Mi-Gyeong Kim

Graphene-based nanosheets for delivery of chemotherapeutics and biological drugs ☆

Graphene-based nanosheets (GNS), including graphenes, graphene oxides and reduced graphene oxides, have properties suitable for delivery of various molecules. With their two-dimensional structures, GNS provide relatively high surface areas and capacity for non-covalent π-π stacking and hydrophobic interactions with various drug molecules. Currently, GNS-based delivery applications extend to chemotherapeutics as well as biological drugs, including nucleic acid drugs, proteins, and peptides. Surfaces of GNS have been modified with various polymers, such as polyethylene glycol and biopolymers, which enhance biocompatibility and increase drug loading. Anticancer drugs are prominent among chemotherapeutic agents tested, and have been loaded onto GNS with relatively high loading capacities compared with other nanocarriers. For enhanced distribution to specific tissues, GNS have been covalently or non-covalently modified with targeting ligands, including folic acid, transferrins, and others. In this review, we cover the current status of GNS for delivery of anticancer chemotherapeutics and biological drugs, with a focus on nucleic acid drugs. Remaining challenges for the application of GNS for drug-delivery systems and future perspectives are also addressed.

Polyaptamer DNA nanothread-anchored, reduced graphene oxide nanosheets for targeted delivery

Here, we report reduced graphene oxide (rGO) nanosheets anchoring receptor-specific polyaptamer nanothreads for targeted drug delivery. DNA polyaptamer nanothreads of protein tyrosine kinase 7 receptor (PTK7) were synthesized by rolling cycle amplification. To strengthen the anchoring of polyaptamer nanothreads onto rGO, oligoT bridge domain was introduced between each repeating PTK7 aptamer sequence. As compared to PTK7 polyaptamer nanothreads alone, PTK7 polyaptamer nanothreads with 22-mer oligoT bridges (PNT) showed higher anchoring capacity onto rGO nanosheets. Nanothread-coated surface morphology of PNTrGO was observed. Coating of PNT did not affect the sizes of rGO, but reduced the zeta potential. In PTK7-negative Ramos cells, the uptake of PNT-anchored rGO (PNTrGO) did not differ from that of oligoT-bridged scrambled polyaptamer-anchored rGO (SNTrGO). However, in CCRF-CEM leukemia cells overexpressing PTK7, the uptake of PNTrGO was 2.1-fold higher than that of SNTrGO after 15 min pulse. In vivo distribution to CCRF-CEM tumor tissues was 2.8-fold higher in PNTrGO than in SNTrGO at 48 h post-injection. In CCRF-CEM xenografted mice, intravenously administered doxorubicin (Dox)-loaded PNTrGO showed the higher antitumor activity than other groups, reducing the tumor weight down to 12% of tumor weights of untreated mice. These results suggest the potential of PNTrGO for target-specific drug delivery nanoplatform.

Double stranded aptamer-anchored reduced graphene oxide as target-specific nano detector

Here, we report a double-stranded, dual-anchored, fluorescent aptamer on reduced graphene oxide (rGO) for the sensitive, selective, and speedy detection of a target protein in biological samples. This nano detector is composed of a target protein-specific fluorescent aptamer with BHQ1 as one anchoring moiety that forms double-stranded sequences with a complementary oligonucleotide sequence with BHQ1 as the other anchoring moiety, anchored to rGO nanosheets. The double-stranded and dual-anchored aptamer on rGO nanosheets (DAGO) exhibited 7.3-fold higher fluorescence intensities compared to a single-stranded, single-anchored fluorescent aptamer on rGO. As a model target protein, interferon-γ was used. DAGO detected the target protein, with linearity over a five-orders-of-magnitude concentration range (0.1 ng/ml-10 μg/ml) in buffer and human serum. DAGO was highly specific for the target protein, exhibiting little changes in fluorescence intensity in response to the non-target proteins, interleukin-2 and tumor necrosis factor-α. Moreover, DAGO allowed rapid quantification of the target protein in human immunodeficiency virus-positive patient serum samples. DAGO-based detection was complete in less than 10 min. Our results indicate that the DAGO provides new opportunities for the rapid and specific detection of target proteins in biological samples and could be widely applied to quantitate various target proteins by replacing the aptamer sequences.

Immunogenicity of a trivalent human papillomavirus L1 DNA-encapsidated, non-replicable baculovirus nanovaccine.

Previously, we developed a non-replicating recombinant baculovirus coated with human endogenous retrovirus envelope protein (AcHERV) for enhanced cellular delivery of human papillomavirus (HPV) 16L1 DNA. Here, we report the immunogenicity of an AcHERV-based multivalent HPV nanovaccine in which the L1 segments of HPV 16, 18, and 58 genes were inserted into a single baculovirus genome of AcHERV. To test whether gene expression levels were affected by the order of HPV L1 gene insertion, we compared the efficacy of bivalent AcHERV vaccines with the HPV 16L1 gene inserted ahead of the 18L1 gene (AcHERV-HP16/18L1) with that of AcHERV with the HPV 18L1 gene inserted ahead of the 16L1 gene (AcHERV-HP18/16L1). Regardless of the order, the bivalent AcHERV DNA vaccines retained the immunogenicity of monovalent AcHERV-HP16L1 and AcHERV-HP18L1 DNA vaccines. Moreover, the immunogenicity of bivalent AcHERV-HP16/18L1 was not significantly different from that of AcHERV-HP18/16L1. In challenge tests, both bivalent vaccines provided complete protection against HPV 16 and 18 pseudotype viruses. Extending these results, we found that a trivalent AcHERV nanovaccine encoding HPV 16L1, 18L1, and 58L1 genes (AcHERV-HP16/18/58L1) provided high levels of humoral and cellular immunogenicity against all three subtypes. Moreover, mice immunized with the trivalent AcHERV-based nanovaccine were protected from challenge with HPV 16, 18, and 58 pseudotype viruses. These results suggest that trivalent AcHERV-HPV16/18/58L1 could serve as a potential prophylactic baculoviral nanovaccine against concurrent infection with HPV 16, 18, and 58.

Current status and regulatory perspective of chimeric antigen receptor-modified T cell therapeutics.

Chimeric antigen receptor-modified T cells (CAR-T) have emerged as a new modality for cancer immunotherapy due to their potent efficacy against terminal cancers. CAR-Ts are reported to exert higher efficacy than monoclonal antibodies and antibody–drug conjugates, and act via mechanisms distinct from T cell receptor-engineered T cells. These cells are constructed by transducing genes encoding fusion proteins of cancer antigen-recognizing single-chain Fv linked to intracellular signaling domains of T cell receptors. CAR-Ts are classified as first-, second- and third-generation, depending on the intracellular signaling domain number of T cell receptors. This review covers the current status of CAR-T research, including basic proof-of-concept investigations at the cell and animal levels. Currently ongoing clinical trials of CAR-T worldwide are additionally discussed. Owing to the lack of existing approved products, several unresolved concerns remain with regard to safety, efficacy and manufacturing of CAR-T, as well as quality control issues. In particular, the cytokine release syndrome is the major side-effect impeding the successful development of CAR-T in clinical trials. Here, we have addressed the challenges and regulatory perspectives of CAR-T therapy.

Biomimetic DNA nanoballs for oligonucleotide delivery

Here, we designed biomimetic DNA nanoballs for delivery of multiple antisense oligonucleotides (ASOs). DNA templates with ASOs-complementary sequences were amplified by rolling circle amplification (RCA). RCA products were loaded with two types of ASOs by hybridization, condensed using adenovirus-derived Mu peptide, and coated with hyaluronic acid (HA) for delivery into CD44-overexpressing tumor cells. HA-coated, Mu peptide-condensed, dual ASO-loaded DNA nanoballs (HMA nanoballs) showed considerable cellular entry of Cy5-incorporated RCA product DNA and fluorescent ASOs, whereas Mu peptide-condensed, dual ASO-loaded DNA nanoballs (MA nanoballs) revealed limited uptake. Dual ASOs, Dz13 and OGX-427, delivered by HMA nanoballs could reduce the levels of protein targets and exert anticancer effects. Enhanced tumor distribution was observed for fluorescent HMA nanoballs than the corresponding MA nanoballs. Upon intravenous co-administration with doxorubicin, HMA nanoballs exerted the greatest anti-tumor effects among the groups. These results suggest HMA nanoballs as a nanoplatform for sequence-specific delivery of multiple ASOs and other functional oligonucleotides.

Immunogenicity of bivalent human papillomavirus DNA vaccine using human endogenous retrovirus envelope-coated baculoviral vectors in mice and pigs.

Human papillomavirus is known to be the major pathogen of cervical cancer. Here, we report the efficacy of a bivalent human papillomavirus type 16 and 18 DNA vaccine system following repeated dosing in mice and pigs using a recombinant baculovirus bearing human endogenous retrovirus envelope protein (AcHERV) as a vector. The intramuscular administration of AcHERV-based HPV16L1 and HPV18L1 DNA vaccines induced antigen-specific serum IgG, vaginal IgA, and neutralizing antibodies to levels comparable to those achieved using the commercially marketed vaccine Cervarix. Similar to Cervarix, AcHERV-based bivalent vaccinations completely blocked subsequent vaginal challenge with HPV type-specific pseudovirions. However, AcHERV-based bivalent vaccinations induced significantly higher cell-mediated immune responses than Cervarix, promoting 4.5- (HPV16L1) and 3.9-(HPV18L1) fold higher interferon-γ production in splenocytes upon stimulation with antigen type-specific pseudovirions. Repeated dosing did not affect the immunogenicity of AcHERV DNA vaccines. Three sequential immunizations with AcHERV-HP18L1 DNA vaccine followed by three repeated dosing with AcHERV-HP16L1 over 11 weeks induced an initial production of anti-HPV18L1 antibody followed by subsequent induction of anti-HPV16L1 antibody. Finally, AcHERV-based bivalent DNA vaccination induced antigen-specific serum IgG immune responses in pigs. These results support the further development of AcHERV as a bivalent human papillomavirus DNA vaccine system for use in preventing the viral infection as well as treating the infected women by inducing both humoral and cell-mediated immune responses. Moreover, the possibility of repeated dosing indicates the utility of AcHERV system for reusable vectors of other viral pathogen vaccines.

Selective Activation of Anticancer Chemotherapy by Cancer-Associated Fibroblasts in the Tumor Microenvironment

Background: The tumor microenvironment has recently emerged as a new target of anticancer chemotherapy. Selective activation of anticancer chemotherapy in the tumor microenvironment would further reduce the toxicity of anticancer drugs toward normal tissues. Fibroblast activation protein (FAP) is known to be selectively overexpressed on cancer-associated fibroblasts (CAFs) in the tumor microenvironment. Here, we designed an anticancer chemotherapeutic system based on promelittin, a peptide toxin that is selectively converted from an inactive form to the pore-forming melittin upon cleavage by FAP in the tumor microenvironment. Methods: We conjugated promelittin-containing FAP-cleavable sequences to pegylated phospholipids and anchored them to reduced graphene oxide (rGO) nanosheets. The resulting nanosheets, PL-rGO, were tested for hemolysis and used for doxorubicin delivery. In vitro cocultures and in vivo tumor growth (n = 5 mice per group) with tissue immunostaining were used to test the selective activation of anticancer chemotherapy by FAP expressed on CAFs. Results: FAP-specific hemolytic activity of PL-rGO was observed in cocultures of CAFs and HT29 cells but not in HT29 cells alone. Doxorubicin-loaded PL-rGO (Dox/PL-rGO) showed 3.4-fold greater cell-killing efficacy (compared with free Dox in the CAF/HT29 coculture system, effects that were not observed in HT29 cells alone). Intravenously administered Dox/PL-rGO reduced the growth of HT29 tumors more effectively than other treatments (Dox/PL-rGO: mean = 200.6 mm3, 95% confidence interval [CI] = 148.7 to 252.5 mm3; free Dox: mean = 697.0 mm3, 95% CI = 646.9 to 747.1 mm3, PL: mean = 565.0 mm3, 95% CI = 550.5 to 579.6 mm3; Dox/rGO: mean = 637.6 mm3, 95% CI = 619.5 to 655.7 mm3; PL-rGO: mean = 464.4 mm3, 95% CI = 433.0 to 495.8 mm3). Immunostaining of tumor tissues revealed that survival of CAFs and HT29 cells was lowest in the group treated with Dox/PL-rGO. Conclusions: The demonstration of selective activation of PL-rGO by FAP on CAFs suggests that PL-rGO may serve as a tumor microenvironment–responsive anticancer chemotherapy system.

Sublingual Immunization of Trivalent Human Papillomavirus DNA Vaccine in Baculovirus Nanovector for Protection against Vaginal Challenge

Here, we report the immunogenicity of a sublingually delivered, trivalent human papillomavirus (HPV) DNA vaccine encapsidated in a human endogenous retrovirus (HERV) envelope-coated, nonreplicable, baculovirus nanovector. The HERV envelope-coated, nonreplicable, baculovirus-based DNA vaccine, encoding HPV16L1, -18L1 and -58L1 (AcHERV-triHPV), was constructed and sublingually administered to mice without adjuvant. Following sublingual (SL) administration, AcHERV-triHPV was absorbed and distributed throughout the body. At 15 minutes and 1 day post-dose, the distribution of AcHERV-triHPV to the lung was higher than that to other tissues. At 30 days post-dose, the levels of AcHERV-triHPV had diminished throughout the body. Six weeks after the first of three doses, 1×108 copies of SL AcHERV-triHPV induced HPV type-specific serum IgG and neutralizing antibodies to a degree comparable to that of IM immunization with 1×109 copies. AcHERV-triHPV induced HPV type-specific vaginal IgA titers in a dose-dependent manner. SL immunization with 1×1010 copies of AcHERV-triHPV induced Th1 and Th2 cellular responses comparable to IM immunization with 1×109 copies. Molecular imaging revealed that SL AcHERV-triHPV in mice provided complete protection against vaginal challenge with HPV16, HPV18, and HPV58 pseudoviruses. These results support the potential of SL immunization using multivalent DNA vaccine in baculovirus nanovector for induction of mucosal, systemic, and cellular immune responses.

Activation of AMPK by berberine induces hepatic lipid accumulation by upregulation of fatty acid translocase CD36 in mice

Abstract Emerging evidence has shown that berberine has a protective effect against metabolic syndrome such as obesity and type II diabetes mellitus by activating AMP‐activated protein kinase (AMPK). AMPK induces CD36 trafficking to the sarcolemma for fatty acid uptake and oxidation in contracting muscle. However, little is known about the effects of AMPK on CD36 regulation in the liver. We investigated whether AMPK activation by berberine affects CD36 expression and fatty acid uptake in hepatocytes and whether it is linked to hepatic lipid accumulation. Activation of AMPK by berberine or transduction with adenoviral vectors encoding constitutively active AMPK in HepG2 and mouse primary hepatocytes increased the expression and membrane translocation of CD36, resulting in enhanced fatty acid uptake and lipid accumulation as determined by BODIPY‐C16 and Nile red fluorescence, respectively. Activation of AMPK by berberine induced the phosphorylation of extracellular signal‐regulated kinases 1/2 (ERK1/2) and subsequently induced CCAAT/enhancer‐binding protein &bgr; (C/EBP&bgr;) binding to the C/EBP‐response element in the CD36 promoter in hepatocytes. In addition, hepatic CD36 expression and triglyceride levels were increased in normal diet‐fed mice treated with berberine, but completely prevented when hepatic CD36 was silenced with adenovirus containing CD36‐specific shRNA. Taken together, prolonged activation of AMPK by berberine increased CD36 expression in hepatocytes, resulting in fatty acid uptake via processes linked to hepatocellular lipid accumulation and fatty liver. HighlightsBerberine increases the expression and membrane translocation of CD36 in hepatocytes.The increase of CD36 results in enhanced fatty acid uptake and lipid accumulation.Berberine‐induced fatty liver is mediated by AMPK‐ERK‐C/EBP&bgr; pathway.CD36‐specific shRNA inhibited berberine‐induced lipid accumulation in liver.