Far-red light-mediated programmable anti-cancer gene delivery in cooperation with photodynamic therapy.

Effective anti-cancer therapy is hurdled by the complicated extracellular and intracellular barriers, and thus a smart gene vector that can enable programmable gene delivery is highly demanded. Photo-manipulation of gene delivery processes features spatial and temporal precision, while majority of current strategies utilizes short-wavelength UV/visible light with poor tissue penetration or high-power-density near-infrared (NIR) light that would cause undesired heat damage. Herein, an ROS-degradable polycation was designed and co-delivered with a photosensitizer (PS), thus realizing photo-programmable gene delivery using far-red light (661 nm) at low optical power density (down to 5 mW cm-2). Thioketal-crosslinked polyethylenimine (TK-PEI) was synthesized to condense p53 gene to form nanocomplexes (NCs), and hyaluronic acid (HA) modified with pheophytin a (Pha) was coated onto NCs to enhance their colloidal stability and enable cancer cell targeting. Short-time (8-min) light irradiation produced non-lethal amount of ROS to disrupt the endosomal membranes and facilitate p53 gene release via degradation of TK-PEI, which collectively enhanced p53 expression levels toward anti-cancer gene therapy. Long-time (30-min) light irradiation at the post-transfection state generated lethal amount of ROS, which cooperatively killed cancer cells to strengthen p53 gene therapy. To the best of our knowledge, this study represents the first example of an "one stone, three birds" approach to realize cooperative anti-cancer gene therapy using low-power-density, long-wavelength visible light as a single stimulus.

Cathepsin B-degradable, NIR-responsive nanoparticulate platform for target-specific cancer therapy.

Stimuli-responsive anticancer formulations can promote drug release and activation within the target tumour, facilitate cellular uptake, as well as improve the therapeutic efficacy of drugs and reduce off-target effects. In the present work, indocyanine green (ICG)-containing polyglutamate (PGA) nanoparticles were developed and characterized. Digestion of nanoparticles with cathepsin B, a matrix metalloproteinase overexpressed in the microenvironment of advanced tumours, decreased particle size and increased ICG cellular uptake. Incorporation of ICG in PGA nanoparticles provided the NIR-absorbing agent with time-dependent altered optical properties in the presence of cathepsin B. Having minimal dark toxicity, the formulation exhibited significant cytotoxicity upon NIR exposure. Combined use of the formulation with saporin, a ribosome-inactivating protein, resulted in synergistically enhanced cytotoxicity attributed to the photo-induced release of saporin from endo/lysosomes. The results suggest that this therapeutic approach can offer significant therapeutic benefit in the treatment of superficial malignancies, such as head and neck tumours.

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.

A Near-Infrared Laser-Activated "Nanobomb" for Breaking the Barriers to MicroRNA Delivery.

A near-infrared laser-activated "nanobomb" is synthesized using lipid and multiple polymers to break the extra-cellular and intracellular barriers to cytosolic delivery of microRNAs. The nanobomb can be used to effectively destroy tumors and cancer stem-like cells in vitro and in vivo with minimal side effects.

Photomediated Reactive Oxygen Species-Generable Nanoparticles for Triggered Release and Endo/Lysosomal Escape of Drug upon Attenuated Single Light Irradiation.

Nanoparticles with "smart" stimuli-responsive materials and multiple therapeutic strategies in a single delivery platform have emerged for highly efficient cancer therapy. Here, photomediated reactive oxygen species (ROS)-generable nanoparticles are designed that can trigger drug release and endo/lysosomal escape upon attenuated single light irradiation, simultaneously, for synergistic chemo-photodynamic ablation. In this study, the self-ROS-generable nanoparticles (SRNs) are prepared from the polymer based on polysaccharide, chlorin e6 as ROS generator and lipoic acid as ROS scavenger covalently conjugated pullulan with anticancer drug (doxorubicin, DOX) through self-assembly, and can disassemble via the ROS-mediated reduction of lipoyl group in response to low level exogenous single light switch. After cellular internalization in hepatic cancer through asialoglycoprotein receptor (ASGPR, as pullulan receptor)-mediated endocytosis, once irradiated, SRNs are able to produce ROS that can simultaneously induce drug release triggering and endo/lysosomal escape of DOX into cytoplasm as well as directly photodynamic therapy for highly efficient chemo-photodynamic cancer therapy. This promising delivery system, which has huge potential in biomedical applications, may be optimal for smart delivery platform.

Photochemical internalisation, a minimally invasive strategy for light-controlled endosomal escape of cancer stem cell-targeting therapeutics.

Despite progress in radio-, chemo- and photodynamic-therapy (PDT) of cancer, treatment resistance still remains a major problem for patients with aggressive tumours. Cancer stem cells (CSCs) or tumour-initiating cells are intrinsically and notoriously resistant to conventional cancer therapies and are proposed to be responsible for the recurrence of tumours after therapy. According to the CSC hypothesis, it is imperative to develop novel anticancer agents or therapeutic strategies that take into account the biology and role of CSCs. The present review outlines our recent study on photochemical internalisation (PCI) using the clinically relevant photosensitiser TPCS2a/Amphinex® as a rational, non-invasive strategy for the light-controlled endosomal escape of CSC-targeting drugs. PCI is an intracellular drug delivery method based on light-induced ROS-generation and a subsequent membrane-disruption of endocytic vesicles, leading to cytosolic release of the entrapped drugs of interest. In different proof-of-concept studies we have demonstrated that PCI of CSC-directed immunotoxins targeting CD133, CD44, CSPG4 and EpCAM is a highly specific and effective strategy for killing cancer cells and CSCs. CSCs overexpressing CD133 are PDT-resistant; however, this is circumvented by PCI of CD133-targeting immunotoxins. In view of the fact that TPCS2a is not a substrate of the efflux pumps ABCG2 and P-glycoprotein (ABCB1), the PCI-method is a promising anti-CSC therapeutic strategy. Due to a laser-controlled exposure, PCI of CSC-targeting drugs will be confined exclusively to the tumour tissue, suggesting that this drug delivery method has the potential to spare distant normal stem cells.

Optogenetic control of organelle transport and positioning.

Proper positioning of organelles by cytoskeleton-based motor proteins underlies cellular events such as signalling, polarization and growth. For many organelles, however, the precise connection between position and function has remained unclear, because strategies to control intracellular organelle positioning with spatiotemporal precision are lacking. Here we establish optical control of intracellular transport by using light-sensitive heterodimerization to recruit specific cytoskeletal motor proteins (kinesin, dynein or myosin) to selected cargoes. We demonstrate that the motility of peroxisomes, recycling endosomes and mitochondria can be locally and repeatedly induced or stopped, allowing rapid organelle repositioning. We applied this approach in primary rat hippocampal neurons to test how local positioning of recycling endosomes contributes to axon outgrowth and found that dynein-driven removal of endosomes from axonal growth cones reversibly suppressed axon growth, whereas kinesin-driven endosome enrichment enhanced growth. Our strategy for optogenetic control of organelle positioning will be widely applicable to explore site-specific organelle functions in different model systems.

APPL proteins modulate DNA repair and radiation survival of pancreatic carcinoma cells by regulating ATM.

Despite intensive multimodal therapies, the overall survival rate of patients with ductal adenocarcinoma of the pancreas is still poor. The chemo- and radioresistance mechanisms of this tumor entity remain to be determined in order to develop novel treatment strategies. In cancer, endocytosis and membrane trafficking proteins are known to be utilized and they also critically regulate essential cell functions like survival and proliferation. On the basis of these data, we evaluated the role of the endosomal proteins adaptor proteins containing pleckstrin homology domain, phosphotyrosine binding domain and a leucine zipper motif (APPL)1 and 2 for the radioresistance of pancreatic carcinoma cells. Here, we show that APPL2 expression in pancreatic cancer cells is upregulated after irradiation and that depletion of APPL proteins by small interfering RNA (siRNA) significantly reduced radiation survival in parallel to impairing DNA double strand break (DSB) repair. In addition, APPL knockdown diminished radiogenic hyperphosphorylation of ataxia telangiectasia mutated (ATM). Activated ATM and APPL1 were also shown to interact after irradiation, suggesting that APPL has a more direct role in the phosphorylation of ATM. Double targeting of APPL proteins and ATM caused similar radiosensitization and concomitant DSB repair perturbation to that observed after depletion of single proteins, indicating that ATM is the central modulator of APPL-mediated effects on radiosensitivity and DNA repair. These data strongly suggest that endosomal APPL proteins contribute to the DNA damage response. Whether targeting of APPL proteins is beneficial for the survival of patients with pancreatic adenocarcinoma remains to be elucidated.

Photocleavable dimerizer for the rapid reversal of molecular trap antagonists.

Herein, we report the development of a photocleavable analog of AP20187, a cell-permeable molecule used to dimerize FK506-binding protein (FKBP) fusion proteins and initiate biological signaling cascades and gene expression or disrupt protein-protein interactions. We demonstrate that this reagent permits the unique ability to rapidly and specifically antagonize a molecular interaction in vitro and follow a biological process due to this acute antagonism (e.g. endosome dispersion) and to release the trap upon photocleavage to follow the cell's return to homeostasis. In addition, this photocleavable AP20187 analog can be used in other systems where the dimerization of FKBP has been used to initiate signaling pathways, offering the ability to correlate the duration of a signaling event and a cellular response.

Photochemical targeting of antigens to the cytosol for stimulation of MHC class-I-restricted T-cell responses.

Tumour chemotherapy with drugs is typically associated with severe systemic and local side effects for which reason immunotherapy represents a safer alternative. However, vaccination often fails to generate the required cytotoxic CD8 T-cell responses due to insufficient access of antigens to the cytosol and the MHC class I pathway of antigen presentation. One important issue of tumour research is therefore to develop strategies that allow cytosolic targeting or endosomal escape of tumour antigens. The objective of the current study was to test whether endocytosed antigen could be delivered to MHC class I by means of photochemical internalisation (PCI). Briefly, the antigen and the photosensitiser Amphinex were loaded in vitro onto bone-marrow-derived murine dendritic cells (DCs). After light activation, which is supposed to cause disruption of OVA- and Amphinex-containing endosomes, the DCs were cultured with OVA-specific CD8 T cells or used for immunisation of mice. PCI facilitated CD8 T-cell responses as measured by IFN-γ secretion in vitro and CD8 T-cell proliferation in vivo. In conclusion, the current proof-of-concept study is the first to describe PCI-mediated immunisation and the results revealed the feasibility of this novel technology in autologous vaccination for stimulation of CD8 T-cell responses.

A point mutation in Semaphorin 4A associates with defective endosomal sorting and causes retinal degeneration.

Semaphorin 4A (Sema4A) has an essential role in photoreceptor survival. In humans, mutations in Sema4A are thought to contribute to retinal degenerative diseases. Here we generate a series of knock-in mouse lines with corresponding mutations (D345H, F350C or R713Q) in the Sema4A gene and find that Sema4A(F350C) causes retinal degeneration phenotypes. The F350C mutation results in abnormal localization of the Sema4A protein, leading to impaired endosomal sorting of molecules indispensable for photoreceptor survival. Additionally, protein structural modelling reveals that the side chain of the 350th amino acid is critical to retain the proper protein conformation. Furthermore, Sema4A gene transfer successfully prevents photoreceptor degeneration in Sema4A(F350C/F350C) and Sema4A(-/-) mice. Thus, our findings not only indicate the importance of the Sema4A protein conformation in human and mouse retina homeostasis but also identify a novel therapeutic target for retinal degenerative diseases.

Conjugation to the cell-penetrating peptide TAT potentiates the photodynamic effect of carboxytetramethylrhodamine.

BACKGROUND: Cell-penetrating peptides (CPPs) can transport macromolecular cargos into live cells. However, the cellular delivery efficiency of these reagents is often suboptimal because CPP-cargo conjugates typically remain trapped inside endosomes. Interestingly, irradiation of fluorescently labeled CPPs with light increases the release of the peptide and its cargos into the cytosol. However, the mechanism of this phenomenon is not clear. Here we investigate the molecular basis of the photo-induced endosomolytic activity of the prototypical CPPs TAT labeled to the fluorophore 5(6)-carboxytetramethylrhodamine (TMR). METHODOLOGY/PRINCIPAL FINDINGS: We report that TMR-TAT acts as a photosensitizer that can destroy membranes. TMR-TAT escapes from endosomes after exposure to moderate light doses. However, this is also accompanied by loss of plasma membrane integrity, membrane blebbing, and cell-death. In addition, the peptide causes the destruction of cells when applied extracellularly and also triggers the photohemolysis of red blood cells. These photolytic and photocytotoxic effects were inhibited by hydrophobic singlet oxygen quenchers but not by hydrophilic quenchers. CONCLUSIONS/SIGNIFICANCE: Together, these results suggest that TAT can convert an innocuous fluorophore such as TMR into a potent photolytic agent. This effect involves the targeting of the fluorophore to cellular membranes and the production of singlet oxygen within the hydrophobic environment of the membranes. Our findings may be relevant for the design of reagents with photo-induced endosomolytic activity. The photocytotoxicity exhibited by TMR-TAT also suggests that CPP-chromophore conjugates could aid the development of novel Photodynamic Therapy agents.

Effects of endosomal photodamage on membrane recycling and endocytosis.

The flux of receptor-independent endocytosis can be estimated by addition of wortmannin to cell cultures. Membrane influx is unaffected but traffic out of late endosomes is impaired, resulting in a substantial enlargement of these organelles. Using the 1c1c7 murine hepatoma, we investigated the effect of endosomal photodamage on this endocytic pathway. We previously reported that photodamage catalyzed by the lysosomal photosensitizer NPe6 prevented wortmannin-induced endosomal swelling, indicating an earlier block in the process. In this study, we show that endosomal photodamage, initiated by photodamage from an asymmetrically substituted porphine or a phthalocyanine also prevents wortmannin-induced endosomal swelling, even when the photodynamic therapy (PDT) dose is insufficient to cause endosomal disruption. As the PDT dose is increased, endosomal breakage occurs, as does apoptosis and cell death. Very high PDT doses result in necrosis. We propose that photodamage to endosomes results in alterations in the endosomal structure such that influx of new material is inhibited and receptor-independent endocytosis is prevented. In an additional series of studies, we found that the swollen late endosomes induced by wortmannin are unable to retain previously accumulated fluorescent probes or photosensitizers.

Effects of photodynamic therapy on the endocytic pathway.

In this report, we describe an effect of photodynamic therapy (PDT) on membrane trafficking in murine 1c1c7 hepatoma cells. A brief exposure of 1c1c7 cells to a 20 nM concentration of the phosphatidylinositol kinase class-3 antagonist wortmannin led to the rapid appearance of cytoplasmic vacuoles. Fluorescence monitoring of plasma membrane-associated 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TDPH) over time demonstrated that the wortmannin-induced vacuoles were derived from endocytosed plasma membrane. Low-dose photodamage catalyzed by the lysosomal photosensitizer NPe6, prior to the addition of wortmannin, prevented formation of these vacuoles. NPe6 was found to suppress for several hours the normal trafficking of TDPH-labeled plasma membrane to the cytosol, and the formation of punctate TDPH-labeled cytoplasmic vesicles. The ability of NPe6-induced photodamage to suppress wortmannin-induced vacuolization occurred under conditions that did not disrupt lysosomes and were at or below the threshold of cytostatic/cytotoxic effects. Furthermore, the suppressive effects of NPe6-PDT were not prevented by inclusion of an agent that stabilized lysosomal membranes, or by E64d, an inhibitor of lysosomal cathepsin proteases. Mitochondrial photodamage was less effective at preventing wortmannin-induced vacuole formation and PDT directed against the ER had no effect. The role of photodamage to the endocytic pathway may be a hitherto unexplored effect on cells that selectively accumulate photosensitizing agents. These results indicate that photodamage directed against endosomes/lysosomes has effects independent of the release of lysosomal proteases.

Photochemical activation of endosomal escape of MRI-Gd-agents in tumor cells.

Endocytosis is a common internalization pathway for cellular labeling with MRI contrast agents. However, the entrapment of the Gd(III) complexes into endosomes results in a "quenching" of the attainable relaxivity when the number of Gd(III) complexes reaches the number of ca. 1 × 10(9)/cell. Herein we show that the use of the newly developed photochemical internalization technique provides an efficient method for attaining the endosomal escape of GdHPDO3A molecules entrapped by pinocytosis into different kind of cells. Furthermore, it has been found that a new "quenching" limit is observed when the number of Gd-HPDO3A complexes is ca. five times higher than the value observed for the endosome entrapped conditions. The observed behavior is explained in terms of the attainment of the conditions in which the difference in proton relaxation rates between the cytoplasmic and the extracellular compartment is higher than the exchange rate of water molecules across the cellular membrane. The experimental data points have been reproduced by using a properly designed theoretical compartment T(1)-relaxation model.

Nonesterified cholesterol content of lysosomes modulates susceptibility to oxidant-induced permeabilization.

Reactive oxygen species (ROS) can induce lysosomal membrane permeabilization (LMP). Photoirradiation of murine hepatoma 1c1c7 cultures preloaded with the photosensitizer NPe6 generates singlet oxygen within acidic organelles and causes LMP and the activation of procaspases. Treatment with the cationic amphiphilic drugs (CADs) U18666A, imipramine, and clozapine stimulated the accumulation of filipin-stainable nonesterified cholesterol/sterols in late endosomes/lysosomes, but not in mitochondria. Concentration-response studies demonstrated an inverse relationship between lysosomal nonesterified cholesterol/sterol contents and susceptibility to NPe6 photoirradiation-induced intracellular membrane oxidation, LMP, and activation of procaspase-9 and -3. Similarly, the kinetics of restoration of NPe6 photoirradiation-induced LMP paralleled the losses of lysosomal cholesterol that occurred upon replating U18666A-treated cultures in CAD-free medium. Consistent with the oxidation of lysosomal cholesterol, filipin staining in U18666A-treated cultures progressively decreased with increasing photoirradiating light dose. U18666A also suppressed the induction of LMP and procaspase activation by exogenously added hydrogen peroxide. However, neither U18666A nor imipramine suppressed the induction of apoptosis by agents that did not directly induce LMP. These studies indicate that lysosomal nonesterified cholesterol/sterol content modulates susceptibility to ROS-induced LMP and possibly does so by being an alternative target for oxidants and lowering the probability of damage to other lysosomal membrane lipids and/or proteins.

Senescence-associated exosome release from human prostate cancer cells.

Males of advanced age represent a rapidly growing population at risk for prostate cancer. In the contemporary setting of earlier detection, a majority of prostate carcinomas are still clinically localized and often treated using radiation therapy. Our recent studies have shown that premature cellular senescence, rather than apoptosis, accounts for most of the clonogenic death induced by clinically relevant doses of irradiation in prostate cancer cells. We show here that this treatment-induced senescence was associated with a significantly increased release of exosome-like microvesicles. In premature senescence, this novel secretory phenotype was dependent on the activation of p53. In addition, the release of exosome-like microvesicles also increased during proliferative senescence in normal human diploid fibroblasts. These data support the hypothesis that senescence, initiated either by telomere attrition (e.g., aging) or DNA damage (e.g., radiotherapy), may induce a p53-dependent increase in the biogenesis of exosome-like vesicles. Ultrastructural analysis and RNA interference-mediated knockdown of Tsg101 provided significant evidence that the additional exosomes released by prematurely senescent prostate cancer cells were principally derived from multivesicular endosomes. Moreover, these exosomes were enriched in B7-H3 protein, a recently identified diagnostic marker for prostate cancer, and an abundance of what has recently been termed "exosomal shuttle RNA." Our findings are consistent with the proposal that exosomes can transfer cargos, with both immunoregulatory potential and genetic information, between cells through a novel mechanism that may be recruited to increase exosome release during accelerated and replicative cellular senescence.

Dynamics of photoinduced endosomal release of polyplexes.

Endosomal escape is a well-known bottleneck for successful delivery of macromolecular drugs and genes. Photochemical disruption of endosomal membranes is an approach to overcome this bottleneck. In this study, we used the photosensitizer disulphonated meso-tetraphenylporphine with sulfonate groups on adjacent phenyl rings (TPPS(2a)) to investigate photoinduced endosomal release in living cells with high resolution fluorescence wide-field microscopy in real time. We studied the release dynamics of 10 kDa dextran and polyplexes consisting of DNA condensed with the cationic polymers linear polyethyleneimine (LPEI), poly-(L)-lysine (PLL) or poly-(D)-lysine (PDL). By means of dual-color microscopy and the use of double-labeled polyplexes DNA and polymer were imaged simultaneously. We show that the characteristics of the cationic polymer significantly influence the release behavior of the polyplexes. The release of dextran occurred within 100 ms. For LPEI/DNA particles, LPEI quickly spread throughout the cytosol similar to dextran, whereas DNA was released slowly (within 4 s) and remained immobile thereafter. In case of PLL particles, both DNA and polymer showed quick release. PDL particles remained condensed upon photosensitizer activation. In addition, we demonstrate that TPPS(2a) has biological side effects. Besides stop of microtubule dynamics in the dark, the movement of endosomes ceased after photosensitizer activation.

Cellular siRNA delivery mediated by a cell-permeant RNA-binding protein and photoinduced RNA interference.

HIV-1 TAT peptide, which is a cell-penetrating peptide (CPP), was fused to the U1A RNA-binding domain (TatU1A) to generate a sequence-specific siRNA delivery system for mammalian cells. The siRNA contained a short 5'-extension that is specifically recognized by the U1A RNA-binding domain (U1AsiRNA). Specific binding of TatU1A to the U1AsiRNA was confirmed using a gel mobility shift assay. The U1AsiRNA was internalized by cells only when it was preincubated with TatU1A before addition to the cells. Although most of the internalized siRNA seemed to be entrapped in endocytic compartments, efficient redistribution of the entrapped siRNAs was achieved by photostimulation of a fluorophore attached to TatU1A. Once in the cytoplasm, the siRNA induced RNAi-mediated gene silencing. We refer to this delivery strategy as CLIP-RNAi. CLIP-RNAi is a promising strategy for RNAi experiments and for pinpoint RNAi therapy.

Light-directed gene delivery by photochemical internalisation.

This article reviews a novel technology, named photochemical internalisation (PCI), for light-directed delivery of transgenes. Most gene therapy vectors are taken into the cell by endocytosis and, hence, are located in the endocytic vesicles. Although viral vectors have developed the means to escape from these vesicles, poor endosomal release is one of the major obstacles for non-viral vectors. PCI is a technology that allows liberation of the entrapped vectors carrying a gene in response to illumination. The method is based on chemical compounds (photosensitisers) that localise specifically in the membranes of endocytic vesicles and, following activation by light, induce the rupture of the vesicular membranes. The released transgenes can further be transferred to the nucleus, transcribed and translated. As gene liberation depends on light, enhancement of gene expression is achieved only at illuminated regions. PCI substantially improves gene transfer in vitro not only with non-viral gene vectors, but, surprisingly, also with adenoviruses and adeno-associated viruses. This article will review the background for the PCI technology and its role for gene delivery using both non-viral and viral vectors. Some aspects of the potential of PCI for site-specific gene delivery in therapeutic situations will also be discussed.