Intracellular Photosensitizer Research Articles

Reversal of drug resistance in breast cancer cells by photodynamic action mediated by curcumin/Ko143/PLGA nanoparticles

Objective: The objective of this study is to prepare Cur/Ko143/PLGA that encapsulate the photosensitizer curcumin (Cur) and the multidrug transporter-specific inhibitor Ko143 to investigate its application in reversing drug resistance in breast cancer and evaluate its properties and therapeutic efficacy. Results: After successful preparation of nanoparticles, the encapsulation rate of Cur/Ko143/PLGA was 73.8% ± 1.8%, and the drug loading rate was 2.8% ± 0.1%. The Cur/Ko143/PLGA were spherical and regular in morphology under transmission electron microscopy. The nanoparticles were morphologically intact, were uniform in size, and did not show significant aggregation under scanning electron microscopy. The average particle size of Cur/Ko143/PLGA was 232.32 ± 10.60 nm, the polymer dispersion index was 0.01, and the zeta potential was −17.25 ± 0.65 mV, as determined by laser particle sizing. In vitro ultrasound observation showed that Cur/Ko143/PLGA were enhanced in both the B-mode and contrast modes. Intracellular Cur fluorescence detection, biocompatibility testing, cell scratch experiment, flow detection of apoptosis, live–dead staining, and actin polymerization test all prove that new nanomaterials have better photodynamic efficacy. Conclusion: Drug-carrying Cur/Ko143/PLGA nanoparticles can increase the concentration of the intracellular photosensitizer Cur and increase the photodynamic efficacy by inhibiting an efflux pump, which is expected to become a new therapeutic strategy.

Carrier-Free, Amorphous Verteporfin Nanodrug for Enhanced Photodynamic Cancer Therapy and Brain Drug Delivery.

Glioblastoma (GBM) is hard to treat due to cellular invasion into functioning brain tissues, limited drug delivery, and evolved treatment resistance. Recurrence is nearly universal even after surgery, chemotherapy, and radiation. Photodynamic therapy (PDT) involves photosensitizer administration followed by light activation to generate reactive oxygen species at tumor sites, thereby killing cells or inducing biological changes. PDT can ablate unresectable GBM and sensitize tumors to chemotherapy. Verteporfin (VP) is a promising photosensitizer that relies on liposomal carriers for clinical use. While lipids increase VP's solubility, they also reduce intracellular photosensitizer accumulation. Here, a pure-drug nanoformulation of VP, termed "NanoVP", eliminating the need for lipids, excipients, or stabilizers is reported. NanoVP has a tunable size (65-150nm) and 1500-fold higher photosensitizer loading capacity than liposomal VP. NanoVP shows a 2-fold increase in photosensitizer uptake and superior PDT efficacy in GBM cells compared to liposomal VP. In mouse models, NanoVP-PDT improved tumor control and extended animal survival, outperforming liposomal VP and 5-aminolevulinic acid (5-ALA). Moreover, low-dose NanoVP-PDT can safely open the blood-brain barrier, increasing drug accumulation in rat brains by 5.5-fold compared to 5-ALA. NanoVP is a new photosensitizer formulation that has the potential to facilitate PDT for the treatment of GBM.

Hypoxia and Singlet Oxygen Dual-Responsive Micelles for Photodynamic and Chemotherapy Therapy Featured with Enhanced Cellular Uptake and Triggered Cargo Delivery.

Combination therapy provides better outcomes than a single therapy and becomes an efficient strategy for cancer treatment. In this study, we designed a hypoxia- and singlet oxygen-responsive polymeric micelles which contain azo and nitroimidazole groups for enhanced cellular uptake, repaid cargo release, and codelivery of photosensitizer Ce6 and hypoxia-activated prodrug tirapazamine TPZ (DHM-Ce6@TPZ), which could be used for combining Ce6-mediated photodynamic therapy (PDT) and PDT-activated chemotherapy to enhance the therapy effect of cancer. The hypoxia- and singlet oxygen-responsive polymeric micelles DHM-Ce6@TPZ were prepared by film hydration method. The morphology, physicochemical properties, stimuli responsiveness, in vitro singlet oxygen production, cellular uptake, and cell viability were evaluated. In addition, the in vivo therapeutic effects of the micelles were verified using a tumor xenograft mice model. The resulting dual-responsive micelles not only increased the concentration of intracellular photosensitizer and TPZ, but also facilitated photosensitizer and TPZ release for enhanced integration of photodynamic and chemotherapy therapy. As a photosensitizer, Ce6 induced PDT by generating toxic singlet reactive oxygen species (ROS), resulting in a hypoxic tumor environment to activate the prodrug TPZ to achieve efficient chemotherapy, thereby evoking a synergistic photodynamic and chemotherapy therapeutic effect. The cascade synergistic therapeutic effect of DHM-Ce6@TPZ was effectively evaluated both in vitro and in vivo to inhibit tumor growth in a breast cancer mice model. The designed multifunctional micellar nano platform could be a convenient and powerful vehicle for the efficient co-delivery of photosensitizers and chemical drugs for enhanced synergistic photodynamic and chemotherapy therapeutic effect of cancer.

Enhanced Intracellular Photosensitizer Uptake and Retention by Targeting Viral Oncoproteins in Human Papillomavirus Infected Cancer Cells and Cancer Stem Cells.

Immunogenic proteins in cancer are relevant targets for drug delivery. In Photodynamic Therapy (PDT), surface antigens have previously been used to deliver the photosensitizer (PS) to the tumor microenvironment for specific targeting. However, can we target intracellular antigens to achieve more than surface recognition? Can we possibly increase PS intracellular localization and prevent drug efflux at the same time? In this study, these questions were addressed by using a compound that can not only specifically recognize and bind to intracellular E6 oncoproteins in Human Papillomavirus (HPV)-Transformed cancer cells, but is also capable of enhancing transmembrane uptake using the cells' own active transport mechanisms. HPV-transformed SiHa cells were cultured in vitro, and the resistant subpopulation was isolated using Magnetic Activated Cell Sorting (MACS). PDT was performed on four different cell types with varying physiognomies in terms of HPV oncoprotein expression and physiological form. Results demonstrated that tagging PSs on a carrier molecule that specifically delivers the PS inside the cells that express the target proteins enhanced both cellular uptake and retention of the PS even in the presence of drug efflux proteins on resistant subpopulations. These findings provide insight into the possibility of preventing cell-mediated resistance to PDT.

Visible-Light-Driven Enhanced Biohydrogen Production by Photo-Biohybrid System Based on Photoelectron Transfer between Intracellular Photosensitizer Gold Nanoparticles and Clostridium butyricum

Biohydrogen is a clean and renewable energy, but the low yield caused by the lack of reducing power in cells greatly restricts the industrialization of biohydrogen production. Photo-biohybrid systems (PBSs) can integrate the high light energy utilization efficiency of photocatalysts with the excellent catalytic performance of microorganisms. Here, gold nanoparticles were targeted into Clostridium butyricum as intracellular photosensitizers to construct a PBS that could efficiently produce biohydrogen under visible light, with the apparent quantum yield as high as 19.31%. Compared with the dark-fermented C. butyricum, the biohydrogen production of PBS increased by 88.74%. The mechanism of photoelectrons from Au NPs to C. butyricum was elucidated by the transcriptome. Compared with the dark-fermented biohybrids, the expressions of biohydrogen generation-related enzymes, such as hydrogenase and pyruvate formate lyase genes, in the PBS were all upregulated more than 2 times. Furthermore, the genes of riboflavin synthase, electron transfer flavoprotein (ETF), and FAD-dependent oxidoreductase, which are closely related to electron transformation, were all significantly upregulated. The photoelectrons were transferred to the hydrogenase via ETF and FAD2+ to enhance biohydrogen production, independent of pyruvate decomposition. This PBS provides theoretical guidance for constructing an efficient light-driven microbial manufacturing system.

The cell-impermeable Ru(II) polypyridyl complex as a potent intracellular photosensitizer under visible light irradiation via ion-pairing with suitable lipophilic counter-anions

Developing the cell-impermeable Ru(II) polypyridyl cationic complexes as effective photosensitizers (PS) which have high cellular uptake and photo-toxicity, but low dark toxicity, is quite challenging. Here we found that the highly reactive singlet oxygen (1O2) can be generated by the irradiation of a typical Ru(II) polypyridyl complex Ru(II)tris(tetramethylphenanthroline) ([Ru(TMP)3]2+) under visible light irradiation by ESR with TEMPO (2,2,6,6-tetramethyl-4-piperidone-N-oxyl) as 1O2 probe. Effective cellular and nuclear delivery of cationic [Ru(TMP)3]2+ was achieved through our recently developed ion-pairing method, and 2,3,4,5-tetrachlorophenol (2,3,4,5-TeCP) was found to be the most effective among all chlorophenols tested. The accelerated cellular, especially nuclear uptake of [Ru(TMP)3]2+ results in the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) and DNA strand breaks, caspase 3/7 activation and cell apoptosis in HeLa cells upon light irradiation. More importantly, compared with other traditional photosensitizers, [Ru(TMP)3]2+ showed significant photo-toxicity but low dark toxicity. Similar effects were observed when 2,3,4,5-TeCP was substituted by the currently clinically used anti-inflammatory drug flufenamic acid. This represents the first report that the cell-impermeable Ru(II) polypyridyl complex ion-paired with suitable lipophilic counter-anions functions as potent intracellular photosensitizer under visible light irradiation mainly via a 1O2-mediated mechanism. These findings should provide new perspectives for future investigations on other metal complexes with similar characteristics as promising photosensitizers for potential photodynamic therapy.

Antimicrobial Blue Light versus Pathogenic Bacteria: Mechanism, Application in the Food Industry, Hurdle Technologies and Potential Resistance.

Blue light primarily exhibits antimicrobial activity through the activation of endogenous photosensitizers, which leads to the formation of reactive oxygen species that attack components of bacterial cells. Current data show that blue light is innocuous on the skin, but may inflict photo-damage to the eyes. Laboratory measurements indicate that antimicrobial blue light has minimal effects on the sensorial and nutritional properties of foods, although future research using human panels is required to ascertain these findings. Food properties also affect the efficacy of antimicrobial blue light, with attenuation or enhancement of the bactericidal activity observed in the presence of absorptive materials (for example, proteins on meats) or photosensitizers (for example, riboflavin in milk), respectively. Blue light can also be coupled with other treatments, such as polyphenols, essential oils and organic acids. While complete resistance to blue light has not been reported, isolated evidence suggests that bacterial tolerance to blue light may occur over time, especially through gene mutations, although at a slower rate than antibiotic resistance. Future studies can aim at characterizing the amount and type of intracellular photosensitizers across bacterial species and at assessing the oxygen-independent mechanism of blue light—for example, the inactivation of spoilage bacteria in vacuum-packed meats.

Uptake of silver, gold, and hybrids silver-iron, gold-iron and silver-gold aminolevulinic acid nanoparticles by MCF-7 breast cancer cells.

Nanoparticles show promise for theranostic applications in cancer. The metal-based nanoparticles can be used both as photosensitizers and delivery vehicles. In bimetallic particles based on gold or silver and iron, a combination of the plasmonic features of the gold or silver components with the magnetic properties of the iron makes these hybrid nanomaterials suitable for both imaging and therapeutic applications. Herein, we discuss toxicity and cell internalization of metallic (silver and gold) and bimetallic (silver-iron, gold-iron, and silver-gold) aminolevulinic acid (ALA) nanoparticles. ALA can control the production of an intracellular photosensitizer, protoporphyrin IX (PpIX), commonly used in photodynamic therapy. Nanoparticles were synthesized by photoreduction method and characterized by UV/Vis spectra, Zeta potential, FTIR, XRD, and transmission electron microscopy. The amount of singlet oxygen generation by a yellow LED, and ultrasound was studied for gold, gold-iron, and silver-gold nanoparticles. Cytotoxicity assays of MCF-7 in the presence of nanoparticles were performed, and PpIX fluorescence was quantified by high content screening (HCS). Red fluorescence observed after 24 h of nanoparticles incubation on MCF-7 cells, indicated that the ALA in surface of nanoparticles was efficiently converted to PpIX. The best results for singlet oxygen generation with LED or ultrasound irradiation were obtained with ALA:AgAuNPs. The studied nanoparticles present the potential to deliver aminolevulinic acid to breast cancer cells efficiently, generate singlet oxygen, and convert ALA into PpIX inside the cells allowing photodiagnosis and therapies such as photodynamic and sonodynamic therapies.

Mitomycin C-induced cell cycle arrest enhances 5-aminolevulinic acid-based photodynamic therapy for bladder cancer

Photodynamic therapy (PDT) and diagnosis (PDD) using 5-aminolevulinic acid (ALA) to control the production of the intracellular photosensitizer protoporphyrin IX (PpIX) are commonly used clinically. Previously, we demonstrated that dormant and drug-induced dormancy-like cancer cells accumulated high PpIX levels, making them sensitive to ALA-PDT. Because EAU Guidelines awarded a level of evidence of 1a to mitomycin C, the drug is widely used to treat bladder cancer. In this study, we investigated that the effect of mitomycin C-induced cell cycle arrest on porphyrin metabolism, including that induced by ALA-PDT. T24 human urinary bladder carcinoma cells were selected for this research. T24 cells were irradiated using a light-emitting diode emitting red light for the ALA-PDT assay. Cell cycle analysis was conducted by flow cytometry using bromodeoxyuridine. Cell viability was confirmed using the MTT or colony formation assay. Furthermore, mRNA gene expression analysis was performed using our previously reported methods. The cell cycle of T24 cells was arrested at G2/M phase by mitomycin C. PpIX accumulation was dramatically increased by mitomycin C treatment. Cell viability after ALA-PDT was remarkably decreased by mitomycin C pretreatment. The gene expression of porphyrin transporters was consistent with the metabolic and morphological results. Finally, we confirmed that ALA-PDT combined with mitomycin C treatment exerted a long-term inhibitory effect on cell proliferation. This study demonstrated a new approach to enhance the effects of ALA-PDT using drugs that induce a dormancy-like status and upregulate porphyrin metabolism.

Verification of 5-Aminolevurinic Radiodynamic Therapy Using a Murine Melanoma Brain Metastasis Model.

Melanoma is a highly aggressive cancer with a propensity for brain metastases. These can be treated by radiotherapy, but the radiation-resistant nature of melanoma makes the prognosis for melanoma patients with brain metastases poor. Previously, we demonstrated that treatment of mice with subcutaneous melanoma with 5-aminolevurinic acid (5-ALA) and X-rays in combination, (“radiodynamic therapy (RDT)”), instead of with 5-ALA and laser beams (“photodynamic therapy”), improved tumor suppression in vivo. Here, using the B16-Luc melanoma brain metastasis model, we demonstrate that 5-ALA RDT effectively treats brain metastasis. We also studied how 5-ALA RDT damages cells in vitro using a B16 melanoma culture. Cell culture preincubated with 5-ALA alone increased intracellular photosensitizer protoporphyrin IX. On X-ray irradiation, the cells enhanced their ∙OH radical generation, which subsequently induced γH2AX, a marker of DNA double-strand breaks in their nuclei, but decreased mitochondrial membrane potential. After two days, the cell cycle was arrested. When 5-ALA RDT was applied to the brain melanoma metastasis model in vivo, suppression of tumor growth was indicated. Therapeutic efficacy in melanoma treatment has recently been improved by molecular targeted drugs and immune checkpoint inhibitors. Treatment with these drugs is now expected to be combined with 5-ALA RDT to further improve therapeutic efficacy.

Preventive Effect of Opuntiol, Isolated from Opuntia ficus indica (L. Mill), Extract Against Ultraviolet A Radiation-Induced Oxidative Damages in NIH/3T3 Cells

Aim: We investigated the role of opuntiol, isolated from Opuntia ficus indica, against ultraviolet A waveband-mediated oxidative damages in the mouse embryonic fibroblast cell lines (NIH‐3T3). Materials and Methods: The antioxidant potential of opuntiol was carried out by hydroxyl radical, superoxide anion and DPPH radical scavenging assays. The preventive effect of opuntiol against UV-mediated cytotoxicity was revealed by MTT assay. Further, the oxidative end points during UV-exposure, in the presence and absence of opuntiol, was analyzed by DCFH-DA staining, rhodamine-123 staining and alkaline comet assay. Results: Opuntiol significantly neutralizes hydroxyl (OH•), superoxide anion (O2•–), hydrogen peroxide (H2O2), and 2,2-diphenyl-2-picrylhydrazyl (DPPH•) radicals in a concentration-dependent manner. In this study, the NIH-3T3 cells were treated with UVA-waveband in the presence and absence of opuntiol and oxidative damage markers were analyzed. We observed that opuntiol pretreatment (5 μM-20 μM) prevented 10 mJ/cm2UVA radiation-induced cytotoxicity in NIH-3T3 cells. Further, single UVA-radiation induces reactive oxygen species (ROS) through intracellular photosensitizers. Conversely, opuntiol pretreatment prevented UVA-mediated ROS generation and subsequent lipid peroxidation and loss of enzymatic antioxidants (superoxide dismutase [SOD], catalase, and glutathione peroxidase) in the NIH-3T3 cells. It has also been observed that the UVA-mediated ROS subsequently induces DNA damage and alters mitochondrial transmembrane potential (MMP). We noticed that opuntiol prevents UVA-radiation-mediated DNA single-strand breaks. Further, it prevents loss of MMPs and apoptotic morphological changes in the NIH-3T3 cells. Conclusion: Thus, these findings illustrate that opuntiol prevents UVA-radiation-mediated oxidative stress-related biochemical changes in the cellular system.

Photoirradiation after aminolevulinic acid treatment suppresses cancer cell proliferation through the HO-1/p21 pathway

Photodynamic therapy (PDT) and diagnosis (PDD) using 5-aminolevulinic acid (ALA) to control the production of an intracellular photosensitizer, protoporphyrin IX (PpIX), are in common clinical use. Although various studies have been published regarding cell death analysis after photoirradiation by ALA-PDT, the changes in gene expressions induced by it are yet unclear. Here, we focused on studying gene expression and cell proliferation changes in cancer cells that survive photoirradiation. HEK293 human embryonic kidney cells, MKN45 human gastric cells, and PC-3 human prostate cancer cells were selected for this research. Cell viability was measured using trypan blue and MTT assays. ALA-PDT experiments were performed using a calibrated LED irradiation module. Furthermore, mRNA and protein gene expression analysis were performed using our previously reported methods. mRNAs of PAI-1, HO-1, and p21 were upregulated after photoirradiation of HEK293, which was suppressed by N-acetyl-L-cysteine, a reactive oxygen species (ROS) scavenger. Primer array results in PC-3 cells and p21 and Ki-67 expression results in both PC-3 and MKN45 cells suggested that photoirradiation suppressed cell proliferation. Cell numbers post-photoirradiation revealed that the proliferation of surviving cells was suppressed in PC-3 and MKN45 cells. ALA-PDD or ALA-PDT can result in rapid ROS-induced cell death and may decrease long-term recurrence rates through several pathways including the HO-1/p21 pathway.

Immobilization of Photo-Immunoconjugates on Nanoparticles Leads to Enhanced Light-Activated Biological Effects.

The past three decades have witnessed notable advances in establishing photosensitizer-antibody photo-immunoconjugates for photo-immunotherapy and imaging of tumors. Photo-immunotherapy minimizes damage to surrounding healthy tissue when using a cancer-selective photo-immunoconjugate, but requires a threshold intracellular photosensitizer concentration to be effective. Delivery of immunoconjugates to the target cells is often hindered by I) the low photosensitizer-to-antibody ratio of photo-immunoconjugates and II) the limited amount of target molecule presented on the cell surface. Here, a nanoengineering approach is introduced to overcome these obstacles and improve the effectiveness of photo-immunotherapy and imaging. Click chemistry coupling of benzoporphyrin derivative (BPD)-Cetuximab photo-immunoconjugates onto FKR560 dye-containing poly(lactic-co-glycolic acid) nanoparticles markedly enhances intracellular photo-immunoconjugate accumulation and potentiates light-activated photo-immunotoxicity in ovarian cancer and glioblastoma. It is further demonstrated that co-delivery and light activation of BPD and FKR560 allow longitudinal fluorescence tracking of photoimmunoconjugate and nanoparticle in cells. Using xenograft mouse models of epithelial ovarian cancer, intravenous injection of photo-immunoconjugated nanoparticles doubles intratumoral accumulation of photo-immunoconjugates, resulting in an enhanced photoimmunotherapy-mediated tumor volume reduction, compared to "standard" immunoconjugates. This generalizable "carrier effect" phenomenon is attributed to the successful incorporation of photo-immunoconjugates onto a nanoplatform, which modulates immunoconjugate delivery and improves treatment outcomes.

Integration of virtual screening and computational simulation identifies photodynamic therapeutics against human Protoporphyrinogen Oxidase IX (hPPO)

Photodynamic therapy (PDT) is a rapidly evolving area of cancer management against solid tumors. PDT is either administrated by injecting photosensitizer (porphyrins) or by accumulation of intracellular protoporphyrin IX via the inhibition of human Protoporphyrinogen Oxidase IX (hPPO). In this study, novel inhibitors of hPPO have been investigated by integrating virtual screening, molecular docking, and molecular dynamics (MD) simulation. A ligand-based pharmacophore was generated from a training set of 22 inhibitors of hPPO. The selected pharmacophore had four chemical features including three hydrogen bond acceptors and one hydrophobic. The pharmacophore was characterized by highest correlation coefficient of 0.96, cost difference of 53.20, and lowest root mean square deviation of 0.73. The resultant pharmacophore was validated by Fischer’s Randomization and Test Set Validation methods. The validated pharmacophore was used as a 3D query to screen chemical databases including NCI, Asinex, Chembridge, and Maybridge. The screening of chemical databases and the subsequent application of Lipinski’s Rule of Five, and ADMET Assessment Test, retrieved 1176 drug-like compounds. The drug-like compounds were subjected to molecular docking studies in the active site of hPPO to eliminate false positive hits and to elucidate their true binding orientation. Top three candidate molecules with high docking scores and hydrogen bond interactions with catalytic active residues were selected as best candidate inhibitors against hPPO. The binding stability of selected candidate inhibitors was evaluated by MD simulation. The MD simulation of hits portrayed strong hydrogen bonds and key hydrophobic interactions with catalytic active residues of hPPO including R59, R97, G159, G332 and flavin moiety of FAD (coenzyme of hPPO). Our study predicts three hit compounds against hPPO, which could possibly accumulate high concentration of protoporphyrinogen-IX, and thereby acting as an intracellular photosensitizer against tumor cells through photodynamic therapy.

Dormant cancer cells accumulate high protoporphyrin IX levels and are sensitive to 5-aminolevulinic acid-based photodynamic therapy.

Photodynamic therapy (PDT) and diagnosis (PDD) using 5-aminolevulinic acid (ALA) to drive the production of an intracellular photosensitizer, protoporphyrin IX (PpIX), are in common clinical use. However, the tendency to accumulate PpIX is not well understood. Patients with cancer can develop recurrent metastatic disease with latency periods. This pause can be explained by cancer dormancy. Here we created uniformly sized PC-3 prostate cancer spheroids using a 3D culture plate (EZSPHERE). We demonstrated that cancer cells exhibited dormancy in a cell density-dependent manner not only in spheroids but also in 2D culture. Dormant cancer cells accumulated high PpIX levels and were sensitive to ALA-PDT. In dormant cancer cells, transporter expressions of PEPT1, ALA importer, and ABCB6, an intermediate porphyrin transporter, were upregulated and that of ABCG2, a PpIX exporter, was downregulated. PpIX accumulation and ALA-PDT cytotoxicity were enhanced by G0/G1-phase arrestors in non-dormant cancer cells. Our results demonstrate that ALA-PDT would be an effective approach for dormant cancer cells and can be enhanced by combining with a cell-growth inhibitor.

Tat/HA2 Peptides Conjugated AuNR@pNIPAAm as a Photosensitizer Carrier for Near Infrared Triggered Photodynamic Therapy.

To achieve an efficiency of intracellular photosensitizers (PSs) delivery and efficacy of photodynamic therapy, we have developed a novel class of PS formulation for encapsulating sulfonated aluminum phthalocyanine (AlPcS4) by taking advantage of the membrane-disruptive peptides Tat/HA2 and the photothermally triggered delivery system using AuNR@pNIPAAm. The coordinated effects of cell penetrating peptide Tat and fusogenic peptide HA2 could enhance the efficient cellular internalization and endo/lysosome escape of PSs delivery systems. Singlet oxygen generation was inhibited due to the reaction between loaded AlPcS4 and Au nanorods, which indicated that the AlPcS4-loaded, AuNR@pNIPAAm delivery system might be nonphototoxic in the circulatory system. However, this PSs-loaded nanosystem became highly phototoxic as it underwent the near-infrared irradiation by using the combined lights of 808 and 680 nm. Upon irradiation, the Tat/HA2 conjugated AuNR@pNIPAAm-Pc elicited an active photodynamic response against the cancer cells, leading to effective cells killing via mitochondria-associated apoptotic pathway. This study also demonstrated improved PDT therapeutic efficacy after intravenous administration of Tat/HA2-AuNR@pNIPAAm-Pc and the subsequent lights irradiations in tumor-bearing mice. We describe here a strategy for enhanced photodynamic eradication of solid tumors by endo/lysosomal escape and highlight the great promise of peptide-based nanocarriers used for cancer therapy.

Suppression of a Specific Intracellular Uptake Pathway by a Saturating Accumulation of Quantum Dots.

Quantum dots (QDs) play an active role in triggering biological effects and should not be viewed as ordinary carriers for biomedical applications; therefore, the aim of this study was to investigate the molecular mechanisms involved in the saturating accumulation of non-targeted, carboxylated QDs, the related specific internalization pathways and the induced changes in the endocytotic cycle in NIH3T3 cells. We determined that the saturating accumulation of QDs suppressed the internalization of subsequently introduced QDs that had an identical chemical composition. However, the reinitiation of uptake was detected in the NIH3T3 cells after 8 h of incubation in medium without QDs. A very small suppressive effect of accumulated QDs was observed on uptake via the clathrin-mediated endocytosis pathway and macropinocytosis. In contrast, uptake via the caveolin-mediated pathway was almost completely prevented. Deeper insight into the suppression mechanism was obtained by transiently transfecting NIH3T3 cells with the plasmid pEGFP-C1-Caveolin-1. In these transfected cells, the usual intracellular presence of Caveolin-1 near the plasma membrane was not observed after long-term incubation with QDs. The putative application of QDs for diagnostic visualization in combination with certain anticancer substances was also evaluated. The QDs did not affect the intracellular photosensitization pattern of the amphiphilic molecule chlorin e6 in the case of photodynamic therapy. However, the saturating accumulation of QDs increased the resistance of NIH3T3 cells to the widely used anticancer drug cisplatin.

Effect of dye localization and self-interactions on the photosensitized generation of singlet oxygen by rose bengal bound to bovine serum albumin

The spectroscopic and photophysical properties of rose bengal (RB) encased in bovine serum albumin (BSA) have been examined to evaluate the photosensitized generation of singlet molecular oxygen (1O2). The results show that RB photophysical and photosensitizing properties are highly modulated by the average number of dye molecules per protein (n). At n≪1, the dye molecule is tightly located into the hydrophobic nanocavity site I of the BSA molecule with a binding constant Kb=0.15±0.01μM−1. The interaction with surrounding amino acids induces heterogeneous decay of both singlet and triplet excited states of RB and partially reduce its triplet quantum yield as compared with that in buffer solution. However, despite of the diffusive barrier imposed by the protein nanocavity to 3O2, the quenching of 3RB∗:BSA generates 1O2 with quantum yield ΦΔ=0.35±0.05. In turns, the intraprotein generated 1O2 is able to diffuse through the bulk solution, where is dynamically quenched by BSA itself with an overall quenching rate constant of 7.3×108M−1s−1. However, at n>1, nonspecific binding of up to ≈6RB molecules per BSA is produced, allowing efficient static quenching of excited states of RB preventing photosensitization of 1O2. These results provide useful information for development of dye-protein adducts suitable for using as potential intracellular photosensitizers.

Far-red fluorescence probe for monitoring singlet oxygen during photodynamic therapy.

Singlet oxygen ((1)O2), molecular oxygen in the lowest excited state, has a critical role in the cell-killing mechanism of photodynamic therapy (PDT). Although (1)O2 phosphorescence measurement has been mainly used to monitor (1)O2 formation during PDT, its intensity is far insufficient to obtain two-dimensional images of intracellular (1)O2 with the subcellular spatial resolution using the currently available near-IR detector. Here, we propose a new far-red fluorescence probe of (1)O2, namely, Si-DMA, composed of silicon-containing rhodamine and anthracene moieties as a chromophore and a (1)O2 reactive site, respectively. In the presence of (1)O2, fluorescence of Si-DMA increases 17 times due to endoperoxide formation at the anthracene moiety. With the advantage of negligible self-oxidation by photoirradiation (ΦΔ < 0.02) and selective mitochondrial localization, Si-DMA is particularly suitable for imaging (1)O2 during PDT. Among three different intracellular photosensitizers (Sens), Si-DMA could selectively detect the (1)O2 that is generated by 5-aminolevulinic acid-derived protoporphyrin IX, colocalized with Si-DMA in mitochondria. On the other hand, mitochondria-targeted KillerRed and lysosomal porphyrins could not induce fluorescence change of Si-DMA. This surprising selectivity of Si-DMA response depending on the Sens localization and photosensitization mechanism is caused by a limited intracellular (1)O2 diffusion distance (∼300 nm) and negligible generation of (1)O2 by type-I Sens, respectively. For the first time, we successfully visualized (1)O2 generated during PDT with a spatial resolution of a single mitochondrial tubule.

Repetitive methylene blue-mediated photoantimicrobial chemotherapy changes the susceptibility and expression of the outer membrane proteins of Pseudomonas aeruginosa

Since bacterial multidrug efflux pumps mediate intracellular photosensitizer methylene blue, a change in the expression alters the susceptibility to photoantimicrobial chemotherapy (PACT) of Pseudomonas aeruginosa, which may occur following repetitive sublethal challenges. We performed 10 consecutive, methylene blue-mediated PACT on one antibiotic-sensitive strain and three antibiotic-resistant strains of P. aeruginosa. Following each therapy, the surviving bacteria were collected for subsequent PACT. The susceptibility was compared for the pre- and the post-treated strains following repetitive PACT. To explore the existence of efflux pumps, one of the inhibitors, namely Phe-Arg β-naphthylamide dihydrochloride (PAβN 25 μg/ml), was added. Profiles of outer membrane proteins were obtained for the pre-treated and the post-treated strains. The susceptibility of PACT did not correlate with the antibiotic sensitivity. Following ten PACT, there was no significant change in susceptibility for three tested strains, except for one antibiotic-resistant strain, for which the 10th generation became less susceptible than the original one. With 2-D electrophoresis, a change in the expression of outer membrane proteins was observed. PAβN successfully increased the phototoxicity in all tested strains, especially the less PACT-susceptible 10th generation of the antibiotic-resistant strain. Following repetitive challenges, PACT had a consistent antimicrobial effect on three strains; however, one antibiotic-resistant strain, which was the most vulnerable to PACT, became more resistant after consecutive challenges. In addition, the post-PACT strain had different expression of outer membrane proteins, providing further evidence view that repetitive PACT with methylene blue could change the expression of efflux pumps.