Su Eun Han

Pegylated poly-L-arginine derivatives of chitosan for effective delivery of siRNA

For delivery of siRNA, chitosan (CS) was derivatized with poly-l-arginine (PLR) and polyethylene glycol (PEG). The formation of polyplexes with siRNA was confirmed by gel retardation. The PLR-grafted CS formed nanosized particles with siRNA. PLR-grafted CS showed higher cellular delivery efficiency of siRNA than did CS, pegylated CS, PLR, or pegylated PLR. The extent of reduction in the expression of fluorescent proteins was highest following treatment of the cells using PLR derivatives of CS in complexes with specific siRNAs. Cell viability was greater in populations treated with pegylated CS-PLR than in those treated with PLR. Hemolysis of erythrocytes was reduced upon conjugation of PLR with CS. The delivery of siRNAs via pegylated CS-PLR revealed little dependence on serum. Molecular imaging techniques revealed that the intratumoral administration of red fluorescent protein-specific siRNA in complexes with pegylated CS-PLR significantly silenced the expression of red fluorescent proteins in tumor tissues in vivo. These results indicate that pegylated CS-PLR might be useful for in vivo delivery of 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.

Tocopheryl oligochitosan-based self assembling oligomersomes for siRNA delivery

Amphiphilic α-tocopherol oligochitosan conjugates were constructed by conjugating α-tocopherol succinate to water soluble oligochitosans with various molecular weights. In aqueous medium, the tocopherol oligochitosan conjugates self-assembled to single layered oligomersomes. The sizes of α-tocopherol-oligochitosan-based oligomersomes (TCOsomes) could be controlled by chain lengths of oligochitosans. The mean sizes of TCOsomes were 220 and 377 nm as the sizes of oligochitosans were 4000 and 12,500, respectively. For all TCOsomes formed in this study, polydispersity indexes were in the ranges of 0.111-0.256. Cryo-TEM images showed clear thickening in the unilamellar layer of TCOsomes upon complexation with siRNAs. Zeta potentials decreased as the ratios of siRNA/TCOsomes increased. TCOsomes self-assembled from tocopherol-oligochitosan 4K (TCOsome(4K)) significantly enhanced the cellular uptake of siRNAs (>98%), and reduced the expression of target proteins more effectively than did Lipofectamine 2000. In tumor xenografted mice, the intratumoral administration of siMcl-1 using TCOsomes substantially silenced the expression of Mcl-1 and prevented the growth of tumor. The hematoxylin-eosin staining showed the apoptosis of cells in the tissues of the mice treated with siMcl-1/TCOsome(4K) complexes, but not with siGL2/TCOsome(4K) complexes. The self-assembling and size-controllable oligomersomes might be suitable for effective in vivo delivery of siRNAs.

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.

In situ dose amplification by apoptosis-targeted drug delivery

When tumor cells undergo apoptosis in response to chemotherapy, the levels of apoptotic biomarkers such as histone H1 are increased at the tumor. This would amplify in situ homing signals and thus drug delivery by apoptosis-targeted drugs. To examine this possibility, we prepared apoptosis-targeted liposomes containing doxorubicin by labeling them with the CQRPPR peptide (ApoPep-1) that recognizes apoptotic cells by binding to histone H1. ApoPep-1-labeled liposomes, but not folate-labeled liposomes, inhibited tumor growth in mice more efficiently than untargeted liposomes, although in vitro cytotoxicities of those liposomes were similar. Fluorescence imaging signals at tumor were increased by the homing of ApoPep-1-labeled, fluorescent liposomes, which was correlated with the increase of apoptosis and the amount of doxorubicin at the tumor and, conversely, with the decrease of tumor volume. These results demonstrate that the apoptosis-targeted drug delivery enables in situ dose amplification and, when combined with imaging of apoptosis, provides a real-time monitoring of treatment response for cancer theragnosis.

Maltosylated polyethylenimine-based triple nanocomplexes of human papillomavirus 16L1 protein and DNA as a vaccine co-delivery system

To improve vaccine delivery, we herein designed a co-delivery system using a protein antigen and its encoding plasmid linked in nanocomplexes via maltosylated PEI (mPEI). Cationic mPEI was electrostatically complexed to a plasmid encoding the human papillomavirus (HPV) type 16L1 protein (pHPV16L1), and further complexed to a maltose binding protein (MBP)-fused human papillomavirus type 16L1 fusion protein (HPV16L1-MBP). The HPV16L1-MBP/mPEI/pHPV16L1 complexes were characterized by gel-retardation properties, zeta potentials and sizes. The intracellular co-delivery of protein and plasmid DNA vaccines was significantly higher for mPEI-based triple nanocomplexes than for a simple physical mixture of the proteins and DNA. Moreover, the cellular delivery of plasmid DNA using mPEI-based triple nanocomplexes resulted in higher expression levels comparable to those obtained using dual complexes of mPEI and the plasmid DNA. In vivo, co-immunization of mice with HPV16L1-MBP/mPEI/pHPV16L1 nanocomplexes triggered the highest levels of humoral immune responses among various vaccination groups. Moreover, the mPEI-based nanocomplexes significantly enhanced the number of interferon-γ producing CD8(+) T cells compared with the use of mixed proteins and plasmid DNA. These results suggest that the effective cellular co-delivery of MBP-fused antigen proteins and plasmid DNA using maltosylated PEI-based triple nanocomplexes could enhance the immunogenicity of HPV16L1 vaccines.

Reduced dose-limiting toxicity of intraperitoneal mitoxantrone chemotherapy using cardiolipin-based anionic liposomes

Intraperitoneal chemotherapy confers limited clinical benefit as a result of the dose-limiting toxicity of anticancer drugs. We aimed to develop optimized liposomes for mitoxantrone (MTO) administration that provide high encapsulation efficiency and increase the therapeutic index. Cationic MTO was loaded onto anionic liposomes by electrostatic surface complexation. The anticancer activity was evaluated in a peritoneal carcinomatosis model. The retention of MTO at the tumor site was monitored by molecular imaging. MTO loading efficiencies by electrostatic complexation were >95% for all anionic liposomes but <5% for neutral liposomes. Among anionic liposomes, cardiolipin liposomes (CLs) exhibited the strongest binding affinity for MTO, the highest anticancer activity, and the lowest toxicity. MTO delivered by CLs showed prolonged retention at tumor sites. Unlike free MTO showing significant cardiotoxicity, MTO administered in CLs provided negligible cardiotoxicity. CL-mediated delivery may increase the therapeutic index of MTO chemotherapy by prolonged retention and reduced cardiotoxicity.

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