Arsenite Regulates Prolongation of Glycan Residues of Membrane Glycoprotein: A Pivotal Study via Wax Physisorption Kinetics and FTIR Imaging.

Arsenic exposure results in several human cancers, including those of the skin, lung, and bladder. As skin cancers are the most common form, epidermal keratinocytes (KC) are the main target of arsenic exposure. The mechanisms by which arsenic induces carcinogenesis remains unclear, but aberrant cell proliferation and dysregulated energy homeostasis play a significant role. Protein glycosylation is involved in many key physiological processes, including cell proliferation and differentiation. To evaluate whether arsenite exposure affected protein glycosylation, the alteration of chain length of glycan residues in arsenite treated skin cells was estimated. Herein we demonstrated that the protein glycosylation was adenosine triphosphate (ATP)-dependent and regulated by arsenite exposure by using Fourier transform infrared (FTIR) reflectance spectroscopy, synchrotron-radiation-based FTIR (SR-FTIR) microspectroscopy, and wax physisorption kinetics coupled with focal-plane-array-based FTIR (WPK-FPA-FTIR) imaging. We were able to estimate the relative length of surface protein-linked glycan residues on arsenite-treated skin cells, including primary KC and two skin cancer cell lines, HSC-1 and HaCaT cells. Differential physisorption of wax adsorbents adhered to long-chain (elongated type) and short-chain (regular type) glycan residues of glycoprotein of skin cell samples treated with various concentration of arsenite was measured. The physisorption ratio of beeswax remain/n-pentacosane remain for KC cells was increased during arsenite exposure. Interestingly, this increase was reversed after oligomycin (an ATP synthase inhibitor) pretreatment, suggesting the chain length of protein-linked glycan residues is likely ATP-dependent. This is the first study to demonstrate the elongation and termination of surface protein-linked glycan residues using WPK-FPA-FTIR imaging in eukaryotes. Herein the result may provide a scientific basis to target surface protein-linked glycan residues in the process of arsenic carcinogenesis.

Improved photodynamic cancer treatment by folate-conjugated polymeric micelles in a KB xenografted animal model.

Photodynamic therapy (PDT) is a light-induced chemical reaction that produces localized tissue damage for the treatment of cancers and various nonmalignant conditions. In the clinic, patients treated with PDT should be kept away from direct sunlight or strong indoor lighting to avoid skin phototoxicity. In a previous study, it was demonstrated that the skin phototoxicity of meta-tetra(hydroxyphenyl)chlorin (m-THPC), a photosensitizer used in the clinic, can be significantly reduced after micellar encapsulation; however, no improvement in antitumor efficacy was observed. In this work, a folate-conjugated polymeric m-THPC delivery system is developed for improving tumor targeting of the photosensitizer, preventing photodamage to the healthy tissue, and increasing the effectiveness of the photosensitizers. The results demonstrate that folate-conjugated m-THPC-loaded micelles with particle sizes around 100 nm are taken up and accumulated by folate receptor-overexpressed KB cells in vitro and in vivo, and their PDT has no significant adverse effects on the body weight of mice. After an extended delivery time, a single dose of folate-conjugated m-THPC-loaded micelles has higher antitumor effects (tumor growth inhibition = 92%) through inhibition of cell proliferation and reduction of vessel density than free m-THPC or m-THPC-loaded micelles at an equivalent m-THPC concentration of 0.3 mg kg(-1) after irradiation. Furthermore, folate-conjugated m-THPC-loaded micelles at only 0.2 mg kg(-1) m-THPC have a similar antitumor efficacy to m-THPC or m-THPC-loaded micelles with the m-THPC concentration at 0.3 mg kg(-1) , which indicates that the folate conjugation on the micellar photosensitizer apparently reduces the requirement of m-THPC for PDT. Thus, folate-conjugated m-THPC-loaded micelles with improved selectivity via folate-folate receptor interactions have the potential to reduce, not only the skin photosensitivity, but also the drug dose requirement for clinical PDT.

Reduced skin photosensitivity with meta-tetra(hydroxyphenyl)chlorin-loaded micelles based on a poly(2-ethyl-2-oxazoline)-b-poly(d,l-lactide) diblock copolymer in vivo.

Photodynamic therapy (PDT) is a light-induced chemical reaction that produces localized tissue damage for the treatment of cancers and other nonmalignant conditions. The activation of photosensitizers in a target tissue is accomplished with a specific light source in the presence of molecular oxygen. In the clinic, patients treated with PDT should be kept away from direct sunlight or strong indoor lighting to avoid skin phototoxicity. In this study, a photosensitizer encapsulated within a micelle was developed to overcome this problem. The pH-sensitive micelles were successfully incorporated with meta-tetra(hydroxyphenyl)chlorin (m-THPC), and the cytotoxicity and antitumor effects were investigated in vitro and in vivo. Our results demonstrated that PDT with m-THPC-loaded micelles had no significant adverse effects on the body weight of mice in vivo. Furthermore, after an extended delivery time, m-THPC-loaded micelles and free m-THPC had similar antitumor effects, but the m-THPC-loaded micelles had less skin phototoxicity. Thus, this strategy could be used as a potential nanocarrier for PDT-mediated cancer therapy.

Photo-Crosslinked Polymeric Matrix with Antimicrobial Functions for Excisional Wound Healing in Mice.

Wound infection extends the duration of wound healing and also causes systemic infections such as sepsis, and, in severe cases, may lead to death. Early prevention of wound infection and its appropriate treatment are important. A photoreactive modified gelatin (GE-BTHE) was synthesized by gelatin and a conjugate formed from the 3,3',4,4'-benzophenone tetracarboxylic dianhydride (BTDA) and the 2-hydroxyethyl methacrylate (HEMA). Herein, we investigated the photocurable polymer solution (GE-BTHE mixture) containing GE-BTHE, poly(ethylene glycol) diacrylate (PEGDA), chitosan, and methylene blue (MB), with antimicrobial functions and photodynamic antimicrobial chemotherapy for wound dressing. This photocurable polymer solution was found to have fast film-forming property attributed to the photochemical reaction between GE-BTHE and PEGDA, as well as the antibacterial activity in vitro attributed to the ingredients of chitosan and MB. Our in vivo results also demonstrated that untreated wounds after 3 days had the same scab level as the GE-BTHE mixture-treated wounds after 20 s of irradiation, which indicates that the irradiated GE-BTHE mixture can be quickly transferred into artificial scabs to protect wounds from an infection that can serve as a convenient excisional wound dressing with antibacterial efficacy. Therefore, it has the potential to treat nonhealing wounds, deep burns, diabetic ulcers and a variety of mucosal wounds.

Enhanced Antitumor Effects of Epidermal Growth Factor Receptor Targetable Cetuximab-Conjugated Polymeric Micelles for Photodynamic Therapy.

Nanocarrier-based delivery systems are promising strategies for enhanced therapeutic efficacy and safety of toxic drugs. Photodynamic therapy (PDT)-a light-triggered chemical reaction that generates localized tissue damage for disease treatments-usually has side effects, and thus patients receiving photosensitizers should be kept away from direct light to avoid skin phototoxicity. In this study, a clinically therapeutic antibody cetuximab (C225) was conjugated to the surface of methoxy poly(ethylene glycol)-b-poly(lactide) (mPEG-b-PLA) micelles via thiol-maleimide coupling to allow tumor-targetable chlorin e6 (Ce6) delivery. Our results demonstrate that more C225-conjugated Ce6-loaded polymeric micelles (C225-Ce6/PM) were selectively taken up than Ce6/PM or IgG conjugated Ce6/PM by epidermal growth factor receptor (EGFR)-overexpressing A431 cells observed by confocal laser scanning microscopy (CLSM), thereby decreasing the IC50 value of Ce6-mediated PDT from 0.42 to 0.173 µM. No significant differences were observed in cellular uptake study or IC50 value between C225-Ce6/PM and Ce6/PM groups in lower EGFR expression HT-29 cells. For antitumor study, the tumor volumes in the C225-Ce6/PM-PDT group (percentage of tumor growth inhibition, TGI% = 84.8) were significantly smaller than those in the Ce6-PDT (TGI% = 38.4) and Ce6/PM-PDT groups (TGI% = 53.3) (p < 0.05) at day 21 through reduced cell proliferation in A431 xenografted mice. These results indicated that active EGFR targeting of photosensitizer-loaded micelles provides a possible way to resolve the dose-limiting toxicity of conventional photosensitizers and represents a potential delivery system for PDT in a clinical setting.

Carbon Nanotube-Mediated Photothermal Disruption of Endosomes/Lysosomes Reverses Doxorubicin Resistance in MCF-7/ADR Cells.

Cancer is the leading cause of human death worldwide. Although many scientists work to fight this disease, multiple drug resistance is a predominant obstacle for effective cancer therapy. In drug-resistant MCF-7/ADR cells, the acidic organelles with lower pH value than normal one can cause the protonation of anthracycline drugs, inducing drug accumulation in these organelles. In this study, single-walled carbon nanotubes with polyethylene glycol phospholipids surface modification (PEGylated SWNTs) were utilized as near infrared-activated drug carriers for doxorubicin (DOX) delivery against MCF-7/ADR cells. Our results showed that a concentration-dependent temperature increase was observed in a solution of PEGylated SWNTs with 808 nm laser irradiation, whereas a water solution showed no significant changes in temperature under a thermal camera using the same irradiation dose. Interestingly, PEGylated DOX-SWNTs enhanced the nuclear accumulation of DOX with 808 nm irradiation whereas free DOX or PEGylated DOX-SWNTs revealed discrete red spots in MCF-7/ADR cells by confocal microscopic observation. Cell viability of PEGylated DOX-SWNTs-treated cells was also significantly decreased after 808 nm laser irradiation. Thus, photothermally activated PEGylated SWNTs can be a potential nanocarrier to deliver DOX into cancer cells and successfully overcome drug-resistant behavior in MCF-7/ADR breast cancer cells.

Real-time vascular imaging and photodynamic therapy efficacy with micelle-nanocarrier delivery of chlorin e6 to the microenvironment of melanoma.

BACKGROUND: Strategies combining anti-vascular therapy and vascular imaging may facilitate the prediction of early response and outcome in cancer treatment. OBJECTIVE: The aim of this study was to investigate the relationship between the tumor-associated vasculature in melanoma and to develop an approach for melanoma treatment by utilizing the free form and micelle form of the photosensitizer (PS) chlorin e6 in photodynamic therapy (PDT). METHODS: Green fluorescence protein (GFP) expressing B16-F10 melanoma cells were implanted into the mouse ear dermis. Ce6 in free form or in micelle form was administered via the tail vein. An OV100 imaging system was used to record the red fluorescence of Ce6 to obtain real-time vascular images in the GFP tumor. RESULTS: Compared to free Ce6, Ce6 linked to the micelle-nanocarrier depicted a much clearer vascular image and had an effective vascular destruction by PDT. Micelle Ce6 was localized in lysosomes and in the endoplasmic reticulum of cultured endothelial cells, implying an active endocytosis of the nano-carrier. CONCLUSION: Micelle Ce6 can serve as a bifunctional PS for vascular imaging and PDT, which facilitates its delivery in the tumor microenvironment.

Metal nanobullets for multidrug resistant bacteria and biofilms.

Infectious diseases were one of the major causes of mortality until now because drug-resistant bacteria have arisen under broad use and abuse of antibacterial drugs. These multidrug-resistant bacteria pose a major challenge to the effective control of bacterial infections and this threat has prompted the development of alternative strategies to treat bacterial diseases. Recently, use of metallic nanoparticles (NPs) as antibacterial agents is one of the promising strategies against bacterial drug resistance. This review first describes mechanisms of bacterial drug resistance and then focuses on the properties and applications of metallic NPs as antibiotic agents to deal with antibiotic-sensitive and -resistant bacteria. We also provide an overview of metallic NPs as bactericidal agents combating antibiotic-resistant bacteria and their potential in vivo toxicology for further drug development.

Bioluminescence resonance energy transfer using luciferase-immobilized quantum dots for self-illuminated photodynamic therapy.

Photodynamic therapy (PDT) is an innovative method for cancer treatment that involves the administration of a photosensitizing agent followed by exposure to visible light. An appreciable amount of a particular light source is a key to activate photosensitizers in PDT. However, the external excitation light source is a problem for clinical application because of the limitation of tissue-penetrating properties. Additionally, the wavelength of laser emission should match the absorption wavelength of each photosensitizer for efficient generation of reactive oxygen species and cell killing. In this study, Renilla luciferase-immobilized quantum dots-655 (QD-RLuc8) was used for bioluminescence resonance energy transfer (BRET)-mediated PDT to resolve these problems. The bioluminescent QD-RLuc8 conjugate exhibits self-illumination at 655 nm after coelenterazine addition, which can activate the photosensitizer, Foscan(®)-loaded micelles for PDT. Our results show that BRET-mediated PDT by QD-RLuc8 plus coelenterazine (20 µg/mL) successfully generated reactive oxygen species (40.8%), killed ~ 50% A549 cells at 2 µg/mL equivalent Foscan(®)in vitro and significantly delayed tumor growth in vivo due to cell apoptosis under TUNEL analysis without obvious weight loss. Based on immunohistochemical observations, the proliferating cell nuclear antigen (PCNA)-negative area of tumor sections after BRET-mediated PDT was obviously increased compared to the PDT-untreated groups without an external light source. We conclude that this nanotechnology-based PDT possesses several clinical benefits, such as overcoming light penetration issues and treating deeper lesions that are intractable by PDT alone.

Facile self-assembly of porphyrin-embedded polymeric vesicles for theranostic applications.

A robust and uniform porphysome, which reveals an efficient photodynamic therapy and contrast-enhanced ultrasonic imaging for theranostic applications, can be fabricated from a 4-armed porphyrin-polylactide conjugate.

Sized controlled synthesis, purification, and cell studies with silicon quantum dots.

This article describes the size control synthesis of silicon quantum dots with simple microemulsion techniques. The silicon nanocrystals are small enough to be in the strong confinement regime and photoluminesce in the blue region of the visible spectrum and the emission can be tuned by changing the nanocrystal size. The silicon quantum dots were capped with allylamine either a platinum catalyst or UV-radiation. An extensive purification protocol is reported and assessed using (1)H NMR to produce ultra pure silicon quantum dots suitable for biological studies. The highly pure quantum dots were used in cellular uptake experiments and monitored using confocal microscopy. The results showed that the amine terminated silicon nanocrystals accumulated in lysosome but not in nuclei and could be used as bio-markers to monitor cancer cells over long timescales.

Reversal of doxorubicin-resistance by multifunctional nanoparticles in MCF-7/ADR cells.

The efficacy of many chemotherapeutic agents is reduced in cells that have developed multiple drug resistance (MDR). To address this important problem, a biodegradable polymer was coupled to a photosensitizer and the resulting photosensitizer-nanoparticles were loaded with the chemotherapeutic agent doxorubicin. The combination of photosensitizer and chemotherapeutic agent had a synergistic action on a doxorubicin-resistant breast cancer MCF-7 cell line. To increase the effectiveness of this combination, d-alpha-tocopheryl poly(ethylene glycol) 1000 succinate (TPGS), an inhibitor of the multidrug transporter overproduced in these resistant cells, was added during the formation of the nanoparticles. The insertion of TPGS decreased the P-glycoprotein activity, increased the intracellular accumulation doxorubicin, and also increased the therapeutic efficacy of the resulting nanoparticles. Both TPGS and irradiation of the photoreactive nanoparticles caused doxorubicin to move from the cytoplasm to the nucleus. This combination of photodynamic activity in a powerful nanocarrier loaded with the chemotherapeutic agent doxorubicin can be used to deliver two types of cancer therapy simultaneously, and the addition of TPGS can further enhance the entry of doxorubicin into the nucleus. Therefore, this innovative delivery system can act as a potential nanomedicine for both drug-sensitive and drug-resistant cancer therapy.

Non-toxic phototriggered gene transfection by PAMAM-porphyrin conjugates.

Development of controllable and non-toxic gene transfection systems is a core issue in gene therapy. Photochemical internalization, an innovative strategy in cytosolic release, provides us with an opportunity to develop a light-inducible gene delivery system. In this study, a novel photochemical internalization (PCI)-mediated gene delivery system was synthesized by surface modification of polyamidoamine (PAMAM) dendrimers via 5,10,15-tri(4-acetamidophenyl)-20-mono(4-carboxyl-phenyl)porphyrin (TAMCPP) conjugated to the generation 4 PAMAM dendrimer (G4). This water-soluble PAMAM-TAMCPP conjugate was characterized for cell viability, phototoxicity, DNA complexation, and in vitro transfection activity. The results show that TAMCPP conjugation did not increase the cytotoxicity of the PAMAM dendrimer below 20 microM, but significantly induced cell death after suitable irradiation. Under almost non-toxic G4-TAMCPP-mediated PCI treatment, the expression of green fluorescent protein determined by flow cytometry could be markedly enhanced in HeLa cells. Therefore, the G4-TAMCPP conjugate had an inducible and effective gene transfection activity, and showed considerable potential as a bimodal biomaterial for PCI-mediated gene therapy.