Genome-Editing-Mediated Restructuring of Tumor Immune Microenvironment for Prevention of Metastasis.

Modulating the tumor immune microenvironment to activate immune cells has been investigated to convert cold to hot tumors. Here, we report that metal-lipid hybrid nanoparticle (MLN)-mediated gene editing of transforming growth factor-ß (TGF-ß) can restructure the tumor microenvironment to an "immune activated" state for subsequent immunotherapy. MLNs with cationic lipids and elemental metallic Au inside were designed to deliver plasmid DNA encoding TGF-ß single guide RNA and Cas9 protein (pC9sTgf) and to convert near-infrared light (NIR) to heat. Upon NIR irradiation, MLNs induced photothermal anticancer effects and calreticulin exposure on B16F10 cancer cells. Lipoplexes of pC9sTgf and MLN (pC9sTgf@MLN) provided gene editing of B16F10 cells and in vivo tumor tissues. In mice treated with pC9sTgf@MLNs and NIR irradiation, the tumor microenvironment showed increases in mature dendritic cells, cytotoxic T cells, and interferon-γ expression. In B16F10 tumor-bearing mice, intratumoral injection of pC9sTgf@MLNs and NIR irradiation resulted in ablation of primary tumors. Application of pC9sTgf@MLNs and NIR irradiation prevented the growth of secondarily challenged B16F10 cells at distant sites and B16F10 lung metastasis. Combined TGF-ß gene editing and phototherapy is herein supported as a modality for restructuring the tumor immune microenvironment and preventing tumor recurrence.

Melanin-loaded CpG DNA hydrogel for modulation of tumor immune microenvironment.

Photothermal immunotherapy has emerged as one of the most potent approaches for cancer treatment, but this strategy has suffered from the lack of biodegradability of the photoresponsive materials. In this study, we aimed to develop biodegradable materials for photothermal immunotherapy. To this end, we designed a DNA CpG hydrogel (DH, generated by rolling-circle amplification), loaded it with bis-(3'-5')-cyclic dimeric guanosine monophosphate (G/DH), and coated the formulation with melanin (Mel/G/DH). Mel/G/DH exhibited a temperature increase upon near infrared (NIR) illumination. In vitro, Mel/G/DH plus NIR (808 nm) irradiation, induced the exposure of calreticulin on CT26 cancer cells, and significantly activated the maturation of dendritic cells (DC). In vivo, local administration of Mel/G/DH (+NIR) exerted photothermal killing of primary tumors and induced maturation of DC in lymph nodes. Treatment of primary tumors with Mel/G/DH(+NIR) prevented the growth of rechallenged tumors at a distant site. Survival was 100% in mice treated with Mel/G/DH(+NIR), 5-fold higher than the group treated with Mel/G(+NIR). Mel/G/DH(+NIR) treatment remodeled the immune microenvironment of distant tumors, increasing cytotoxic T cells and decreasing Treg cells. Taken together, the results of this study suggest the potential of Mel/G/DH as a platform for modulating tumor immune microenvironment aimed at preventing the recurrence of distant tumors.

Photosensitizer-Trapped Gold Nanocluster for Dual Light-Responsive Phototherapy.

Photoresponsive nanomaterials have recently received great attention in the field of cancer therapy. Here, we report a photosensitizer-trapped gold nanocluster that can facilitate dual light-responsive cancer therapy. We utilized methylene blue (MB) as a model photosensitizer, gold nanocluster as a model photothermal agent, and a polymerized DNA as the backbone of the nanocluster. We synthesized MB-intercalated gold DNA nanocluster (GMDN) via reduction and clustering of gold ions on a template consisting of MB-intercalated long DNA. Upon GMDN treatment, cancer cells revealed clear cellular uptake of MB and gold clusters; following dual light irradiation (660 nm/808 nm), the cells showed reactive oxygen species generation and increased temperature. Significantly higher cancer cell death was observed in cells treated with GMDN and dual irradiation compared with non-irradiated or single light-irradiated cells. Mice systemically injected with GMDN showed enhanced tumor accumulation compared to that of free MB and exhibited increased temperature upon near infrared irradiation of the tumor site. Tumor growth was almost completely inhibited in GMDN-treated tumor-bearing mice after dual light irradiation, and the survival rate of this group was 100% over more than 60 days. These findings suggest that GMDN could potentially function as an effective phototherapeutic for the treatment of cancer disease.

Biochemical reprogramming of tumors for active modulation of receptor-mediated nanomaterial delivery.

Here we report that reactive oxygen species (ROS) can reprogram cancer cells to increase the expression of specific receptors and modulate the delivery of nanomaterials. Gold and γ-polyglutamic acid (γ-PGA) hybrid nanoparticles (PGANP) were prepared via a facile single-step process. Gold nanoclusters in PGANP were dispersed within the tangled γ-PGA matrix of the nanoparticles. The condensed assembly of gold nanoclusters in γ-PGA matrix enabled the interparticle plasmon coupling effect, which lacks in single gold nanoparticles. Compared with gold nanoparticles of the similar sizes, PGANP showed significantly higher absorbance at near infrared (NIR) wavelength and light-to-heat converting ratios, resulting in greater temperature increase upon NIR light irradiation. Pretreatment of HeLa cancer cells with methylene blue (MB) generated reactive oxygen species. The ROS reprogrammed the cancer cells to express higher cell membrane levels of gamma glutamyl transferase (GGT), which is known to bind to γ-PGA of PGANP. MB pretreatment significantly enhanced delivery of PGANP to cancer cells. Cancer cells internalized PGANP to a greater extent and, were highly susceptible to irradiation with NIR light, which reduced cell viability to near zero. In vivo, MB pretreatment of HeLa xenograft mice increased the expression of GGT in tumor tissues. In mice pretreated with MB and exposed to NIR irradiation, PGANP treatment resulted in complete tumor ablation. The strategy of actively reprogramming tumor membrane levels of target receptors could be widely applied to overcome the heterogeneity of cancer cells. Although we used interparticle plasmon coupling effect-based PGANP for proving the concept of receptor-modulated delivery, this strategy could be broadly applicable to the active modulation of the receptor-mediated delivery of anticancer nanomaterials.

Noncovalent tethering of nucleic acid aptamer on DNA nanostructure for targeted photo/chemo/gene therapies.

Here, we report various therapeutic cargo-loadable DNA nanostructures that are shelled in polydopamine and noncovalently tethered with cancer cell-targeting DNA aptamers. Initial DNA nanostructure was formed by rolling-circle amplification and condensation with Mu peptides. This DNA nanostructure was loaded with an antisense oligonucleotide, a photosensitizer, or an anticancer chemotherapeutic drug. Each therapeutic agent-loaded DNA nanostructure was then shelled with polydopamine (PDA), and noncovalently decorated with a poly adenine-tailed nucleic acid aptamer (PA) specific for PTK7 receptor, resulting in PA-tethered and PDA-shelled DNA nanostructure (PA/PDN). PDA coating shell enabled photothermal therapy. In the cells overexpressing PTK7 receptor, photosensitizer-loaded PA/PDN showed greater photodynamic activity. Doxorubicin-loaded PA/PDN exerted higher anticancer activity than the other groups. Antisense oligonucleotide-loaded PA/PDN provided selective reduction of target proteins compared with other groups. Our results suggest that the PA-tethered and PDA-shelled DNA nanostructures could enable the specific receptor-targeted phototherapy, chemotherapy, and gene therapy against cancer cells.

A Microbial Siderophore-Inspired Self-Gelling Hydrogel for Noninvasive Anticancer Phototherapy.

Microbial carboxyl and catechol siderophores have been shown to have natural iron-chelating abilities, suggesting that hyaluronic acid (HA) and the catechol compound, gallic acid (GA), may have iron-coordinating activities. Here, a photoresponsive self-gelling hydrogel that was both injectable and could be applied to the skin was developed on the basis of the abilities of HA and GA to form coordination bonds with ferric ions (Fe3+). The conjugate of HA and GA (HA-GA) instantly formed hydrogels in the presence of ferric ions and showed near-infrared (NIR)-responsive photothermal properties. Following their subcutaneous injection into mice, HA-GA and ferric ion formed a hydrogel, which remained at the injection site for at least 8 days. Intratumoral injection of HA-GA/Fe hydrogel into mice allowed repeated exposure of the tumor to NIR irradiation. This repeated NIR irradiation resulted in complete tumor ablation in KB carcinoma cell-xenografted mice and suppressed lung metastasis of 4T1-Luc orthotopic breast tumors. Application of HA-GA/Fe hydrogel to the skin of A375 melanoma-xenografted tumor sites, followed by NIR irradiation, also resulted in complete tumor ablation. These findings demonstrate that single applications of HA-GA/Fe hydrogel have photothermal anticancer effects against both solid tumors and skin cancers. SIGNIFICANCE: These findings provide new insights into noninvasive anticancer phototherapy using self-gelling hydrogels. Application of these hydrogels in preclinical models reduces the sizes of solid tumors and skin cancers without surgery, radiation, or chemotherapy.

Claudin 4-targeted nanographene phototherapy using a Clostridium perfringens enterotoxin peptide-photosensitizer conjugate.

In this study we designed a claudin 4-directed dual photodynamic and photothermal system, in which a 30-amino acid claudin 4-binding peptide, Clostridium perfringens enterotoxin (CPE), was linked to a photodynamic agent chlorin e6 (Ce6) through a polyethylene glycol spacer (CPC) and anchored onto reduced graphene oxide (rGO) nanosheets to form CPC/rGO nanosheets. For comparison, a conjugate of polyethylene glycol and Ce6 (PC) was anchored onto the rGO nanosheets to generate PC/rGO. Both PC and CPC generated reactive oxygen species upon irradiation at 660 nm. Application of CPC/rGO to claudin 4-overexpressing U87 glioblastoma cells in vitro resulted in a significantly higher cellular uptake compared to application of PC/rGO. Upon irradiation at 660 and 808 nm, the CPC/rGO-treated U87 cells generated significantly higher reactive oxygen species and caused significantly higher temperature increase, and showed most potent anticancer effect compared to the other groups. Taken together, these results suggest that CPC/rGO is potentially useful as a tumor-specific combined phototherapy.

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.

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.

Pharmaceutical applications of graphene-based nanosheets.

Graphene-based nanosheets (GNS) are atomic-thickness monolayers of hexagonally arranged, graphite-derived carbon atoms that may be composed of graphene, graphene oxide, or reduced graphene oxide. They have attracted tremendous interest for their potential in pharmaceutical applications, due to their unique physical, chemical, and mechanical properties GNS exhibit highly uniform surface areas and may have hydroxyl (-OH), epoxide (-O-), and carboxyl functional groups at their basal surfaces and plane edges, depending on their oxidized and reduced surface properties. GNS show high-level optical absorption of near infrared (NIR) light and elevate the temperature of nearby environments. Furthermore, they can be loaded with anticancer drugs via hydrophobic interactions, π-π stacking, or electrostatic binding. Given these properties, GNS can be used in chemotherapy, photodynamic therapy, photothermal therapy, and theranostics. However, although GNS appear to have far-reaching potential in the field of biomedical research, their widespread pharmaceutical application has been limited by issues such as poor stability in physiological buffers, undefined mechanisms of cellular uptake, toxicity problems, and a lack of standard preparation methods. Here, we review the current pharmaceutical applications of GNS, focusing on chemotherapy, phototherapy, combo therapy and theranostic applications with challenging issues.

Topical application of porcine placenta extract inhibits the progression of experimental contact hypersensitivity.

AIM OF STUDY: Placenta extract features as a composition of ointments used for skin beautification, dermatological diseases and skin dryness. However, little evidence has been cited about its underlying mechanisms of action by which it exerts a beneficial role in dermatological diseases in vivo. In this study, we intended to test the effect of topical application of porcine placenta extract in mouse model of contact hypersensitivity and elucidate its mechanism of action. MATERIALS AND METHODS: To test the in vitro effect of porcine placenta extract, RAW 264.7 cells were cocultured with porcine placenta extract and stimulated with LPS (1 µg/ml) and the expression of inflammatory mediator TNF-α was estimated by RT-PCR at the mRNA level and by intracellular staining at the protein level. To further test in vivo efficacy, porcine placenta extract was topically applied to the mice with experimental skin hypersensitivity. For in vivo studies placenta extract in gel form was topically applied to ear of DNCB-induced contact hypersensitivity mouse model everyday for 2 weeks and progression of the disease was estimated by following criteria: (a) ear thickness, (b) serum IgE level by ELISA, (c) histological examination of ear tissue by H&E staining and (d) cytokine profile of total cells and CD4(+) T cells by real time PCR. RESULTS: Topical application of porcine placenta extract on mouse ears with contact hypersensitivity decreased the severity and progression of the disease manifested by reducing ear swelling, inflammation and edema. Histological evaluation showed that placenta extract treatment reduced lymphocyte infiltration in the ear tissues. Protective effect of placenta extract is also associated with down-regulation of serum IgE level and inflammatory cytokine production (IL-1ß, IFN-γ, TNF-α, IL-4, IL-12 and IL-17) in total lymph node cells and CD4(+) T cells. CONCLUSIONS: Our data indicate that protective effect of porcine placenta extract in contact hypersensitivity is mediated by inhibition of the inflammatory responses and IgE production, suggesting a potential therapeutic application of porcine placenta extract to modulate skin inflammation.

Enhanced humoral and cellular immune responses after sublingual immunization against human papillomavirus 16 L1 protein with adjuvants.

Needle-free nonparenteral vaccines offer a number of practical advantages, especially in developing countries. To address the effects of vaccine administration route, we tested mucosal and systemic immune responses against human papillomavirus 16 L1(HPV16L1) protein using intranasal, intravaginal, transdermal, sublingual (SL) and intramuscular routes. The SL route provided the most effective mucosal secretory IgA (sIgA) and serum IgG responses. After a 150 microg antigen dose via the SL route, saliva sIgA levels were 7.2- and 5.8-fold higher than those achieved via intravaginal and transdermal routes, respectively. Notably, SL administration even produced 4.6-fold higher levels of vaginal sIgA levels than did intravaginal delivery of 150 microg antigen. To enhance the immunogenicity of SL vaccines, we tested the adjuvanticity of nine molecules: three toll-like receptor agonists, three nucleotide-binding oligomerization-domain agonists, vitamin D3, poly-gamma-glutamic acid and cholera toxin subunit B (CTB). Among the molecules tested, CTB provided the most enhanced mucosal sIgA and systemic IgG induction. SL-applied CTB enhanced the production of interleukin-4 and interferon-gamma from stimulated CD4+ T cells. Moreover, interferon-gamma-producing CD8+ T cell responses were increased 1.7-fold after co-treatment with SL CTB and HPV16L1. These results suggest the potential of the SL route for delivery of HPV16L1 vaccines using CTB as an adjuvant.

Antitumor activity of EGFR targeted pH-sensitive immunoliposomes encapsulating gemcitabine in A549 xenograft nude mice.

Immunoliposomes directed by monoclonal antibodies are promising vehicles for tumor targeted drug delivery. Development of a long-circulating formulation of pH-sensitive liposomes (PSLs) with epidermal growth factor receptor (EGFR) antibody attached was designed and tested using A549 cells and BALB/c-nu/nu mouse tumor model. PSL formulation was prepared using small unilamellar vesicles of DOPE and CHEMS (6:4 molar ratio) by REV method. The average size and zeta-potential of the formulation measured by dynamic laser-light scattering were approximately 146+/-43.9 nm (PDI=0.09+/-0.02) and -1.77+/-0.03 mV, respectively. A549 cells were xenotransplanted into BALB/c-nu/nu mice and various formulations of gemcitabine (gem), such as in its free form, PSLs or Ab-PSLs, were injected intravenously via a tail vein. The rate of tumor volume increment in Ab-PSLs with gem-treated group was remarkably slower than that of other drug-treated group. The tumor from Ab-PSLs with gem 160 mg/kg-injected group exhibited a markedly lowest account of PCNA labeled cells and had highest TUNEL-positive cells among tested. This suggests that treatment of Ab-PSLs with gem resulted in an increased apoptosis of tumor cells, leading to tumor growth inhibition. These results demonstrate that PSLs provide an efficient and targeted delivery of gemcitabine and may represent a useful new treatment approach for tumors which overexpress the EGFR.

Mimosine prevents the death of glucose-deprived immunostimulated astrocytes by scavenging peroxynitrite.

Immunostimulated astrocytes become highly vulnerable to glucose deprivation (Choi and Kim: J Neurosci Res 54:870-875, 1998a). The increased vulnerability is caused by the enhanced level of peroxynitrite endogenously produced in glucose-deprived immunostimulated astrocytes. In the present study, we report that the plant amino acid mimosine can attenuate the increased death by scavenging peroxynitrite. Treatment with mimosine blocked the increase of nitrotyrosine immunoreactivity, a marker of peroxynitrite, in glucose-deprived immunostimulated astrocytes. Furthermore, mimosine directly inhibited the nitration of tyrosine residues of bovine serum albumin and the oxidation of dihydrorhodamine-123 to rhodamine-123 by peroxynitrite. Mimosine has been used experimentally as a cell cycle G1/S phase transition blocker (Lalande: Exp Cell Res 186:332-339, 1990; Hoffman et al.: Cytometry 12:26-32, 1991). Flow cytometry analysis, however, showed that the cytoprotective effect of mimosine was not attributed to its inhibition of cell cycle progression. Furthermore, under our experimental conditions, mimosine did not alter the levels of cell cycle regulatory proteins, including p21(WAF1/CIP1), cyclins D1 and E, and proliferating cell nuclear antigen. In addition, cyclin-dependent kinase inhibitors olomoucine and roscovitine did not block the increased death. These results indicate that mimosine inhibits the augmented death of glucose-deprived immunostimulated astrocytes by scavenging peroxynitrite rather than suppressing the cell cycle progression.