Sangbin 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.

Cationic solid lipid nanoparticles for co-delivery of paclitaxel and siRNA

In this study, we formulated cationic solid lipid nanoparticles (cSLN) for co-delivery of paclitaxel (PTX) and siRNA. 1,2-Dioleoyl-sn-glycero-3-ethylphosphocholine-based cSLN were prepared by emulsification solidification methods. PTX-loaded cSLN (PcSLN) were characterized by zeta potential and gel retardation of complexes with small interfering RNA (siRNA). The sizes of PcSLN did not significantly differ from those of empty cSLN without PTX (EcSLN). The use of cSLN increased the cellular uptake of fluorescent dsRNA in human epithelial carcinoma KB cells, with PcSLN complexed to fluorescence-labeled dsRNA promoting the greatest uptake. For co-delivery of therapeutic siRNA, human MCL1-specific siRNA (siMCL1) was complexed with PcSLN; luciferase-specific siRNA (siGL2) complexed to EcSLN or PcSLN was used as a control. MCL1 mRNA levels were significantly reduced in KB cells treated with siMCL1 complexed to PcSLN, but not in groups treated with siMCL alone or siGL2 complexed to PcSLN. siMCL1 complexed to PcSLN exerted the greatest in vitro anticancer effects in KB cells, followed by siMCL1 complexed to EcSLN, siGL2 complexed to PcSLN, PTX alone, and siMCL1 alone. In KB cell-xenografted mice, intratumoral injection of PcSLN complexed to siMCL1 significantly reduced the growth of tumors. Taken together, our results demonstrate the potential of cSLN for the development of co-delivery systems of various lipophilic anticancer drugs and therapeutic siRNAs.

Trilysinoyl oleylamide-based cationic liposomes for systemic co-delivery of siRNA and an anticancer drug.

Oligolysine-based cationic lipid derivatives were synthesized for delivery of siRNA, and formulated into cationic liposomes. Among various oligolysine-based lipid derivatives differing in lysine residue number and lipid moiety, trilysinoyl oleylamide (TLO)-based liposomes (TLOL) showed the highest delivery efficiency combined with minimal cytotoxicity. Delivery of siRNA using TLOL silenced target genes both in vitro and in vivo. In green fluorescent protein (GFP)-expressing tumor tissue, a significant reduction of fluorescence was observed after intratumoral administration of siGFP using TLOL compared with control siGL2. Intravenous administration of siMcl1 employing pegylated TLOL (pTLOL) reduced the expression of human Mcl1 protein in KB-xenografted tumor tissue. Despite the reduction in target protein Mcl1 expression following such systemic delivery, tumor growth was only slightly reduced compared to a siGL2-treated control group. To potentiate the anticancer activity of siMcl1, the anticancer drug suberoylanilide hydroxamic acid (SAHA) was additionally encapsulated in pTLOL. After intravenous administration of siMcl1 using SAHA-loaded pTLOL (pSTLOL), a significant reduction in tumor growth was observed compared to that seen in animals treated with free SAHA or siGL2 complexed with pSTLOL. The results indicate that pTLOL could be further developed as a systemic delivery system for synergistic anticancer siRNA and a drug.

Cationic drug-derived nanoparticles for multifunctional delivery of anticancer siRNA

Combined treatment of anticancer drugs and small interfering RNAs (siRNAs) have emerged as a new modality of anticancer therapy. Here, we describe a co-delivery system of anticancer drugs and siRNA in which anticancer drug-derived lipids form cationic nanoparticles for siRNA complexation. The anticancer drug mitoxantrone (MTO) was conjugated to palmitoleic acid, generating two types of palmitoleyl MTO (Pal-MTO) lipids: monopalmitoleyl MTO (mono-Pal-MTO) and dipalmitoleyl MTO (di-Pal-MTO). Among various lipid compositions of MTO, nanoparticles containing mono-Pal-MTO and di-Pal-MTO at a molar ratio of 1:1 (md11-Pal-MTO nanoparticles) showed the most efficient cellular delivery of siRNA, higher than that of Lipofectamine 2000. Delivery of red fluorescence protein-specific siRNA into B16F10-RFP cells using md11-Pal-MTO nanoparticles reduced the expression of RFP at both mRNA and protein levels, demonstrating silencing of the siRNA target gene. Moreover, delivery of Mcl-1-specific anticancer siRNA (siMcl-1) using md11-Pal-MTO enhanced antitumor activity in vitro, reducing tumor cell viability by 81% compared to a reduction of 68% following Lipofectamine 2000-mediated transfection of siMcl-1. Intratumoral administration of siMcl-1 using md11-Pal-MTO nanoparticles significantly inhibited tumor growth, reducing tumor size by 83% compared to untreated controls. Our results suggest the potential of md11-Pal-MTO multifunctional nanoparticles for co-delivery of anticancer siRNAs for effective combination therapy.

Enhanced Intrapulmonary Delivery of Anticancer siRNA for Lung Cancer Therapy Using Cationic Ethylphosphocholine-based Nanolipoplexes

Here, we report a cationic nanolipoplex as a pulmonary cellular delivery system for small-interfering RNA (siRNA). Six nanoliposomes differing in cationic lipids were formulated and screened in vitro and in vivo for cellular delivery functions in lung cells/tissues. Although the six nanoliposomes showed similar siRNA delivery efficiency in vitro, they exhibited significant differences in pulmonary cellular delivery functions in vivo. Among the various nanoliposomes, cationic dioleoyl-sn-glycero-3-ethylphosphocholine and cholesterol (ECL)-based nanoliposomes showed the highest pulmonary cellular delivery in vivo and the lowest cytotoxicity in vitro. The delivery efficiency of fluorescent siRNA in ECL nanoliposomes was 26.2-fold higher than that of naked siRNA in vivo. Treatment with Mcl1 (myeloid cell leukemia sequence 1)-specific siRNA (siMcl1) using ECL nanolipoplexes reduced target expression in B16F10 cell lines, whereas control, luciferase-specific siGL2 in ECL nanolipoplexes did not. In metastatic lung cancer mouse models induced by B16F10 or Lewis lung carcinoma (LLC) cells, intratracheal administration of siMcl1 in ECL nanolipoplexes significantly silenced Mcl1 mRNA and protein levels in lung tissue. Reduced formation of melanoma tumor nodules was observed in the lung. These results demonstrate the utility of ECL nanoliposomes for pulmonary delivery of therapeutic siRNA for the treatment of lung cancers and potentially for other respiratory diseases.

Tetraiodothyroacetic acid-tagged liposomes for enhanced delivery of anticancer drug to tumor tissue via integrin receptor.

Nanoparticles have demonstrated potential for promoting drug delivery to tumor sites and enhancing uptake. Here, we report tetraiodothyroacetic acid (tetrac) as a promising new targeting moiety for delivery of anticancer drugs to tumor tissues. Tetrac, an antagonist that blocks the binding of thyroid hormone to integrin αvβ3, was covalently linked to the activated end of pegylated lipid and used to formulate tetrac-tagged pegylated liposomes (TPL). After incubating with TPL for 9h, cellular accumulation efficiency into A375 human melanoma cells, which express integrin αvβ3 at high density, was high (98.5%± 0.5% of cells), whereas that in KB cells, which express integrin at a very low density, was much lower (35.1%± 4.5%). Molecular imaging revealed that TPL preferentially distributed to tumor tissues after systemic administration in mice, where as non-targeting pegylated liposomes were distributed to tumors at background levels. Treatment with the alkyl lysophospholipid anticancer drug edelfosine, encapsulated in TPL, significantly reduced the survival of A375 tumor cells compared to cells treated with edelfosine in pegylated liposomes or with lysophosphatidylcholine encapsulated in TPL. Moreover, intravenous administration of edelfosine in TPL significantly reduced the growth of tumors and prolonged the survival of A375-xenografted mice, providing 100% protection for up to 50 days and some protection until 66 days (0% survival endpoint). In contrast, no untreated mice or mice treated with edelfosine-loaded pegylated liposomes survived up to 50 or 48 days, respectively, after tumor inoculation. These results suggest the potential of tetrac as a new ligand moiety for enhancing the delivery of anticancer drug-loaded nanoparticles to tumors and enhancing the therapeutic efficacy of encapsulated anticancer drugs.

Enhanced tumor localization and retention of chlorin e6 in cationic nanolipoplexes potentiate the tumor ablation effects of photodynamic therapy

Here we report the tumor ablation effects of the negatively charged photosensitizer chlorin e6 (Ce6) in nanocomplexes. Ce6 was complexed to cationic 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine-based liposomes, forming cationic nanolipoplexes. The loading efficiency of Ce6 to cationic nanolipoplexes was greater than 90%. The degree of enhancement of cellular uptake of Ce6 by treatment in cationic nanolipoplexes increased with the concentration of Ce6, showing 18.3-fold higher uptake than free Ce6 at 15 µM. Molecular imaging revealed the preferential distribution and retention of Ce6 in SCC7 tumor tissues after intravenous administration of Ce6 in cationic nanolipoplexes. Moreover, localized illumination of mice receiving Ce6 in cationic nanolipoplexes resulted in the formation of thick scabs over tumor regions, and complete ablation of tumors after scab detachment. In contrast, continuous growth of tumors was observed in the group treated with free Ce6. Our results suggest that the cationic nanolipoplexes of Ce6 improve the therapeutic effects of photodynamic cancer therapy as compared to free Ce6.

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.

Staphylococcus aureus-mimetic control of antibody orientation on nanoparticles

We designed a bacterio-mimetic nanoparticle that can noncovalently control the orientation of attached antibodies. Liposomes with Fc-binding peptide (FcBP), formulated using FcBP-conjugated PEGylated lipid, were used as model nanoparticles. Compared with control nanoparticles surface-modified with antibody covalently attached via maleimide functional groups (Mal-NPs), FcBP-capped nanoparticles (FcBP-NPs) exhibited greater binding affinity to the target protein. Human epidermal growth factor receptor 2 (HER2)-specific antibody-modified FcBP-NPs (HER2/FcBP-NPs) showed 5.3-fold higher binding affinity to HER2 than isotype IgG antibody-modified NPs, and 2.6-fold higher affinity compared with anti-HER2 antibody-conjugated Mal-NPs. Cellular uptake of HER2/FcBP-NPs in HER2-positive cells was significantly higher than that of other formulations. The biodistribution of HER2/FcBP-NPs was higher than that of antibody-conjugated NPs in HER2-positive tumor tissues, but not in HER2-negative tumors. Our findings suggest the potential of bacteriomimetic nanoparticles for controlling the orientation of antibody attachment. These nanoparticles may have diverse applications in nanomedicine, including drug delivery, molecular imaging, and diagnosis.