesol derivatives on the atRA metabolism showed similar tendency as the results in the microsome experiment, even if the effect was somewhat decreased. Effects of farnesoic acid and farnesol, however, were not significant. This research suggests that carboxylic end groups, such as atRA and hydrophobicity, might be important factors causing the higher inhibition effect, and that derivatization of farnesol can be 1 method to develop new inhibitors of atRA metabolism.
Journal of Biomaterials Science, Polymer Edition, 2001
The aggregation of poly(®-hydroxy acid) microspheres during ethylene oxide (EO) gas sterilization... more The aggregation of poly(®-hydroxy acid) microspheres during ethylene oxide (EO) gas sterilization makes it dif cult for the microspheres to be used in clinical applications. In this study, six kinds of PLLA-PEG-PLLA triblock copolymers (TriPLE) were synthesized with various composition ratios of PEG / PLLA in the range of 0.012 to 0.103. TriPLE microspheres were prepared by the oil-in-water emulsion method. TriPLE microspheres were characterized by using 1 H-NMR, gel permeation chromatography (GPC), and differential scanning calorimetry (DSC). After sterilization by EO gas at 55 ± C, the microspheres were analyzed by scanning electron microscope (SEM), laser diffractometry, standard sieves, X-ray diffraction (XRD), GPC, and DSC. When the composition ratio of PEG / PLLA was above 0.02, the initial crystallinity of TriPLE in microspheres was as high as 50%, and the microspheres were suitable to be sterilized by EO gas. On the other hand, TriPLE microspheres, which had composition ratios of PEG / PLLA below 0.02, had low initial crystallinities of about 30%, and aggregated during EO gas sterilization. For these microspheres, crystallinity increased up to 50% during the sterilization, whereas other TriPLE microspheres did not show any changes in crystallinity. Therefore, the aggregation of TriPLE microspheres during EO gas sterilization was markedly reduced as the initial crystallinity of TriPLE in the microspheres was increased.
Journal of Biomaterials Science, Polymer Edition, 2002
Biocompatibility studies of all-rrnns-retinoic acid (RA)-loaded microspheres were carried out ati... more Biocompatibility studies of all-rrnns-retinoic acid (RA)-loaded microspheres were carried out atier they were subcutaneously injected into rats. To characterize the inflammatory response to these microspheres, tissue reactions at the implantation site and cell types in the interstices of thc microsphercs were evaluated for 180 days. On the 15th day, the cross-sectional area of the fibrous capsules surrounding the implantation site of the RA-loaded microspheres was ftrur times larger than that of the control microspheres. The size of the fibrous capsules surrounding the implantation site of the RA-loaded microspheres decreased significantly over a period of 75 days. while the size of the fibrous capsules surrounding the implantation site of the control microspheres rernained alnrt'rst constant throughout the entire course of I 80 days. The tissue response to the RA-loaded microspheres was more intensified by the increased extensive cellular infiltration of rnacrophages, granulation tissue, and librosis than that to the control microspheres. The difference in the inflarnrnatory responsc between thc RA-loaded microspheres and the control microspheres was significant for 7-5 days afier implantation. lt was suggested that the released RA from the microspheres stimulated inflammatory responses. However, no further enhanced inflammation reactions were detectcd after RA had been completely released tiorn the microspheres.
All‐trans retinoic acid (atRA)‐loaded microspheres severely induce inflammatory responses after m... more All‐trans retinoic acid (atRA)‐loaded microspheres severely induce inflammatory responses after microsphere implantation. Fibroblasts and a thick band of fibrous capsule resulting from the inflammatory responses could hamper drug permeation to the bloodstream because fibroblasts actively metabolize atRA into polar metabolites and the thick fibrous capsule acts as a diffusion barrier. In the present study, we investigated whether the fibroblast proliferation and collagen deposition induced by atRA released from microspheres might affect the atRA concentration in plasma and atRA metabolism with or without treatment of dexamethasone as an anti‐inflammatory drug. After subcutaneous injection of atRA‐loaded microspheres in rats, it was observed that atRA‐loaded microspheres induced severe inflammatory responses and stimulated fibroblast proliferation and collagen deposition in fibrous capsules. On the other hand, the orally treated dexamethasone effectively prevented inflammatory respons...
Previously, poly(L‐lactide) microspheres containing retinoic acid (RA) had been prepared for pare... more Previously, poly(L‐lactide) microspheres containing retinoic acid (RA) had been prepared for parenteral administration in order to overcome “acute retinoic acid resistance.” In this study, acute toxicities of RA‐loaded microspheres were investigated in mice after the microspheres were injected intramuscularly. The maximum dose of RA in the microspheres was determined by considering both the injection volume and dispersion of microspheres in the vehicle media, and the dose levels were determined to be 0, 90, 180, and 360 mg RA/kg. Mice were carefully observed for any clinical signs for 14 days, and gross observation of the organs was performed at 14 days. When a dose of 90 mg RA/kg was administered, no severe toxicities were observed; however, with the administration of 180 and 360 mg RA/kg, abnormal appearances of organs, such as discolor, atrophy, hemorrhage, etc., were observed. There were no mortalities or bone fractures observed at any of the doses. In our previous study of canc...
Biodegradable microspheres containing all‐trans‐retinoic acid (atRA) were prepared previously to ... more Biodegradable microspheres containing all‐trans‐retinoic acid (atRA) were prepared previously to enhance the clinical efficacy of atRA for chemoprevention. In this study, subacute toxicity of atRA in sustained release was evaluated after subcutaneous administration of 0, 25, 50, and 100 mg atRA/kg of atRA‐loaded microspheres. After the subcutaneous injection of atRA‐loaded microspheres, the atRA concentration in plasma was maintained in the range of 3.5–25 ng/ml for 2–3 weeks. When 100 mg atRA/kg was administered, the atRA concentration in plasma was maintained at levels greater than 10 ng/ml and severe toxic effects were observed. When the dose of 50 mg atRA/kg was administered, the plasma concentration of atRA was maintained below 10 ng/ml over 3 weeks and only mild signs of toxicity were observed. Therefore, it was concluded that the dosage of atRA‐loaded microspheres should be less than 50 mg atRA/kg for chemopreventive applications against carcinogenesis. Considering recovery f...
A folate receptor-specific activatable probe for near-infrared fluorescence imaging of ovarian cancer
Chem. Commun., 2014
A folate receptor-specific activatable probe for in vivo near-infrared fluorescence imaging with ... more A folate receptor-specific activatable probe for in vivo near-infrared fluorescence imaging with a high target-to-background ratio was synthesized.
The present study was performed to investigate the stability of W/O/W emulsions containing 4-␣glu... more The present study was performed to investigate the stability of W/O/W emulsions containing 4-␣glucanotransferase (4␣GTase)-treated starch against environmental stresses such as heating, shearing, and repeated freeze-thawing. W/O/W emulsions were subjected to thermal processing at different temperatures ranging from 30 to 90 • C for 30 min, constant shear for 0-7 min, and freeze-thaw cycling between −20 • C and 30 • C, respectively, and followed by encapsulation efficiency (EE) measurement. As for the case of thermal stress, it was clearly shown that addition of 4␣GTase-treated starch in the internal aqueous phase of emulsions helped to maintain higher EE during thermal processing. However, at lower PGPR level (2%), the addition of 4␣GTase-treated starch dramatically reduced EE at temperatures higher than 70 • C, which was probably related to the melting of 4␣GTase-treated starch gel. The incorporation of 4␣GTase-treated starch improved the stability of emulsions during shearing process, but could not prevent W/O/W emulsions from creaming and destabilizing during freeze-thaw cycling.
Background: Prediction of resistance mechanisms for epidermal growth factor receptor-tyrosine kin... more Background: Prediction of resistance mechanisms for epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) remains challenging. Thus, we investigated whether resistant cancer cells that expand shortly after EGFR-TKI treatment would eventually cause the resistant phenotype. Methods: We generated two EGFR-mutant lung cancer cell lines resistant to gefitinib (PC9GR and HCC827GR). The parent cell lines were exposed to short-term treatment with gefitinib or paclitaxel and then were assessed for EGFR T790M mutation and C-MET expression. These experiments were repeated in vivo and in clinically relevant patient-derived cell (PDC) models. For validation in clinical cases, we measured these gene alterations in plasma circulating tumor DNA (ctDNA) before and 8 weeks after starting EGFR-TKIs in four patients with EGFR-mutant lung cancer. Results: T790M mutation was only detected in the PC9GR cells, whereas C-MET amplification was detected in the HCC827GR cells. The T790M mutat...
We developed a single-camera-based near-infrared (NIR) fluorescence imaging device using indocyan... more We developed a single-camera-based near-infrared (NIR) fluorescence imaging device using indocyanine green (ICG) NIR fluorescence contrast agents for image-induced surgery. In general, a fluorescent imaging system that simultaneously provides color and NIR images uses two cameras, which is disadvantageous because it increases the imaging head of the system. Recently, a single-camera-based NIR optical imaging device with quantum efficiency partially extended to the NIR region was developed to overcome this drawback. The system used RGB_NIR filters for camera sensors to provide color and NIR images simultaneously; however, the sensitivity and resolution of the infrared images are reduced by 1/4, and the exposure time and gain cannot be set individually when acquiring color and NIR images. Thus, to overcome these shortcomings, this study developed a compact fluorescent imaging system that uses a single camera with two complementary metal–oxide semiconductor (CMOS) image sensors. Sensit...
Materials and methods Materials Pyrrole (98 %), polyvinylpyrrolidone (PVP, MW ~29,000), and iron(... more Materials and methods Materials Pyrrole (98 %), polyvinylpyrrolidone (PVP, MW ~29,000), and iron(III) chloride hexahydrate (FeCl 3 •6H 2 O), trypsin from porcine pancreas (13,100 U/mg), and hyaluronidase (1,228 U/mg) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hyaluronic acid (HA, Mw 39,000 Da) was obtained from Lifecore Biomedical (Chaska, MN, USA) and used as received. Chlorin e4 (Ce4) was purchased from Frontier Scientific, Inc. (Logan, UT, USA). Singlet oxygen sensor green (SOSG), which is an agent for detecting singlet oxygens, was obtained from Invitrogen Corp. (Carlsbad, CA, USA). Methods Synthesis and characterization of positively charged-Ce4 (PCe4) Positively charged photosensitizer (PCe4) was prepared by conjugating Ce4 with RRK peptide. Briefly, the peptide (RRK) was synthesized using a standard solid-phase peptide synthesis protocol. Coupling was carried out using Fmoc amino acid (8 eq), Nhydroxybenzotriazole (HOBt, 8 eq), N,N-diisopropylethylamine (DIPEA,16 eq) in DMF for 2 h. After each step, resin washing was performed using DMF and methanol 2 times each. The Fmoc protecting group was removed by rocking in 20% piperidine in DMF for 10 min (twice). Ce4 and HOBt/DIPEA in DMF were added to the peptide-attached resin. The mixture was reacted for 12 h and suctioned, and then the resin was washed with DMF, MeOH, and DMF. When the reaction was completed, crude peptide was cleaved from the resin using a mixture of TFA/EDT/thioanisole/TIS/deionized water (90/2.5/2.5/2.5/2.5 volume) for 2 h. The solution was precipitated with cold ether and pelleted using a centrifuge. The solid was collected and air-dried. Crude peptide was dissolved in deionized water and purified by prep-LC, lyophilized, and preserved in a refrigerator before use. The molecular weights of PCe4 were measured using LC/MSD
Quantitative Imaging in Medicine and Surgery, 2020
Background: Accurate identification of tumor sites and boundaries is of paramount importance duri... more Background: Accurate identification of tumor sites and boundaries is of paramount importance during minimally invasive surgery. Although laparoscopic resection is being increasingly and widely performed for early gastric and colorectal cancers, the detection of tumors located inside the stomach and intestine is difficult owing to the lack of tactile sensation. Here, we propose the application of an indocyanine green (ICG)-loaded alginate hydrogel system as a fluorescence surgical marker for precise laparoscopic operations. Methods: A physical complex of ICG and human serum albumin (HSA) was mixed with sodium alginate to form an injectable hydrogel system. Calcium carbonate and D-gluconic acid (GA) were added to the gel to control its strength and gelation time, respectively. The optimal conditions for the preparation of injectable hydrogels were determined by analyzing the fluorescence spectra and sol-gel transition time of the prepared samples at various concentrations and compositions. Next, the aqueous solutions of ICG, ICG-HSA, and ICG-HSA-loaded alginate were subcutaneously injected into nude mice (three mice per group), and nearinfrared (NIR) fluorescence images of the mice (λ ex. =780 nm, λ em. =845 nm) were obtained at different points in time for 8 days. Then, fluorescence intensities at the injection sites, target-to-background ratio, and areas of ICG fluorescence were analyzed. Finally, the potential utility of ICG-HSA-loaded alginate hydrogel as a surgical marker was evaluated in a porcine model. The ICG-HSA-loaded alginate solution was injected into three sites in the submucosal space of the porcine stomach via a catheter. A fluorescent laparoscopic system was installed on the abdomen of the pig 3 days post-injection, and the fluorescence signal generated from the fluorescence surgical marker located inside the stomach was evaluated using the fluorescence laparoscope system (λ ex. =785 nm, λ em. =805 nm). Results: The optimal concentration of ICG-HSA complex was determined to be 30 μM, and maximum fluorescence intensity of the complex was obtained at a 1:1 mole ratio of HSA to ICG. The subcutaneous injection of ICG or ICG-HSA solution in mice resulted in the rapid spread of the fluorescence signal around the injection site in 3 h, and a weak fluorescence was detected at the injection site 24 h post-injection. In contrast, the fluorescence detection time was effectively prolonged up to 96 h post-injection in the case of ICG-HSA-loaded alginate gel, while diffusion of the injected ICG from the injection site was effectively prevented. In the laparoscopic operation, injection sites of the hydrogel in porcine stomach could be accurately detected in real time even after 3 days. Conclusions: This alginate hydrogel system may be potentially useful as an effective surgical marker in terms of accuracy and persistence for laparoscopic operation.
Epidermal growth factor receptor (EGFR) is overexpressed in many types of cancers, which is assoc... more Epidermal growth factor receptor (EGFR) is overexpressed in many types of cancers, which is associated with metastatic potential and poor prognosis in cancer patients. Therefore, development of EGFR-targeted sensitive imaging probes has been a challenge in tumor targeting, image-guided cancer surgery, patient-selective anti-EGFR therapy, and efficient targeted therapies. Methods: We synthesized a zwitterionic near-infrared fluorophore (ATTO655)-conjugated epidermal growth factor (EGF) as a novel activatable molecular probe. Fluorescence OFF/ON property and EGFR-targeting specificity of EGF-ATTO655 as well as its utility in real-time near-infrared (NIR) fluorescence imaging of EGFR-positive cancers were evaluated using in vitro and in vivo studies. Results: When conjugated to EGF, the fluorescence of ATTO655 quenched efficiently by photo-induced electron transfer (PET) mechanism between the conjugated dyes and nearby amino acid quenchers (tryptophan/tyrosine residues), which was stably maintained at physiological pH and in the presence of serum for at least 17 h. The fluorescence of EGF-ATTO655 turned on by receptor-mediated endocytosis and subsequent disintegration of EGF in EGFR-positive A431 cancer cells, thereby enabling specific and real-time fluorescence imaging of EGFR-positive cancer cells. Consequently, EGFR-positive tumors could be clearly visualized 3 h post-injection with a significantly high tumor-to-background ratio (TBR = 6.37). Conclusion: This PET mechanism-based OFF/ON type of EGF probe showed great potential for rapid, real-time, and target-cell-specific imaging of EGFR-overexpressing cancers in vitro and in vivo.
In this study, we synthesized manganese dioxide nanoparticles (MnO 2 NPs) stabilized with biocomp... more In this study, we synthesized manganese dioxide nanoparticles (MnO 2 NPs) stabilized with biocompatible polymers (polyvinylpyrrolidone and polyacrylic acid) and analyzed their effect on non-small cell lung cancer (NSCLC) cells with or without gefitinib resistance in vitro. MnO 2 NPs showed glutathione (GSH)-responsive dissolution and subsequent enhancement in magnetic resonance (MR) imaging. Of note, treatment with MnO 2 NPs induced significant cytotoxic effects on NSCLC cells, and additional dose-dependent therapeutic effects were obtained upon X-ray irradiation. Normal cells treated with MnO 2 NPs were viable at the tested concentrations. In addition, increased therapeutic efficacy could be achieved when the cells were treated with MnO 2 NPs in hypoxic conditions. Therefore, we conclude that the use of MnO 2 NPs in MR imaging and combination radiotherapy may be an efficient strategy for the imaging and therapy of NSCLC.
Gastric cancer is the third most common cause of cancer mortality, and the survival rate of stage... more Gastric cancer is the third most common cause of cancer mortality, and the survival rate of stage IV advanced gastric cancer (AGC) patients with distant metastasis is very low. Thus, the detection and eradication of disseminated cancer cells by targeting cell surface molecules in AGC would improve patient survival. The hyaluronic acid receptor, CD44, has various isoforms generated by alternative splicing, and some isoforms are known to be correlated to gastric cancer. In this study, to find out the most appropriate CD44v for targeting AGC, we analysed the expression differences of CD44 isoforms at the mRNA level in stomach cancer cell lines as well as in 74 patients with AGC by using exon-specific qRT-PCR. Among the CD44v isoforms, CD44v8-10 was determined as the most promising biomarker for the development of theranostic agents of gastric cancer. Next, we synthesised the conjugate of anti-CD44v9 antibody with near-infrared fluorophore or photosensitiser, and then demonstrated its f...
Antibody-fluorophore conjugates have high potential for the specific fluorescence detection of ta... more Antibody-fluorophore conjugates have high potential for the specific fluorescence detection of target cancer cells in vitro and in vivo. However, the antibody-fluorophore conjugates described to date are inappropriate for real-time imaging of target cells because removal of unbound antibody is required to reduce background fluorescence before quantifiable analysis by microscopy. In addition, clinical applications of the conjugates have been limited by persistent background retention due to their long systemic circulation and nonspecific uptake. Here we report fast and real-time near-infrared fluorescence imaging of target cancer cells using an antigen-responsive molecular "on-off" sensor: the fluorescence of trastuzumab-ATTO680 conjugate is dark (i.e., turned off) in the extracellular region, while it becomes highly fluorescent (i.e., turned on) upon binding to the target antigen HER2 on cancer cell surface. This molecular switch enables fast and real-time imaging of targe...
Proceedings of the 2nd World Congress on New Technologies, 2016
Indocyanine green (ICG) is a FDA-approved near-infrared (NIR) fluorophore that is widely used in ... more Indocyanine green (ICG) is a FDA-approved near-infrared (NIR) fluorophore that is widely used in in vivo nearinfrared (NIR) fluorescence imaging of cancers . However, since ICG is "always on" agent, it has the potential problem of high background fluorescence when circulating in the blood stream, which can result in a low target-to-background ratio (TBR) in in vivo imaging. In addition, non-specific uptake by normal cells and tissues of ICG further reduced TBR. Due to these reasons, its applications for fluorescence imaging as well as phototherapy of cancers have been limited. Here we developed ICG-loaded hollow mesoporous silica nanoparticles (HMSNP) for selective NIR fluorescence imaging and phototherapy of cancers. We expected that NIR fluorescence and phototoxicity are turned off in normal cells or in the extracelluar regions but becomes highly fluorescent and phototoxic inside cancer cells, thereby enabling selective fluorescence imaging and therapy of cancers. The size of the prepared nanoparticles was about 180 nm in diameter. ICG was loaded into HMSNP by 12 w/w%. Comparison of the UV/Vis absorption spectra of ICG-loaded HMSNP and free ICG in aqueous solution confirmed aggregation of the loaded ICGs inside the nanoparticles. As expected, both NIR fluorescence and singlet oxygen generation of ICG-loaded HMSNP were significantly inhibited in comparison with free, ICG. In the in vitro cell study, strong fluorescence was observed in the cancer cells treated with ICG-loaded HMSNP indicating turn-on of its fluorescence inside cancer cells. Upon light irradiation, ICG-loaded HMSNP showed significant cytotoxicity higher than that of free ICG molecules. These features make HMSNP a promising platform for utilizing ICG in biomedical applications.
Sterilization procedure is one of the most important obstacles in the clinical applications of bi... more Sterilization procedure is one of the most important obstacles in the clinical applications of biodegradable microspheres. The microspheres prepared with poly( -hydroxy acid) were severely aggregated during ethylene oxide (EO) gas sterilization, and could not be used in clinical applications. In this study, the e!ects of EO gas sterilization on the poly(L-lactide) (PLLA) microspheres were analyzed by nuclear magnetic resonance spectroscopy (H-NMR), di!erential scanning calorimetry (DSC), gel permeation chromatography (GPC), scanning electron microscope (SEM) and size fractionation. The aggregation between the microspheres might be stimulated by high mobility of amorphous regions of PLLA on the microsphere surfaces since both water vapor and gas mixture can reduce glass transition temperature (¹ ) of PLLA below the sterilization temperature. During EO gas sterilization, there were no changes in the molecular structure and the molecular weight of PLLA in microspheres, but there were changes in the crystallinity of PLLA in microspheres. In this study, poly(L-lactide)-poly(ethylene glycol) diblock copolymers (PLE) were blended with PLLA homopolymers in various ratios to design the microsphere suitable for EO gas sterilization. Aggregation of PLLA microspheres was markedly prevented when more than 4 wt% of PLE was blended in the microspheres. This inhibition e!ect on aggregation may be due to the increased initial crystallinity of the microspheres, which help to maintain the microsphere morphology during EO gas sterilization.
All-trans-retinoic acid (atRA) has been proved to be effective against several malignancies in hu... more All-trans-retinoic acid (atRA) has been proved to be effective against several malignancies in human clinical trials. However, in many patients who were treated with atRA, the cancer relapsed after a brief remission. One reason for such relapse is that atRA is metabolized by specific P450s that are induced in the liver during prolonged atRA treatments. In order to overcome such a drawback of atRA, we prepared biodegradable microspheres to provide continuous release of atRA for a long period of time. These biodegradable microspheres were prepared by poly(L-lactide) (PLLA) and polyethylene glycol (PEG)-PLLA diblock copolymers (PLE) in various blending ratios to control the release rate of atRA. As the PLE content in microsphere was increased, the density of the hydrophilic PEG block of PLE on microsphere surfaces increased and the microspheres were dispersed well in PBS without any surfactants. Various release patterns of atRA were obtained according to PLE and atRA contents in the microspheres. Especially, the pseudo-zero-order release profiles were observed for 5 weeks when the contents of PLE and atRA in the microspheres were above 4 wt.%.
All-trans-retinoic acid (atRA) is a promising anticancer and antiwrinkle drug. However, its clini... more All-trans-retinoic acid (atRA) is a promising anticancer and antiwrinkle drug. However, its clinical application is limited because it is rapidly metabolized by the induced cytochrome P450 (P450). In this study, farnesol derivatives are proposed as new inhibitors to prevent P450-mediated metabolism. The farnesol derivatives were suc-farnesol and mal-farnesol, which were synthesized by the chemical conjugation of farnesol with succinic anhydride and maleic anhydride, respectively. The inhibition effects of farnesol, farnesoic acid, and farnesol derivatives on the atRA metabolism were evaluated in microsome and in AMC-HN-6 cells. In the microsome experiment, suc-farnesol and mal-farnesol strongly inhibited atRA metabolism at 10 -4 mol/L concentration by as much as 61% and 77%, respectively. In the cell experiment, the inhibition effects of farnesol derivatives on the atRA metabolism showed similar tendency as the results in the microsome experiment, even if the effect was somewhat decreased. Effects of farnesoic acid and farnesol, however, were not significant. This research suggests that carboxylic end groups, such as atRA and hydrophobicity, might be important factors causing the higher inhibition effect, and that derivatization of farnesol can be 1 method to develop new inhibitors of atRA metabolism.
Journal of Biomaterials Science, Polymer Edition, 2001
The aggregation of poly(®-hydroxy acid) microspheres during ethylene oxide (EO) gas sterilization... more The aggregation of poly(®-hydroxy acid) microspheres during ethylene oxide (EO) gas sterilization makes it dif cult for the microspheres to be used in clinical applications. In this study, six kinds of PLLA-PEG-PLLA triblock copolymers (TriPLE) were synthesized with various composition ratios of PEG / PLLA in the range of 0.012 to 0.103. TriPLE microspheres were prepared by the oil-in-water emulsion method. TriPLE microspheres were characterized by using 1 H-NMR, gel permeation chromatography (GPC), and differential scanning calorimetry (DSC). After sterilization by EO gas at 55 ± C, the microspheres were analyzed by scanning electron microscope (SEM), laser diffractometry, standard sieves, X-ray diffraction (XRD), GPC, and DSC. When the composition ratio of PEG / PLLA was above 0.02, the initial crystallinity of TriPLE in microspheres was as high as 50%, and the microspheres were suitable to be sterilized by EO gas. On the other hand, TriPLE microspheres, which had composition ratios of PEG / PLLA below 0.02, had low initial crystallinities of about 30%, and aggregated during EO gas sterilization. For these microspheres, crystallinity increased up to 50% during the sterilization, whereas other TriPLE microspheres did not show any changes in crystallinity. Therefore, the aggregation of TriPLE microspheres during EO gas sterilization was markedly reduced as the initial crystallinity of TriPLE in the microspheres was increased.
Journal of Biomaterials Science, Polymer Edition, 2002
Biocompatibility studies of all-rrnns-retinoic acid (RA)-loaded microspheres were carried out ati... more Biocompatibility studies of all-rrnns-retinoic acid (RA)-loaded microspheres were carried out atier they were subcutaneously injected into rats. To characterize the inflammatory response to these microspheres, tissue reactions at the implantation site and cell types in the interstices of thc microsphercs were evaluated for 180 days. On the 15th day, the cross-sectional area of the fibrous capsules surrounding the implantation site of the RA-loaded microspheres was ftrur times larger than that of the control microspheres. The size of the fibrous capsules surrounding the implantation site of the RA-loaded microspheres decreased significantly over a period of 75 days. while the size of the fibrous capsules surrounding the implantation site of the control microspheres rernained alnrt'rst constant throughout the entire course of I 80 days. The tissue response to the RA-loaded microspheres was more intensified by the increased extensive cellular infiltration of rnacrophages, granulation tissue, and librosis than that to the control microspheres. The difference in the inflarnrnatory responsc between thc RA-loaded microspheres and the control microspheres was significant for 7-5 days afier implantation. lt was suggested that the released RA from the microspheres stimulated inflammatory responses. However, no further enhanced inflammation reactions were detectcd after RA had been completely released tiorn the microspheres.
All‐trans retinoic acid (atRA)‐loaded microspheres severely induce inflammatory responses after m... more All‐trans retinoic acid (atRA)‐loaded microspheres severely induce inflammatory responses after microsphere implantation. Fibroblasts and a thick band of fibrous capsule resulting from the inflammatory responses could hamper drug permeation to the bloodstream because fibroblasts actively metabolize atRA into polar metabolites and the thick fibrous capsule acts as a diffusion barrier. In the present study, we investigated whether the fibroblast proliferation and collagen deposition induced by atRA released from microspheres might affect the atRA concentration in plasma and atRA metabolism with or without treatment of dexamethasone as an anti‐inflammatory drug. After subcutaneous injection of atRA‐loaded microspheres in rats, it was observed that atRA‐loaded microspheres induced severe inflammatory responses and stimulated fibroblast proliferation and collagen deposition in fibrous capsules. On the other hand, the orally treated dexamethasone effectively prevented inflammatory respons...
Previously, poly(L‐lactide) microspheres containing retinoic acid (RA) had been prepared for pare... more Previously, poly(L‐lactide) microspheres containing retinoic acid (RA) had been prepared for parenteral administration in order to overcome “acute retinoic acid resistance.” In this study, acute toxicities of RA‐loaded microspheres were investigated in mice after the microspheres were injected intramuscularly. The maximum dose of RA in the microspheres was determined by considering both the injection volume and dispersion of microspheres in the vehicle media, and the dose levels were determined to be 0, 90, 180, and 360 mg RA/kg. Mice were carefully observed for any clinical signs for 14 days, and gross observation of the organs was performed at 14 days. When a dose of 90 mg RA/kg was administered, no severe toxicities were observed; however, with the administration of 180 and 360 mg RA/kg, abnormal appearances of organs, such as discolor, atrophy, hemorrhage, etc., were observed. There were no mortalities or bone fractures observed at any of the doses. In our previous study of canc...
Biodegradable microspheres containing all‐trans‐retinoic acid (atRA) were prepared previously to ... more Biodegradable microspheres containing all‐trans‐retinoic acid (atRA) were prepared previously to enhance the clinical efficacy of atRA for chemoprevention. In this study, subacute toxicity of atRA in sustained release was evaluated after subcutaneous administration of 0, 25, 50, and 100 mg atRA/kg of atRA‐loaded microspheres. After the subcutaneous injection of atRA‐loaded microspheres, the atRA concentration in plasma was maintained in the range of 3.5–25 ng/ml for 2–3 weeks. When 100 mg atRA/kg was administered, the atRA concentration in plasma was maintained at levels greater than 10 ng/ml and severe toxic effects were observed. When the dose of 50 mg atRA/kg was administered, the plasma concentration of atRA was maintained below 10 ng/ml over 3 weeks and only mild signs of toxicity were observed. Therefore, it was concluded that the dosage of atRA‐loaded microspheres should be less than 50 mg atRA/kg for chemopreventive applications against carcinogenesis. Considering recovery f...
A folate receptor-specific activatable probe for near-infrared fluorescence imaging of ovarian cancer
Chem. Commun., 2014
A folate receptor-specific activatable probe for in vivo near-infrared fluorescence imaging with ... more A folate receptor-specific activatable probe for in vivo near-infrared fluorescence imaging with a high target-to-background ratio was synthesized.
The present study was performed to investigate the stability of W/O/W emulsions containing 4-␣glu... more The present study was performed to investigate the stability of W/O/W emulsions containing 4-␣glucanotransferase (4␣GTase)-treated starch against environmental stresses such as heating, shearing, and repeated freeze-thawing. W/O/W emulsions were subjected to thermal processing at different temperatures ranging from 30 to 90 • C for 30 min, constant shear for 0-7 min, and freeze-thaw cycling between −20 • C and 30 • C, respectively, and followed by encapsulation efficiency (EE) measurement. As for the case of thermal stress, it was clearly shown that addition of 4␣GTase-treated starch in the internal aqueous phase of emulsions helped to maintain higher EE during thermal processing. However, at lower PGPR level (2%), the addition of 4␣GTase-treated starch dramatically reduced EE at temperatures higher than 70 • C, which was probably related to the melting of 4␣GTase-treated starch gel. The incorporation of 4␣GTase-treated starch improved the stability of emulsions during shearing process, but could not prevent W/O/W emulsions from creaming and destabilizing during freeze-thaw cycling.
Background: Prediction of resistance mechanisms for epidermal growth factor receptor-tyrosine kin... more Background: Prediction of resistance mechanisms for epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) remains challenging. Thus, we investigated whether resistant cancer cells that expand shortly after EGFR-TKI treatment would eventually cause the resistant phenotype. Methods: We generated two EGFR-mutant lung cancer cell lines resistant to gefitinib (PC9GR and HCC827GR). The parent cell lines were exposed to short-term treatment with gefitinib or paclitaxel and then were assessed for EGFR T790M mutation and C-MET expression. These experiments were repeated in vivo and in clinically relevant patient-derived cell (PDC) models. For validation in clinical cases, we measured these gene alterations in plasma circulating tumor DNA (ctDNA) before and 8 weeks after starting EGFR-TKIs in four patients with EGFR-mutant lung cancer. Results: T790M mutation was only detected in the PC9GR cells, whereas C-MET amplification was detected in the HCC827GR cells. The T790M mutat...
We developed a single-camera-based near-infrared (NIR) fluorescence imaging device using indocyan... more We developed a single-camera-based near-infrared (NIR) fluorescence imaging device using indocyanine green (ICG) NIR fluorescence contrast agents for image-induced surgery. In general, a fluorescent imaging system that simultaneously provides color and NIR images uses two cameras, which is disadvantageous because it increases the imaging head of the system. Recently, a single-camera-based NIR optical imaging device with quantum efficiency partially extended to the NIR region was developed to overcome this drawback. The system used RGB_NIR filters for camera sensors to provide color and NIR images simultaneously; however, the sensitivity and resolution of the infrared images are reduced by 1/4, and the exposure time and gain cannot be set individually when acquiring color and NIR images. Thus, to overcome these shortcomings, this study developed a compact fluorescent imaging system that uses a single camera with two complementary metal–oxide semiconductor (CMOS) image sensors. Sensit...
Materials and methods Materials Pyrrole (98 %), polyvinylpyrrolidone (PVP, MW ~29,000), and iron(... more Materials and methods Materials Pyrrole (98 %), polyvinylpyrrolidone (PVP, MW ~29,000), and iron(III) chloride hexahydrate (FeCl 3 •6H 2 O), trypsin from porcine pancreas (13,100 U/mg), and hyaluronidase (1,228 U/mg) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hyaluronic acid (HA, Mw 39,000 Da) was obtained from Lifecore Biomedical (Chaska, MN, USA) and used as received. Chlorin e4 (Ce4) was purchased from Frontier Scientific, Inc. (Logan, UT, USA). Singlet oxygen sensor green (SOSG), which is an agent for detecting singlet oxygens, was obtained from Invitrogen Corp. (Carlsbad, CA, USA). Methods Synthesis and characterization of positively charged-Ce4 (PCe4) Positively charged photosensitizer (PCe4) was prepared by conjugating Ce4 with RRK peptide. Briefly, the peptide (RRK) was synthesized using a standard solid-phase peptide synthesis protocol. Coupling was carried out using Fmoc amino acid (8 eq), Nhydroxybenzotriazole (HOBt, 8 eq), N,N-diisopropylethylamine (DIPEA,16 eq) in DMF for 2 h. After each step, resin washing was performed using DMF and methanol 2 times each. The Fmoc protecting group was removed by rocking in 20% piperidine in DMF for 10 min (twice). Ce4 and HOBt/DIPEA in DMF were added to the peptide-attached resin. The mixture was reacted for 12 h and suctioned, and then the resin was washed with DMF, MeOH, and DMF. When the reaction was completed, crude peptide was cleaved from the resin using a mixture of TFA/EDT/thioanisole/TIS/deionized water (90/2.5/2.5/2.5/2.5 volume) for 2 h. The solution was precipitated with cold ether and pelleted using a centrifuge. The solid was collected and air-dried. Crude peptide was dissolved in deionized water and purified by prep-LC, lyophilized, and preserved in a refrigerator before use. The molecular weights of PCe4 were measured using LC/MSD
Quantitative Imaging in Medicine and Surgery, 2020
Background: Accurate identification of tumor sites and boundaries is of paramount importance duri... more Background: Accurate identification of tumor sites and boundaries is of paramount importance during minimally invasive surgery. Although laparoscopic resection is being increasingly and widely performed for early gastric and colorectal cancers, the detection of tumors located inside the stomach and intestine is difficult owing to the lack of tactile sensation. Here, we propose the application of an indocyanine green (ICG)-loaded alginate hydrogel system as a fluorescence surgical marker for precise laparoscopic operations. Methods: A physical complex of ICG and human serum albumin (HSA) was mixed with sodium alginate to form an injectable hydrogel system. Calcium carbonate and D-gluconic acid (GA) were added to the gel to control its strength and gelation time, respectively. The optimal conditions for the preparation of injectable hydrogels were determined by analyzing the fluorescence spectra and sol-gel transition time of the prepared samples at various concentrations and compositions. Next, the aqueous solutions of ICG, ICG-HSA, and ICG-HSA-loaded alginate were subcutaneously injected into nude mice (three mice per group), and nearinfrared (NIR) fluorescence images of the mice (λ ex. =780 nm, λ em. =845 nm) were obtained at different points in time for 8 days. Then, fluorescence intensities at the injection sites, target-to-background ratio, and areas of ICG fluorescence were analyzed. Finally, the potential utility of ICG-HSA-loaded alginate hydrogel as a surgical marker was evaluated in a porcine model. The ICG-HSA-loaded alginate solution was injected into three sites in the submucosal space of the porcine stomach via a catheter. A fluorescent laparoscopic system was installed on the abdomen of the pig 3 days post-injection, and the fluorescence signal generated from the fluorescence surgical marker located inside the stomach was evaluated using the fluorescence laparoscope system (λ ex. =785 nm, λ em. =805 nm). Results: The optimal concentration of ICG-HSA complex was determined to be 30 μM, and maximum fluorescence intensity of the complex was obtained at a 1:1 mole ratio of HSA to ICG. The subcutaneous injection of ICG or ICG-HSA solution in mice resulted in the rapid spread of the fluorescence signal around the injection site in 3 h, and a weak fluorescence was detected at the injection site 24 h post-injection. In contrast, the fluorescence detection time was effectively prolonged up to 96 h post-injection in the case of ICG-HSA-loaded alginate gel, while diffusion of the injected ICG from the injection site was effectively prevented. In the laparoscopic operation, injection sites of the hydrogel in porcine stomach could be accurately detected in real time even after 3 days. Conclusions: This alginate hydrogel system may be potentially useful as an effective surgical marker in terms of accuracy and persistence for laparoscopic operation.
Epidermal growth factor receptor (EGFR) is overexpressed in many types of cancers, which is assoc... more Epidermal growth factor receptor (EGFR) is overexpressed in many types of cancers, which is associated with metastatic potential and poor prognosis in cancer patients. Therefore, development of EGFR-targeted sensitive imaging probes has been a challenge in tumor targeting, image-guided cancer surgery, patient-selective anti-EGFR therapy, and efficient targeted therapies. Methods: We synthesized a zwitterionic near-infrared fluorophore (ATTO655)-conjugated epidermal growth factor (EGF) as a novel activatable molecular probe. Fluorescence OFF/ON property and EGFR-targeting specificity of EGF-ATTO655 as well as its utility in real-time near-infrared (NIR) fluorescence imaging of EGFR-positive cancers were evaluated using in vitro and in vivo studies. Results: When conjugated to EGF, the fluorescence of ATTO655 quenched efficiently by photo-induced electron transfer (PET) mechanism between the conjugated dyes and nearby amino acid quenchers (tryptophan/tyrosine residues), which was stably maintained at physiological pH and in the presence of serum for at least 17 h. The fluorescence of EGF-ATTO655 turned on by receptor-mediated endocytosis and subsequent disintegration of EGF in EGFR-positive A431 cancer cells, thereby enabling specific and real-time fluorescence imaging of EGFR-positive cancer cells. Consequently, EGFR-positive tumors could be clearly visualized 3 h post-injection with a significantly high tumor-to-background ratio (TBR = 6.37). Conclusion: This PET mechanism-based OFF/ON type of EGF probe showed great potential for rapid, real-time, and target-cell-specific imaging of EGFR-overexpressing cancers in vitro and in vivo.
In this study, we synthesized manganese dioxide nanoparticles (MnO 2 NPs) stabilized with biocomp... more In this study, we synthesized manganese dioxide nanoparticles (MnO 2 NPs) stabilized with biocompatible polymers (polyvinylpyrrolidone and polyacrylic acid) and analyzed their effect on non-small cell lung cancer (NSCLC) cells with or without gefitinib resistance in vitro. MnO 2 NPs showed glutathione (GSH)-responsive dissolution and subsequent enhancement in magnetic resonance (MR) imaging. Of note, treatment with MnO 2 NPs induced significant cytotoxic effects on NSCLC cells, and additional dose-dependent therapeutic effects were obtained upon X-ray irradiation. Normal cells treated with MnO 2 NPs were viable at the tested concentrations. In addition, increased therapeutic efficacy could be achieved when the cells were treated with MnO 2 NPs in hypoxic conditions. Therefore, we conclude that the use of MnO 2 NPs in MR imaging and combination radiotherapy may be an efficient strategy for the imaging and therapy of NSCLC.
Gastric cancer is the third most common cause of cancer mortality, and the survival rate of stage... more Gastric cancer is the third most common cause of cancer mortality, and the survival rate of stage IV advanced gastric cancer (AGC) patients with distant metastasis is very low. Thus, the detection and eradication of disseminated cancer cells by targeting cell surface molecules in AGC would improve patient survival. The hyaluronic acid receptor, CD44, has various isoforms generated by alternative splicing, and some isoforms are known to be correlated to gastric cancer. In this study, to find out the most appropriate CD44v for targeting AGC, we analysed the expression differences of CD44 isoforms at the mRNA level in stomach cancer cell lines as well as in 74 patients with AGC by using exon-specific qRT-PCR. Among the CD44v isoforms, CD44v8-10 was determined as the most promising biomarker for the development of theranostic agents of gastric cancer. Next, we synthesised the conjugate of anti-CD44v9 antibody with near-infrared fluorophore or photosensitiser, and then demonstrated its f...
Antibody-fluorophore conjugates have high potential for the specific fluorescence detection of ta... more Antibody-fluorophore conjugates have high potential for the specific fluorescence detection of target cancer cells in vitro and in vivo. However, the antibody-fluorophore conjugates described to date are inappropriate for real-time imaging of target cells because removal of unbound antibody is required to reduce background fluorescence before quantifiable analysis by microscopy. In addition, clinical applications of the conjugates have been limited by persistent background retention due to their long systemic circulation and nonspecific uptake. Here we report fast and real-time near-infrared fluorescence imaging of target cancer cells using an antigen-responsive molecular "on-off" sensor: the fluorescence of trastuzumab-ATTO680 conjugate is dark (i.e., turned off) in the extracellular region, while it becomes highly fluorescent (i.e., turned on) upon binding to the target antigen HER2 on cancer cell surface. This molecular switch enables fast and real-time imaging of targe...
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Papers by Yongdoo Choi