Soondong Lee

Safety and tumor tissue accumulation of pegylated graphene oxide nanosheets for co-delivery of anticancer drug and photosensitizer

Here, we report the safety, tumor accumulation and potential of polyethylene glycol-grafted graphene oxide (pGO) as a multimodal nanocarrier of photosensitizers and synergistic anticancer agents. First, both graphene oxide (GO) and pGO were synthesized, and their in vitro and in vivo toxicities were tested. When 80 mg/kg was injected intravenously into mice, there was 100% fatality in the GO-treated group, but 100% survival among mice treated with pGO nanosheets. Treatment of cells with a photosensitizer chlorin e6 (Ce6) in pGO nanophysisorplexes significantly enhanced cellular delivery compared to that seen with Ce6 alone. The combination and dose reduction indexes revealed that combining doxorubicin (Dox) with Ce6 with at a molar ratio of 1:2 provided the highest synergism. The Ce6- and Dox-loaded pGO nanophysisorplexes (Ce6/Dox/pGO) were 148.0 ± 18.0 nm in size. Molecular imaging of mice showed that Ce6/Dox/pGO could accumulate in tumor tissues over 3 days. Moreover, in SCC tumor-bearing mice, the photodynamic anticancer effects of Ce6/Dox/pGO were higher than those of Ce6/pGO or Dox/pGO. Moreover, tumor sections from illuminated mice treated with Ce6/Dox/pGO showed substantial disruption of tumor nuclei, whereas the other groups did not. Our results suggest that pGO nanosheets have superior in vivo safety relative to GO, and that it is possible to enhance the tumor tissue distribution and photodynamic anticancer effects of systemically administered Ce6 by forming multimodal nanophysisorplexes with pGO and synergistic anticancer chemotherapeutics such as Dox.

Cholesteryl hyaluronic acid-coated, reduced graphene oxide nanosheets for anti-cancer drug delivery

Here, we report hyaluronyl reduced graphene oxide (rGO) nanosheets as a tumor-targeting delivery system for anticancer agents. Hyaluronyl-modified rGO nanosheets were prepared by synthesizing cholesteryl hyaluronic acid (CHA) and using it to coat rGO nanosheets, yielding CHA-rGO. Compared with rGO, CHA-rGO nanosheets showed increased colloidal stability under physiological conditions and improved in vivo safety, with a survival rate of 100% after intravenous administration of 40 mg/kg in mice. The doxorubicin (Dox) loading capacity of CHA-rGO was 4-fold greater than that of rGO. Uptake of Dox by CD44-overexpressing KB cells was higher for CHA-rGO than for rGO, and was decreased in the presence of hyaluronic acid through competition for CD44 receptor binding. After intravenous administration in tumor-bearing mice, CHA-rGO/Dox showed higher tumor accumulation than rGO/Dox. The in vivo antitumor efficacy of Dox delivered by CHA-rGO was significantly increased compared with free Dox or rGO/Dox. In CHA-rGO/Dox-treated mice, tumor weights were reduced to 14.1% ± 0.1% of those in untreated mice. Our findings indicate that CHA-rGO nanosheets possess greater stability, safety, drug-loading capacity, and CD44-mediated delivery of Dox than rGO nanosheets. These beneficial properties of CHA-rGO improved the distribution of Dox to tumors and facilitated the cellular uptake of Dox by CD44-overexpressing tumor cells, resulting in enhanced anticancer effects.

Image-guided synergistic photothermal therapy using photoresponsive imaging agent-loaded graphene-based nanosheets.

We report the image-guided synergistic photothermal antitumor effects of photoresponsive near-infrared (NIR) imaging agent, indocyanine green (ICG), by loading onto hyaluronic acid-anchored, reduced graphene oxide (HArGO) nanosheets. Loading of ICG onto either rGO (ICG/rGO) or HArGO (ICG/HArGO) substantially improved the photostability of photoresponsive ICG upon NIR irradiation. After 1min of irradiation, the NIR absorption peak of ICG almost disappeared whereas the peak of ICG on rGO or HArGO was retained even after 5min of irradiation. Compared with plain rGO, HArGO provided greater cellular delivery of ICG and photothermal tumor cell-killing effects upon laser irradiation in CD44-positive KB cells. The temperature of cell suspensions treated with ICG/HArGO was 2.4-fold higher than that of cells treated with free ICG. Molecular imaging revealed that intravenously administered ICG/HArGO accumulated in KB tumor tissues higher than ICG/rGO or free ICG. Local temperatures in tumor tissues of laser-irradiated KB cell-bearing nude mice were highest in those intravenously administered ICG/HArGO, and were sufficient to trigger thermal-induced complete tumor ablation. Immunohistologically stained tumors also showed the highest percentages of apoptotic cells in the group treated with ICG/HArGO. These results suggest that photoresponsive ICG-loaded HArGO nanosheets could serve as a potential theranostic nano-platform for image-guided and synergistic photothermal antitumor therapy.

Reduced graphene oxide nanosheets coated with an anti-angiogenic anticancer low-molecular-weight heparin derivative for delivery of anticancer drugs.

Here, we report reduced graphene oxide (rGO) nanosheets coated with an anti-angiogenic anticancer taurocholate derivative of low-molecular-weight heparin (LHT7) as a tumor-targeting nanodelivery platform for anticancer drugs. Surface coating of LHT7 onto rGO was confirmed using fluorescein isothiocyanate-labeled LHT7, monitored as fluorescence quenching due to associated rGO. Unlike plain rGO, LHT7-coated rGO (LHT-rGO) nanosheets maintained a stable dispersion under physiological conditions for at least 24h. Moreover, LHT-rGO provided greater loading capacity for doxorubicin (Dox) compared with uncoated rGO nanosheets. Following intravenous administration into KB tumor-bearing mice, in vivo tumor accumulation of LHT-rGO/Dox was 7-fold higher than that of rGO/Dox 24h post dosing. In tumor tissues, LHT-rGO/Dox was shown to localize not to the tumor vasculature, but rather to tumor cells. Intravenously administered LHT-rGO/Dox showed the greatest anti-tumor effect in KB-bearing mice, reducing tumor volume by 92.5%±3.1% compared to the untreated group 25days after tumor inoculation. TUNEL assays revealed that the population of apoptotic cells was highest in the group treated with LHT-rGO/Dox. Taken together, our results demonstrate that LHT-rGO nanosheets confer improved dispersion stability, tumor distribution and in vivo antitumor effects, and may be further developed as a potential active nanoplatform of various anticancer drugs.

Structure-dependent photothermal anticancer effects of carbon-based photoresponsive nanomaterials

Here, we report the effect of structure on the biological properties of photoresponsive carbon nanomaterials. Poloxamer 407-functionalized single-walled carbon nanotubes (PSWCNT) and poloxamer 407-functionalized graphene nanosheets (PGNS) exhibited similar physical stability and heating capacities after irradiation with an 808 nm near-infrared (NIR) laser. Despite sharing common physical properties, the cellular uptake of the PSWCNT and PGNS differed significantly. Cancer cells treated with PGNS took up a higher quantity of the nanosheets than of the PSWCNT and displayed a higher rate of cancer cell killing upon laser irradiation. Structure of carbon nanomaterials also affected the in vivo behaviors. PGNS could circulate in the blood 2.2 times longer than that of the PSWCNT. PGNS accumulated in the SCC tumor tissues to a greater degree than did PSWCNT over 7 days. NIR irradiation resulted in the complete ablation of tumor tissues in the PGNS-treated group but not in the other groups. After NIR irradiation, 100% of the PGNS-treated and NIR-irradiated mice survived until day 70. These results suggest the importance of structure in controlling the in vivo behaviors of carbon nanomaterials. Moreover, the results indicate the structural advantages of nanosheets over nanotubes in the enhancement of photothermal anticancer effects.

Enhanced survival of transplanted human adipose-derived stem cells by co-delivery with liposomal apoptosome inhibitor in fibrin gel matrix.

To improve the survival of transplanted human adipose-derived stem cells (ADSCs), a liposome preparation containing the apoptosome inhibitor, NS3694, was formulated and co-delivered with ADSCs in fibrin gel scaffolds. Liposomes provided enhanced effect on ADSC proliferation in vitro as compared to free drug. Exposure of ADSCs to liposomal NS3694 for 7 days did not affect the surface marker expression profile. NS3694 encapsulated in negatively charged liposomes composed of phosphatidylcholine, phosphatidylglycerol, and cholesterol was evaluated in vivo following subcutaneous transplantation in mice. Survival of ADSCs co-delivered with liposomal NS3694 was significantly higher than that of untreated ADSCs or ADSCs treated with free NS3694 or empty liposomes. An immunohistochemical analysis revealed a higher number of human nucleus-positive cells after treatment with liposomal NS3694 than following treatment with free NS3694. Similarly, liposomal NS3694 significantly enhanced survival of transplanted ADSCs in rabbits compared to other treatments. Taken together, our results indicate the potential of liposomal NS3694 co-delivered with ADSCs using fibrin gel systems as an in vivo-survival enhancer.

Liposomal Co-Delivery of Omacetaxine Mepesuccinate and Doxorubicin for Synergistic Potentiation of Antitumor Activity

PurposeAnticancer chemotherapy usually involves the administration of several anticancer drugs that differ in their action mechanisms. Here, we aimed to test whether the combination of omacetaxine mepesuccinate (OMT) and doxorubicin (DOX) could show synergism, and whether the liposomal co-delivery of these two drugs could enhance their antitumor effects in cervical carcinoma model.MethodOMT-loaded liposomes (OL) were prepared by loading the drug in the lipid bilayers. OL were then electrostatically complexed with DOX, yielding double-loaded liposomes (DOL). DOX-loaded liposomes (DL) were formulated by electrostatic interaction with negatively charged empty liposomes (EL). The combination index (CI) values were calculated to evaluate the synergism of two drugs. In vitro antitumor effects against HeLa cells were measured using CCK-8, calcein staining, and crystal violet staining. In vivo antitumor effects of various liposomes were tested using HeLa cell-bearing mice.ResultsCombination of DOX and OMT had ratio-dependent synergistic activities, with very strong synergism observed at a molar ratio of 4:1 (DOX:OMT). The sizes of EL, DL, OL, and DOL did not significantly differ, but the zeta potentials of DL and DOL were slightly higher than those of OL and EL. In vitro, DOL showed higher antitumor activity than OL, DL or EL in cervical carcinoma HeLa cells. In vivo, unlike other liposomes, DOL reduced the tumor growths by 98.6% and 97.3% relative to the untreated control on day 15 and 25 after the cessation of treatment, respectively.ConclusionsThese results suggest that liposomal co-delivery of DOX and OMT could synergistically potentiate antitumor effects.

Initial preclinical safety of non‐replicating human endogenous retrovirus envelope protein‐coated baculovirus vector‐based vaccines against human papillomavirus

Human endogenous retrovirus (HERV) envelope protein‐coated, baculovirus vector‐based HPV 16 L1 (AcHERV‐HPV16L1) is a non‐replicating recombinant baculoviral vaccine. Here, we report an initial evaluation of the preclinical safety of AcHERV‐HPV16L1 vaccine. In an acute toxicity study, a single administration of AcHERV‐HPV16L1 DNA vaccine given intramuscularly (i.m.) to mice at a dose of 1 × 108 plaque‐forming units (PFU) did not cause significant changes in body weight compared with vehicle‐treated controls. It did cause a brief increase in the weights of some organs on day 15 post‐treatment, but by day 30, all organ weights were not significantly different from those in the vehicle‐treated control group. No hematological changes were observed on day 30 post‐treatment. In a range‐finding toxicity study with three doses of 1 × 107, 2 × 107 and 5 × 107 PFU once daily for 5 days, the group treated with 5 × 107 PFU showed a transient decrease in the body weights from day 5 to day 15 post‐treatment, but recovery to the levels similar to those in the vehicle‐treated control group by post‐treatment day 20. Organ weights were slightly higher for lymph nodes, spleen, thymus and liver after repeated dosing with 5 × 107 PFU on day 15, but had normalized by day 30. Moreover, repeated administration of AcHERV‐HPV16L1 did not induce myosin‐specific autoantibody in serum, and did not cause immune complex deposition or tissue damage at injection sites. Taken together, these results provide preliminary evidence of the preclinical safety of AcHERV‐based HPV16L1 DNA vaccines in mice. Copyright