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Targeted intracellular delivery of photosensitizers to enhance photodynamic efficiency
Abstract
Photodynamic therapy (PDT) is a novel treatment, used mainly for anticancer therapy, that depends on the retention of photosensitizers (PS) in tumour cells and irradiation of the tumour with appropriate wavelength light. Photosensitizers are molecules such as porphyrins and chlorins that, on photoactivation, effect strongly localized oxidative damage within target cells. The PS used for PDT localize in various cytoplasmic membranous structures, but are not found in the most vulnerable intracellular sites for reactive oxygen species, such as the cell nucleus. The experimental approaches discussed in the present paper indicate that it is possible to design highly efficient molecular constructs, PS carriers, with specific modules conferring cell-specific targeting, internalization, escape from intracellular vesicles and targeting to the most vulnerable intracellular compartments, such as the nucleus. Nuclear targeting of these PS-carrying constructs results in enhanced photodynamic activity, maximally about 2500-fold that of free PS. Future work is intended to optimize this approach to the point at which tumour cells can be killed rapidly and efficiently, while minimizing normal cell and tissue damage.
... The idea of using the combination of several amino acid sequences and transport modules for the delivery of a drug into the nuclei of target cells was originated by A.S. Sobolev and first published in 2000 [148]. The development of this approach has shown that the delivery of locally acting cytotoxic agents such as photosensitizers [149,150], α-emitters [151] and Auger electron emitters [152][153][154] greatly increases their effectiveness in destroying cancer target cells. ...
... In general, this is a non-trivial task; however, the already created molecules have demonstrated that its solution is quite possible (Figure 4). The idea of using the combination of several amino acid sequences and transport modules for the delivery of a drug into the nuclei of target cells was originated by A.S. Sobolev and first published in 2000 [148]. The development of this approach has shown that the delivery of locally acting cytotoxic agents such as photosensitizers [149,150], α-emitters [151] and Auger electron emitters [152][153][154] greatly increases their effectiveness in destroying cancer target cells. ...
A large number of proteins are successfully used to treat various diseases. These include natural polypeptide hormones, their synthetic analogues, antibodies, antibody mimetics, enzymes, and other drugs based on them. Many of them are demanded in clinical settings and commercially successful, mainly for cancer treatment. The targets for most of the aforementioned drugs are located at the cell surface. Meanwhile, the vast majority of therapeutic targets, which are usually regulatory macromolecules, are located inside the cell. Traditional low molecular weight drugs freely penetrate all cells, causing side effects in non-target cells. In addition, it is often difficult to elaborate a small molecule that can specifically affect protein interactions. Modern technologies make it possible to obtain proteins capable of interacting with almost any target. However, proteins, like other macromolecules, cannot, as a rule, freely penetrate into the desired cellular compartment. Recent studies allow us to design multifunctional proteins that solve these problems. This review considers the scope of application of such artificial constructs for the targeted delivery of both protein-based and traditional low molecular weight drugs, the obstacles met on the way of their transport to the specified intracellular compartment of the target cells after their systemic bloodstream administration, and the means to overcome those difficulties.
... When the liposome enters the spores, it releases the encapsulated photosensitizers. In this way, the carrier system protects the encapsulated photosensitizers from enzymes in the honey bee's body and from any substances that would reduce photodynamic activation (Rosenkranz et al., 2000). Phthalocyanine, which easily crosses the spore coat, can then disrupt the structural integrity of the spores, leading to deformations and, ultimately, the destruction of the entire spore. ...
... Higher localization of the photosensitizer in the bacterial cells, leads to improved therapeutic efficacy at a reduced dose (Lee and Kopelman 2011). Initially, researchers have attempted to develop new photosensitizers by changing the physicochemical properties such as logP value to improve their specificity (Rosenkranz et al., 2000). Hence, second-generation photosensitizers had a better efficacy compared to first-generation photosensitizers. ...
Resistance to antimicrobial drugs is an impending healthcare problem of growing significance. In the post-antibiotic era, there is a huge push to develop new tools for effectively treating bacterial infections. Photodynamic therapy involves the use of a photosensitizer that is activated by the use of light of an appropriate wavelength in the presence of oxygen. This results in the generation of singlet oxygen molecules that can kill the target cells, including cancerous cells and microbial cells. Photodynamic therapy is shown to be effective against parasites, viruses, algae, and bacteria. To achieve high antimicrobial activity, a sufficient concentration of photosensitizer should enter the microbial cells. Generally, photosensitizers tend to aggregate in aqueous environments resulting in the weakening of photochemical activity and lowering their uptake into cells. Nanocarrier systems are shown to be efficient in targeting photosensitizers into microbial cells and improve their therapeutic efficiency by enhancing the internalization of photosensitizers into microbial cells. This review aims to highlight the basic principles of photodynamic therapy with a special emphasis on the use of nanosystems in delivering photosensitizers for improving antimicrobial photodynamic therapy.
... Thus, it is not necessary for IR700-based TSPCs to be internalized by cells, nor do they necessarily rely on reactive oxygen species for their anticancer activity [10]. In contrast, conventional PSs, which are generally hydrophobic, must be internalized by targeted cells, thus, hindering their target selectivity and antitumor efficacy [1,11]. Third, IR700 molecules can be modified with an N-hydroxysuccinimide ester (NHS) group, which facilitates conjugation with antibodies or other tumor-homing species. ...
Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer treatment modality based on a target-specific photosensitizer conjugate (TSPC) composed of an NIR phthalocyanine photosensitizer and an antigen-specific recognition system. NIR-PIT has predominantly been used for targeted therapy of tumors via local irradiation with NIR light, following binding of TSPC to antigen-expressing cells. Physical stress-induced membrane damage is thought to be a major mechanism underlying NIR-PIT-triggered photokilling. Notably, NIR-PIT can rapidly induce immunogenic cell death and activate the adaptive immune response, thereby enabling its combination with immune checkpoint inhibitors. Furthermore, NIR-PIT-triggered "super-enhanced permeability and retention" effects can enhance drug delivery into tumors. Supported by its potential efficacy and safety, NIR-PIT is a rapidly developing therapeutic option for various cancers. Hence, this review seeks to provide an update on the (i) broad range of target molecules suitable for NIR-PIT, (ii) various types of receptor-selective ligands for designing the TSPC "magic bullet," (iii) NIR light parameters, and (iv) strategies for enhancing the efficacy of NIR-PIT. Moreover, we review the potential application of NIR-PIT, including the specific design and efficacy in 19 different cancer types, and its clinical studies. Finally, we summarize possible NIR-PIT applications in noncancerous conditions, including infection, pain, itching, metabolic disease, autoimmune disease, and tissue engineering.
... The surface modification of nanoparticles with ligands greatly enhances cell uptake process due to specific interaction of ligands with cell surface receptors. However, therapeutic efficacy is still limited owing to receptor density and overexpressed receptor-ligand binding (Rosenkranz et al., 2000;Wang & Thanou, 2010). Using PS, the possibility of efficient cancer cell death is more due to diverse subcellular location of PS in lysosome, Golgi apparatus, cell membrane, mitochondrion, and nucleus (Benov, 2015). ...
Porphyrins have been explored as emerging theranostic agents in the therapeutic intervention of cancer. It has been potentially implicated in the treatment of cancer radiotherapy, photodynamic therapy, chemical sonosensitizer, fluorescence magnetic resonance, and other biomedical imaging. Due to the hydrophobic property of porphyrin, it forms insoluble aggregate in aqueous medium and is not easy to work. However, several work has been taken place and isolated new porphyrin but due to the lack of tissue selectivity, high aggregation in skin only few of them has real clinical efficacy. Despite this, encapsulating porphyrin in nanoparticles through rational design make it promising because of transformation into suitable forms such as lipophilic, immune tolerance, prolonged tissue lifetime, and improved delivery features. Additionally the various surface modifying components (viz., ligands, targeting moiety/protein/peptides, and imaging agent) could be introduced into nanoparticulate platforms for cancer theranostics. This chapter briefly acquainted the insight into current porphyrin-based nanoparticles delivery in multimodal imaging and diagnosis of tumor and simultaneous therapeutic utility in pharmaceutical and biomedical sciences.
... The surface modification of nanoparticles with ligands greatly enhances cell uptake process due to specific interaction of ligands with cell surface receptors. However, therapeutic efficacy is still limited owing to receptor density and overexpressed receptor-ligand binding (Rosenkranz et al., 2000;Wang & Thanou, 2010). Using PS, the possibility of efficient cancer cell death is more due to diverse subcellular location of PS in lysosome, Golgi apparatus, cell membrane, mitochondrion, and nucleus (Benov, 2015). ...
Nanotechnology is playing a very vital role in the medical field. Nanoparticles (NPs) are utilized in the field of nanomedicine which partakes in diagnosis and therapy of different diseases including cancer. The interesting distinguishing features of NPs, for example, are their tall exterior to volume proportion which empowers them to stalemate, absorb, and convey small biomolecule like RNA, DNA, proteins, drugs, and dissimilar atoms to embattled site and consequently improves the adequacy of therapeutic agents. In this chapter, common principles of drug target to malignant growth, intracellular mechanism, NP-based detailing in market, and ongoing application in medication as diagnostic and therapeutic aspects are examined based on bibliographic information, as reported in research article on nanotechnology. These features offer different methodologies of tumor nanotechnology in the progression of cancer therapy.
... It is well-known that PDT works by way of a photochemical reaction between photoexcited photosensitizers (PS) and O 2 molecules. The excited photosensitizer molecules can interact with nearby oxygen molecules, generating singlet oxygen 1 O 2 , causing damage to tumor cells and ultimately their death [2,3]. Regardless of the promising future of PDT in the field of cancer treatment, there are still problems to be solved. ...
Photodynamic therapy (PDT) has been reported as a possible pathway for the treatment of tumors. The exploration for promising PDT systems thus attracts continuous research efforts. This work focused on an ordered core–shell structure encapsulated by mesoporous SiO2 with the upconverting emission property following a surfactant-assisted sol–gel technique. The mesoporous silica shell possessed a high surface area-to-volume ratio and uniform distribution in pore size, favoring photosensitizer (rose bengal) loading. Simultaneously, upconverting nanocrystals were synthesized and used as the core. After modification via hydrophobic silica, the hydrophobic upconverting nanocrystals became hydrophilic ones. Under near-infrared (NIR) light irradiation, the nanomaterials exhibited strong green upconverting luminescence so that rose bengal could be excited to produce singlet oxygen. The photodynamic therapy (PDT) feature was evaluated using a 1O2 fluorescent indicator. It was found that this core–shell structure generates 1O2 efficiently. The novelty of this core–shell structure was the combination of upconverting nanocrystals with a mesoporous SiO2 shell so that photosensitizer rose bengal could be effectively adsorbed in the SiO2 shell and then excited by the upconverting core.
... In tumor cells, where the ATP intake is rather limited, the ATP boost given by PBM can allow tumor cells to respond to pro-apoptotic cytotoxic stimuli with cell death programs (apoptosis), which are highly energy-dependent (i.e., they require a lot of ATP). On the contrary, in normal healthy cells with an adequate ATP intake, the effect of PBM produces an explosion of ROS that could induce protective mechanisms and reduce the harmful effects of cancer therapy on healthy tissues [17,[46][47][48][49][50][51][52][53][54][55][56][57][58][59][60][61][62][63]. ...
The treatment of oral squamous cell carcinoma (OSCC) is particularly complex due to its aggressive behavior, location, the patient’s age, and its spread at diagnosis. In recent years, photobiomodulation (PBM) has been introduced in different medical fields; however, its application, in patients suffering from OSCC for palliative support or to induce analgesia, has been hotly debated due to the possibility that the cell growth stimuli induced by PBM could lead to a worsening of the lesions. The aim of this study is to review the literature to observe the available data investigating the effect of PBM on cancer cells in vitro and in vivo. A review was conducted on the PubMed and Scopus databases. A total of twelve studies met the inclusion criteria and were therefore included for quality assessment and data extraction. The analysis showed that the clinical use of PBM is still only partially understood and is, therefore, controversial. Some authors stated that it could be contraindicated for clinical use in patients suffering from SCC, while others noted that it could have beneficial effects. According to the data that emerged from this review, it is possible to hypothesize that there are possibilities for PBM to play a beneficial role in treating cancer patients, but further evidence about its clinical efficacy and the identification of protocols and correct dosages is still needed.
... This triggers photodynamic reactions result ing in generation of ROS that exert powerful damaging effect. A distance the most reactive ROS (singlet oxygen, hydroxyl radical) can travel is limited by several dozens of nanometers, which makes them most efficient in the case they enter the nucleus of target cells [227,228]. MNTs including those targeting EGFR have been examined as delivery vehicles of PS into the nuclei of target cells. It was shown that PS conjugated to MNTs preserve their potential to generate ROS upon illumination [202]. ...
Epidermal growth factor receptor (EGFR) is an integral surface protein mediating cellular response to a number of growth factors. Its overexpression and increased activation due to mutations is one of the most common traits of many types of cancer. Development and clinical use of the agents, which block EGFR activation, became a prime example of the personalized targeted medicine. However, despite the obvious success in this area, cancer cure remains unattainable in most cases. Because of that, as well as the result of the search for possible ways to overcome the difficulties of treatment, a huge number of new treatment methods relying on the use of EGFR overexpression and its changes to destroy cancer cells. Modern data on the structure, functioning, and intracellular transport of EGFR, its natural ligands, as well as signaling cascades triggered by the EGFR activation, peculiarities of the EGFR expression and activation in oncological disorders, as well as applied therapeutic approaches aimed at blocking EGFR signaling pathway are summarized and analyzed in this review. Approaches to the targeted delivery of various chemotherapeutic agents, radionuclides, immunotoxins, photosensitizers, as well as the prospects for gene therapy aimed at cancer cells with EGFR overexpression are reviewed in detail. It should be noted that increasing attention is being paid nowadays to the development of multifunctional systems, either carrying several different active agents, or possessing several environment-dependent transport functions. Potentials of the systems based on receptor-mediated endocytosis of EGFR and their possible advantages and limitations are discussed.
... Many biologically active agents can be transported into a specific cell compartment in order to exert their activity or to achieve higher activity. There are drugs, like photosensitizers (Rosenkranz et al., 2000), radionuclides emitting short-range particles (Sobolev, 2018;Rosenkranz et al., 2020), anticancer, antimicrobial, and antiviral drugs (Torchilin, 2014), that can exert their maximum effect within a certain compartment. In spite of considerable progress in development of subcellular delivery approaches, many types of biologically active molecules, potentially exploitable in clinical settings, are on the waiting list. ...
... De ce fait, l'adressage subcellulaire est un facteur important pour augmenter l'efficacité thérapeutique des PS [104]. En fonction de leurs caractéristiques physico-chimiques, les PS peuvent avoir plusieurs cibles subcellulaires, telles que les mitochondries, les lysosomes, la membrane plasmique, etc. [105]. ...
Les photosensibilisateurs les plus utilisés dans la Photothérapie dynamique (PDT) sont des porphyrines et leurs dérivés. Cependant, ces composés souffrent souvent d'une faible solubilité dans les milieux physiologiques et d'un manque de sélectivité envers les cellules cancéreuses, ce qui limite leurs utilisations cliniques. L’utilisation des nanoparticules comme vecteurs de photosensibilisateurs (PS) constitue une des stratégies les plus prometteuses pour surmonter ces problèmes. Dans ce contexte, nous avons développé des nanoparticules à base de xylane pour une délivrance ciblée de porphyrines. Deux types de nanoparticules ont été étudiées : des nanoparticules hybrides, noyau-coquille, et des nanoparticules organiques. Dans une première étude, les xylanesporphyrines ont été utilisés pour enrober des nanoparticules de silice (SNPs). En effet, la présence de groupes acides glucuroniques sur le xylane permet la formation de liaisons ioniques à la surface des SNPs rendues cationiques par des sels d’ammonium. L’évaluation biologique de ces nanoobjets a montré que l’association des porphyrines avec les nanoparticules augmente leur efficacité thérapeutique. De plus, l’adressage mitochondrial de ces photosensibilisateurs avec le cation triphénylphosphonium améliore encore de plus l’efficacité thérapeutique. Dans une deuxième approche, nous avons montré que les xylanes-porphyrines pouvaient conduire, par un autoassemblage, à des nanoparticules 100% organiques. Différents nano-objets à degré de substitution variable en porphyrine ont été obtenus et caractérisés. En parallèle, des nanoparticules 100% naturelles formées de xylane extrait de bois de châtaignier, et d’une chlorine naturelle, le phéophorbide a ont été obtenus et caractérisées. L’évaluation biologique de ces nanoparticules est en cours de réalisation et les premiers résultats sont très encourageants.
... Intracellular localization of the photosensitizer is of primary importance as the produced ROS are characterized by very short lifetimes and their targets should be very close to their production site. [51][52][53] Therefore the subcellular localization of the photosensitizer essentially determines the localization of the primary targets. There was no specific cellular localization observed for curcumin 1 in LNCaP cells, contrasting previous findings where mitochondrial curcumin localization was reported in non-small-cell lung cancer A549 cells, and membrane preference was observed in MCF7 breast cancer cells. ...
Photodynamic therapy (PDT) is a minimally invasive approach to treat various forms of cancer, based on the ability of certain non-toxic molecules (photosensitizers) to generate reactive oxygen species (ROS) after excitation by light of a certain wavelength and eventually induce strong phototoxic reactions against malignant cells and other pathogens. Curcumin is one of the most extensively investigated phytochemicals with a wide range of therapeutic properties and has been shown to induce strong photocytotoxic effects in micromolar concentrations against a variety of cancer cell lines. Curcumin (1) is comparatively evaluated with the naturally occurring bisdemethoxy Curcumin (2), which lacks the two methoxy groups, as well as two newly synthesized curcuminoids, the cinnamaldehyde derivative (3) and the dimethylamino one (4), designed to increase the absorption maximum and hence the tissue penetration. The synthetic curcuminoids were successfully synthesized in sufficient amounts and their photophysical properties such as absorption, fluorescence, photobleaching and free radical generation were investigated. Compound 4 exhibited a significant increase in peak absorption (497 nm) and strong fluorescent emission signals were recorded for all curcuminoids. Photobleaching of 4 was comparable to 1 whereas 2 and 3 showed more extended photobleaching but much higher ROS production in very short irradiation times. Compounds 2 and 4 exhibited specific intracellular localization. After dark and light cytotoxicity experiments against LNCaP prostate cancer cell line for all curcuminoids, concentration of 3 μΜ and irradiance of 6 mW / cm² were selected for the PDT application which resulted in remarkable results with very short LD50. Curcuminoids 2 and 4 exhibited a significant dose-dependent PDT effect. The biphasic dose-response photodynamic effect observed for 1 and 3 may provide a strategy against prolonged and sustained photosensitivity.
... It could be treated by photodynamic therapy (PDT), which consists in intravenous injection of a photosensitizer, and subsequent local illumination of the tumor at an appropriate wavelength (Teixo et al. 2015). However, the efficiency of this therapy seems directly related to (i) the capacity of the photosensitizer to penetrate into cancer cells, its transport pathway, (ii) subcellular localization, and (iii) physical state (monomer, aggregate) in the cytoplasma (Rosenkranz et al. 2000;. Considering the poor intracellular penetration efficiency of the tetraphenylporphyrins (TPPs), attributed to their low solubility in aqueous medium and lack of specificity, glycoconjugated derivatives have been specifically designed to improve their amphipathic properties and address sugar receptors overexposed at the surface of retinoblastoma cells (Griegel et al. 1989). ...
La photothérapie dynamique (PDT) consiste à injecter un photosensibilisateur (PSr) au patient, puis à illuminer sa tumeur. En présence d’oxygène, le PSr activé entraîne la formation d’oxygène singulet cytotoxique. Nos collaborateurs à l’Institut Curie ont synthétisé des dérivés porphyriniques glycoconjugués(TPP) pour traiter le rétinoblastome par PDT.Des études de caractérisation de ces TPP in vitro ont montré une internalisation dans la cellule par voie passive. C’est dans ce contexte que nous avons analysé l’interaction de certaines TPP avec les lipides membranaires.Dans un premier temps, cette interaction a été étudiée par une approche chromatographique sur des colonnes C18/C8, PolarTec, HILIC et IAM. Nous avons démontré une variation de l’interaction selon la structure des TPP. Par la suite, nous avons mis en évidence par DSC, les changements d’organisation de bicouches phospholipidiques produits par deux TPP d’intérêt, et déterminé par FTIR-ATR, la localisation de la perturbation au niveau des têtespolaires ou des chaines aliphatiques des lipides.Cette approche a été poursuivie par l’évaluation de la localisation des TPP à l’échelle cellulaire,par microspectroscopie IR couplée au rayonnement synchrotron. Une discrimination des TPP a été mise en évidence par des outils chimiométriques pour les cellules Y79, mais pas pour les lignées WERI-Rb1 ni ARPE-19. Afin de développer un modèle membranaire artificielde rétinoblastome, nous avons réalisé par spectrométrie de masse (Orbitrap) une analyse lipidomique approfondie des phospholipides des membranes plasmiques et mitochondriales des lignées Y79 et ARPE-19,. Nous avons analysé les propriétés visco-élastiques des extraits membranaires et proposé un modèle artificiel complexe mimant au moins partiellement ces propriétés. Ce modèle pourrait permettre le criblage in vitro des TPP.
... These vehicles can be obtained as a single polypeptide chain from an artificial gene biosynthetically (e.g. in bacteria), allowing production of the desired product at a scale that will ultimately be needed for clinical use. This idea and its first experimental verification were first published in the early 2000s and described its use for the delivery of photosensitizers as emitters of short-range reactive oxygen species [173]. The full functional polypeptide components have included at least four different "modules": the ligand module for cancer cell recognition and receptor-mediated internalization, the endosomolytic module for pH-dependent endosomal escape, a module with an NLS for transport into the nucleus via NPC, and a carrier module for combining all transport modules together in proper orientation and in some cases, the attachment of the active component (photosensitizer, radionuclide). ...
The presence of Auger electrons (AE) among the decay products of a number of radionuclides makes these radionuclides an attractive means for treating cancer because these short-range electrons can cause significant damage in the immediate vicinity of the decomposition site. Moreover, the extreme locality of the effect provides a potential for selective eradication of cancer cells with minimal damage to adjacent normal cells provided that the delivery of the AE emitter to the most vulnerable parts of the cell can be achieved. Few cellular compartments have been regarded as the desired target site for AE emitters, with the cell nucleus generally recognized as the preferred site for AE decay due to the extreme sensitivity of nuclear DNA to direct damage by radiation of high linear energy transfer. Thus, the advantages of AE emitters for cancer therapy are most likely to be realized by their selective delivery into the nucleus of the malignant cells. To achieve this goal, delivery systems must combine a challenging complex of properties that not only provide cancer cell preferential recognition but also cell entry followed by transport into the cell nucleus. A promising strategy for achieving this is the recruitment of natural cell transport processes of macromolecules, involved in each of the aforementioned steps. To date, a number of constructs exploiting intracellular transport systems have been proposed for AE emitter delivery to the nucleus of a targeted cell. An example of such a multifunctional vehicle that provides smart step-by-step delivery is the so-called modular nanotransporter, which accomplishes selective recognition, binding, internalization, and endosomal escape followed by nuclear import of the delivered radionuclide. The current review will focus on delivery systems utilizing various intracellular transport pathways and their combinations in order to provide efficient targeting of AE to the cancer cell nucleus.
... Hence, research were directed towards two main approaches; (1) the generation of new PS with the hope that the newly developed molecule will accumulate in the targeted cells, and (2) the use of carriers such as nanoparticles (NP), liposomes and microspheres to enable site-specific delivery of the encapsulated or conjugated PS into the targeted cells (Rosenkranz et al. 2000). The latter is an example of third generation PS, in which the delivery of PS is designed to be specific and targeted with the help of carriers or certain targeting agents. ...
... microseconds in tissues; therefore, the diffusion range of the ROS is limited [68][69]. The targeting effect from a photosensitizer is also of great importance in tumor treatments [70][71]. PDT-induced ROS must be generated in an aerobic environment, so the applicability of the treatment is subject to certain restrictions. ...
Phototherapy is a non-invasive or minimally invasive therapeutic strategy. Immunotherapy uses different immunological approaches, such as antibodies, vaccines, immunoadjuvants, and cytokines to stimulate the host immune system to fight against diseases. In cancer treatment, phototherapy not only destroys tumor cells, but also induces immunogenic tumor cell death to initiate a systemic anti-tumor immune response. When combined with immunotherapy, the effectiveness of phototherapy can be enhanced. Because of their special physical, chemical, and sometimes immunological properties, nanomaterials have also been used to enhance phototherapy. In this article, we review the recent progress in nanotechnology-based phototherapy, including nano-photothermal therapy, nano-photochemical therapy, and nano-photoimmunological therapy in cancer treatment. Specifically, we focus on the immunological responses induced by nano-phototherapies.
... Photodynamic therapy (PDT) is a treatment modality that uses a light sensitive drug which is known as a photosensitizer along with near infrared (NIR) visible light of the appropriate wavelength to damage malignant or abnormal tissues [1,2]. PDT has been ...
Photodynamic therapy explicit dosimetry is recently developed to better correlate with clinical outcome of photodynamic therapy (PDT), which is a triple-element photochemical interaction involving light, photosensitizer (PS), and oxygen. In this chapter, we give a short recap of the theory of reactive oxygen species explicit dosimetry (ROSED) that explains the mechanism of photochemical interactions, followed by examples of applications. For PDT, we present explicit dosimetry methods of in-vivo light fluence (rate), in-vivo PS concentration (or uptake), and oxygen concentration in tissue. In the medical physics context, ROSED provides a systemic approach to predict the PDT outcome, which cannot otherwise be achieved with existing dosimetrical methods. Finally, a comparison of dose metrics vs. PDT outcome is present to show the clinical impact of the ROSED.
... Subcellular distribution is an important factor determining the efficacy of a PDT agent due to the short lifetime and limited diffusion range of ROS (31). Previous studies have shown that lipophilic, charged metal complexes may accumulate in the mitochondria because of the negative potential difference across the mitochondrial membrane (16,24,28). ...
Significance
Photodynamic therapy is a noninvasive medical procedure in which malignant tissue is damaged by reactive oxygen species generated from targeted light irradiation on a photosensitizer. Herein, we incorporate an Ru(II)-based two-photon photosensitizer into a macrocyclic structure via coordination-driven self-assembly. Properties favorable for photodynamic therapy arise upon formation of the metallacycle, including near-infrared luminescence, strong two-photon absorption, high reactive oxygen species generation efficiency, and selective accumulation in mitochondria and nucleus that are hypersensitive to reactive oxygen species, resulting in good photodynamic therapy efficacy in both in vitro and in vivo investigations. This study shows that supramolecular coordination complexes provide a platform for efficient tuning on the photophysical and biological properties of molecules with biomedical interest.
... However, patient outcomes have been only modestly improved when naked mAb is used as monotherapy or in combination with other therapies [2][3][4]. Combinations of conventional photosensitizers and mAbs have been tested but the improvement in selectivity is still limited [5][6][7][8]. Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed specific cancer treatment that employs a targeted monoclonal antibody (mAb)-photo-absorber conjugate (mAC) [9]. ...
As a novel low-side-effect cancer therapy, photo-immunotherapy (PIT) is based on conjugating monoclonal antibody (mAb) with a near-infrared (NIR) phthalocyanine dye IRDye700DX (IR 700). IR700 is not only fluorescent to be used as an imaging agent, but also phototoxic. When illuminating with NIR light, PIT can induce highly-selective cancer cell death while leaving most of tumor blood vessels unharmed, leading to an effect termed super-enhanced permeability and retention (SUPR), which can significantly improve the effectiveness of anti-cancer drug. Currently, the therapeutic effects of PIT are monitored using 2D macroscopic fluorescence reflectance imager, which lacks the resolution and depth information to reveal the 3D distribution of mAb-IR700. In the study, we applied a multi-modal optical imaging approach including high-resolution optical coherence tomography (OCT) and high-sensitivity fluorescence laminar optical tomography (FLOT), to provide 3D tumor micro-structure and micro-distribution of mAb-IR700 in the tumor simultaneously during PIT in situ and in vivo. The multi-wavelength FLOT can also provide the blood vessels morphology of the tumor. Thus, the 3D FLOT reconstructed images allow us to evaluate the IR700 fluorescence distribution change with respect to the blood vessels and at different tumor locations/depths non-invasively, thereby enabling evaluation of the therapeutic effects in vivo and optimization of treatment regimens accordingly. The mAb-IR700 can access more tumor areas after PIT treatment, which can be explained by increased vascular permeability immediately after NIR-PIT. Two-photon microscopy was also used to record the mAb-IR700 on the tumor surface near the blood vessels to verify the results.
... Initial attempts to increase photosensitizers delivery fo- cused on improving targeted to non-targeted tissue ratios. However, it was demonstrated that elevated tumor to normal tissue ratios did not ensure improved efficiency in vivo [61]. Photoactivated photosensitizer generates reactive oxygen species (singlet oxygen 1 O 2 , and free radicals such as OH . ...
La thérapie photodynamique (PDT) est une modalité de traitement des petites tumeurs localisées, reposant sur l?action conjuguée d'un photosensibilisateur (PS), de la lumière et de l'oxygène. Dans le cadre d'un nouveau mode de PDT, la VTP (vascular targeted photodynamic therapy), notre stratégie a consisté à favoriser l?effet anti-vasculaire du traitement par ciblage de la néo-vascularisation tumorale. Pour cela, nous avons étudié in vitro et in vivo un PS de type chlorine (TPC) couplé, via le bras espaceur (Ahx), à un heptapeptide (ATWLPPR) spécifique d?un co-récepteur du VEGF, la neuropiline-1 (NRP-1). Une étude comparative de la TPC versus le PS conjugué (TPC-Ahx-ATWLPPR), a permis de mettre en évidence in vitro, grâce à une technique d'ARN interférence visant à éteindre l'expression de NRP-1, une incorporation cellulaire récepteur-dépendante du conjugué. In vivo, l'accumulation préférentielle de la TPC-Ahx-ATWLPPR au niveau de l'endothélium vasculaire de la tumeur ainsi que son effet anti-vasculaire après PDT ont été mises en évidence. Une étude de stabilité in vitro et in vivo du conjugué a été réalisée. In vivo, la séquence peptidique est dégradée 4 h après injection par voie intra-veineuse. Des études de pharmacocinétique et de biodistribution tissulaire de la TPC-Ahx-ATWLPPR et de son principal produit de dégradation, TPC-Ahx-A ont été réalisées chez la souris nude xénogreffée. La dégradation de la partie peptidique est majoritaire dans les organes du système réticulo-endothélial où l'accumulation du conjugué est la plus importante. Dans le but d'augmenter la stabilité in vivo du peptide adresseur, de nouveaux peptides ont été synthétisés, puis couplés à la TPC et testés. Le pseudopeptide A[psi] [CH2NH]TWLPPR est prometteur car il reste affin vis-à-vis de NRP-1 et après couplage au PS, il ne subit aucune dégradation dans le 8plasma in vivo 4 h après injection par voie intra-veineuse.
... However, the contribution of a particular oxidant species to cell death is usually unclear. Current approaches to optimize the effectiveness of photodynamic therapy include improving selective targeting, intra-cellular localization of the photosensitizer to an oxidative-vulnerable compartment and designing photosensitizers that generate large yields of oxidants [27,32]. ...
Oxidants play an important role in the cell and are involved in many redox processes. Oxidant concentrations are maintained through coordinated production and removal systems. The dysregulation of oxidant homeostasis is a hallmark of many disease pathologies. The local oxidant microdomain is crucial for the initiation of many redox signaling events; however, methods to control oxidant product are limited. Some fluorescent proteins, including GFP, TagRFP, KillerRed, miniSOG, and their derivatives, generate oxidants in response to light. These genetically-encoded photosensitizers produce singlet oxygen and superoxide upon illumination and offer spatial and temporal control over oxidant production. In this review, we will examine the photosensitization properties of fluorescent proteins and their application to redox biology. Emerging concepts of selective oxidant species production via photosensitization and the impact of light on biological systems are discussed.
... In particular, immunotoxins utilize this approach for delivery of catalytic subunits of bacterial toxins for cancer treatment [260]. It is important to note that all these transport functions, including binding to the specific cell, endocytosis, endosome escape, transport to the nucleus, can be combined in the same polypeptide macromolecule [261][262][263]. These multifunctional constructs or modular nanotransporters consisting of four Taz Human mesenchymal stem cell (hMSC) [242] Abbreviations: CPP -Cell-penetrating peptide; DARTs -DNA assembled recombinant transcription factors; HA2peptide based on influenza A virus hemagglutinin protein; NLS -Nuclear localization signal; PTD -Protein transduction domain; TAT-Transactivator of transcription; VP22 -type I herpes simplex virus; 3R, 9R, or 11Rpolyarginines sequence consisting of 3, 9, or 11 arginines. ...
Transcription factors (TFs) are at the center of the broad regulatory network orchestrating gene expression programs that elicit different biological responses. For a long time, TFs have been considered as potent drug targets due to their implications in the pathogenesis of a variety of diseases. At the same time, TFs, located at convergence points of cellular regulatory pathways, are powerful tools providing opportunities both for cell type change and for managing the state of cells. This task formulation requires the TF modulation problem to come to the fore. We review several ways to manage TF activity (small molecules, transfection, nanocarriers, protein-based approaches), analyzing their limitations and the possibilities to overcome them. Delivery of TFs could revolutionize the biomedical field. Whether this forecast comes true will depend on the ability to develop convenient technologies for targeted delivery of TFs.
... While this technology can provide detailed images of tissue architecture and cellular morphology, a very small field of view and limited penetration depth of 250 -500 μm considerably reduce the clinical usefulness of this approach [43]. The ability of the photosensitisers to accumulate in cancer cells and to fluoresce under specific wavelengths can be used to identify and delineate areas of microscopic changes [44,45]. Multi-photon laser scanning microscopy (MLSM) is one technique, in which the fluorophore excitation results from visible or longwave infrared range lasers. ...
For over half a century, laser technology has undergone a technological revolution. These technologies, particularly semiconductor lasers, are employed in a myriad of fields. Optical medical diagnostics, one of the emerging areas of laser application, are on the forefront of application around the world. Optical methods of non- or minimally invasive bio-tissue investigation offer significant advantages over alternative methods, including rapid real-time measurement, non-invasiveness and high resolution (guaranteeing the safety of a patient). These advantages demonstrate the growing success of such techniques.In this review, we will outline the recent status of laser technology applied in the biomedical field, focusing on the various available approaches, particularly utilising compact semiconductor lasers. We will further consider the advancement and integration of several complimentary biophotonic techniques into single multimodal devices, the potential impact of such devices and their future applications. Based on our own studies, we will also cover the simultaneous collection of physiological data with the aid a multifunctional diagnostics system, concentrating on the optimisation of the new technology towards a clinical application. Such data is invaluable for developing algorithms capable of delivering consistent, reliable and meaningful diagnostic information, which can ultimately be employed for the early diagnosis of disease conditions in individuals from around the world.
... (mono-aspartyl chlorin e6) 可通过内吞作用主要定位 于溶酶体 [51] 。酞菁类光敏剂可分布在线粒体 [52] 。 苯并卟啉衍生物 (benzoporphyrin derivative, BpD) 主 要定位于高尔基器 [53] But note the significant multiple localizations of LCI, SnOPA, SnET2 and AIPC. Re-drawn from Kessel [58] . ...
Photodynamic action, due to the rather limited lifetime (1 μs) and effective reactive distance of singlet oxygen (< 10 nm), could subcellular-specifically regulate different cellular functions. Photodynamic action could activate permanently cholecystokinin (CCK) 1 receptors, and sensitize or desensitize other G protein-coupled receptors. The emergence in recent years of genetically- encoded protein photosensitisers has enabled more precisely-targeted photodynamic modulation of subcellular organelles and functional proteins. Protein photosensitisers (such as KillerRed, miniSOG or SOPP) expressed on the plasma membrane, mitochondria, lysosomes or endoplasmic reticulum can modulate photodynamically subcellular functions and fine-tune protein activity by targeted photooxidation. With the newly emerged active illumination technique, simultaneous photodynamic action localized at multiple sites is now possible, and the contribution of subcellular regions to the whole cell or individual cells to a cell cluster could be quantitated. Photodynamic action with protein photosensitiser will be a powerful tool for nano-manipulation in cell physiology research.
... Results of our experiment revealed that the BNCE exhibits relatively high toxicity effect in U937 cells against SK-HEP-1 cells. This corresponds to a previous study reporting that the effects of photosensitizers depend on tissue distribution, affinity for certain cell types, penetration into various subcellular compartments, and accumulation [16]. Further, there was the difference between cells due to the various mechanisms involved in cell death [3,17,18]. ...
Brassica napus is the most common feedstock for biodiesel production, and its cultivation area has been rapidly increased. Thus, B. napus residues left in the field after harvest are valuable resources. However, there have been few studies on biologically active substances from B. napus plant. The objective of this study is to evaluate cytotoxicity/photodynamic activity and antioxidant activity of B. napus plant extracts. B. napus plants were sequentially extracted with organic solvents (hexane, chloroform, ethanol, and water) and then screened for antioxidant activity and cytotoxicity against leukemia U937 and human liver cancer SK-HEP-1 cells. Among the solvent extracts, the cytotoxicity was the highest when cells treated with chloroform extract and irradiated. Degree of apoptosis substantially increased in both cell types in concentration-dependent manner, and non-irradiated cells showed similar results as the control cells. For the highest concentrations (100 μg/ml), toxicity effect in U937 and SK-HEP-1 cells was 94.62 ± 0.15% and 74.16 ± 1.54%, respectively. We observed the number of cells significantly decreased, and vesicles were floating in B. napus chloroform extract (BNCE) and light condition. BNCE induced DNA laddering pattern (between 300 and 1000 bp) and caspase-3/7 activation in both U937 and SK-HEP-1 cells. Total apoptotic U937 and SK-HEP-1 cells following BNCE 100 μg/ml and light treatment were significantly increased (92.62 ± 2.07% and 59.71 ± 4.38%, respectively) compared with control. Our results showed that U937 cells were more sensitive than SK-HEP-1 cells. For the antioxidant activity, B. napus ethanol extract was the highest (IC50 = 0.52 mg/ml).
... However, patient outcomes have been only modestly improved when naked mAbs are used as monotherapies or in combination with other therapies [2][3][4]. Combinations of conventional photosensitizers and mAbs have been tested, but the improvement in selectivity is still limited [5][6][7][8]. Photoimmunotherapy (PIT) is an emerging low side effect cancer therapy based on a targeted monoclonal antibody-photo absorber conjugate (APC) [mAb conjugated with a near-infrared (NIR) phthalocyanine dye (IR700)] that induces rapid cellular necrosis after exposure to NIR light [9]. ...
Photoimmunotherapy (PIT) is an emerging low side effect cancer therapy based on a monoclonal antibody (mAb) conjugated with a near-infrared (NIR) phthalocyanine dye IRDye 700DX. IR700 is fluorescent, can be used as an imaging agent, and also is phototoxic. It induces rapid cell death after exposure to NIR light. PIT induces highly selective cancer cell death, while leaving most of tumor blood vessels unharmed, leading to an effect called super-enhanced permeability and retention (SUPR). SUPR significantly improves the effectiveness of the anticancer drug. Currently, the therapeutic effects of PIT are monitored using the IR700 fluorescent signal based on macroscopic fluorescence reflectance imagery. This technique, however, lacks the resolution and depth information to reveal the intratumor heterogeneity of mAb-IR700 distribution. We applied a minimally invasive two-channel fluorescence fiber imaging system by combining the traditional fluorescence imaging microscope with two imaging fiber bundles (~ 0.85 mm). This method monitored mAb-IR700 distribution and therapeutic effects during PIT at different intratumor locations (e.g., tumor surface vs. deep tumor) in situ and in real time simultaneously. This enabled evaluation of the therapeutic effects in vivo and treatment regimens. The average IR700 fluorescence intensity recovery after PIT to the tumor surface is 91.50%, while it is 100.63% in deep tumors. To verify the results, two-photon microscopy combined with a microprism was also used to record the mAb-IR700 distribution and fluorescence intensity of green fluorescent protein (GFP) at different tumor depths during PIT. After PIT treatment, there was significantly higher IR700 fluorescence recovery in deep tumor than in the tumor surface. This phenomenon can be explained by increased vascular permeability immediately after NIR-PIT. Fluorescence intensity of GFP at the tumor surface decreased significantly more compared to that of deep tumor and in controls (no PIT).
... However, patient outcomes have been only modestly improved when naked mAbs are used as monotherapies or in combination with other therapies [2][3][4]. Combinations of conventional photosensitizers and mAbs have been tested, but the improvement in selectivity is still limited [5][6][7][8]. Photoimmunotherapy (PIT) is an emerging low side effect cancer therapy based on a targeted monoclonal antibody-photo absorber conjugate (APC) [mAb conjugated with a near-infrared (NIR) phthalocyanine dye (IR700)] that induces rapid cellular necrosis after exposure to NIR light [9]. ...
The effectiveness of phototherapy using photosensitizers is limited by the challenges in their delivery at the site of irradiation. Here, we demonstrate the localized application of a photosensitizer-loaded microneedle patch for effective photodynamic and photothermal therapy in oral carcinoma. Indocyanine green (ICG) was studied as a photosensitizer for its effect on oral carcinoma, FaDu cells. Different parameters including concentration, near-infrared (NIR) laser irradiation intensity and irradiation time were optimized while measuring temperature increase and reactive oxygen species (ROS) generation in FaDu cells. A dissolvable microneedle (DMN) patch made of sodium carboxymethyl cellulose and sodium alginate was fabricated by the micromolding technique. DMN showed sufficient mechanical strength for insertion in the excised porcine buccal mucosa. DMN dissolved within 30 s in phosphate buffer and 30 min in the excised buccal mucosa. Confocal microscopy studies revealed DMN penetration up to a depth of 300 µm within the buccal mucosa. ICG-DMN applied on the back of the rat was found to be localized at the application site before and after irradiation using an 808 nm NIR laser. ICG-DMN was applied on the FaDu xenografted tumor model in athymic nude mice. The localized temperature increase and ROS generation significantly (P<0.05) decreased the tumor volume after ICG-DMN application compared with the control group. In conclusion, DMN can be developed for the localized administration of photosensitizers for phototherapy in oral carcinoma.
Photodynamic therapy (PDT) is a clinically approved cancer treatment that requires a photosensitizer (PS), light, and molecular oxygen─a combination which produces reactive oxygen species (ROS) that can induce cancer cell death. To enhance the efficacy of PDT, dual-targeted strategies have been explored where two photosensitizers are administered and localize to different subcellular organelles. To date, a single small-molecule conjugate for dual-targeted PDT with light-controlled nuclear localization has not been achieved. We designed a probe composed of a DNA-binding PS (Br-DAPI) and a photosensitizing photocage (WinterGreen). Illumination with 480 nm light removes WinterGreen from the conjugate and produces singlet oxygen mainly in the cytosol, while Br-DAPI localizes to nuclei, binds DNA, and produces ROS using one- or two-photon illumination. We observe synergistic photocytotoxicity in MCF7 breast cancer cells, and a reduction in size of three-dimensional (3D) tumor spheroids, demonstrating that nuclear/cytosolic photosensitization using a single agent can enhance PDT efficacy.
Therapeutic resistance caused by hypoxia tumor microenvironment (TME) is one of the main challenges for the development of nanoscale delivery systems, particular for the photodynamic agents. In this work, porphyrin-based metal-organic frameworks composite [email protected]@MnO2-PEG (APM) with the mimetic sea cucumber morphology was post-synthesized for using in photodynamic therapy (PDT) against hypoxia. Besides the good colloidal stability, as-synthesized APM nanoparticles also exhibited the well biocompatibility and H2O2-triggered drug releasing. After being reacted with endogenous H2O2, the amorphous MnO2 layer on the APM outersurface could quickly decompose to produce abundant oxygen, self-compensating for the consumption of photodynamic process. Meanwhile, the highly loaded hypoxia inhibitors acriflavine (ACF) of 18.7 wt% in APM was released to down-regulate the expression level of hypoxia-inducible factors (HIF). Combining with the self-generating oxygen and ACF inhibitors, the tumor hypoxia could be greatly relieved and the better tumor suppression effect by PDT have been demonstrated in vivo. This self-suppling oxygen nanosystem triggered by endogenous tumor markers proves a feasible way for remodeling the hypoxia TME and to achieve better therapeutic performances.
Poly(oxanorbornene)s with zinc(II) phthalocyanine side chains have been synthesized by ring-opening metathesis polymerization. The incorporation of zinc(II) phthalocyanine into cationic polymer has given poly(oxanorbornene)s noteworthy photophysicochemical properties and the capacity to generate singlet oxygen under light irradiation. To investigate photosensitizer’s properties of the newly synthesized polymers P6 and P7: fluorescence (ΦF), singlet oxygen (ΦΔ) and triplet (ΦT) quantum yields of polymers have been measured in dimethyl sulfoxide and aqueous medium. Singlet oxygen quantum yields of P6 and P7 have been found to be 0.22 and 0.20 in dimethyl sulfoxide, respectively. Then, photodynamic therapy activities of polymers (P1-P7) against human breast adenocarcinoma cell line (MCF-7 cells) have been investigated. The copolymer P5 bearing pendant zinc(II) phthalocyanine and triethyl phosphonium functionalities has showed enhanced PDT activity with less than 10% viable cells at 60 μg/mL.
Tanshinone IIA (TanIIA) has multiple biological functions and already been clinically used to treat many cardiovascular diseases. TanIIA is a photoactive molecule and can be excited by light to generate 3TanIIA*. Generation of 3TanIIA* by TanIIA photosensitization indicates that TanIIA may serve as a photosensitizer to bring photodynamic damage to organisms. Therefore, human choroidal melanoma MUM‐2B cell was chosen as a superficial tumor model and the photodynamic effect of TanIIA on tumor cells was evaluated in this study. The results showed that TanIIA photosensitization could generate singlet oxygen in non‐cellular system. MTT, clone formation and wound‐healing assays showed that the survival and migration of MUM‐2B cells could be efficiently inhibited by TanIIA photosensitization. And then, laser confocal microscope and flow cytometry were used to try to elucidate related mechanism. It was found that TanIIA could pass through cellular membrane and preferably accumulate in nucleus. TanIIA photosensitization could efficiently induce cell apoptosis and necrosis, increase intracellular ROS levels, decrease mitochondria membrane potential, and lead to cell cycle arrest in G2/M phase. Our findings indicate that TanIIA photosensitization can exert remarkable toxicity on choroidal melanoma cells.
Cancer remains a main public health issue and the second cause of mortality worldwide. Photodynamic therapy is a clinically approved therapeutic option for several conditions and can induce damages in localized tumors. Effective photodynamic therapy induces cancer damage and death through a multifactorial manner including reactive oxygen species-mediated direct cancer damage and killing, tumor vasculature damage, and immune defense activation. Its anticancer efficiency depends on the improvement of photosensitizers, drugs used in photodynamic therapy, considering their selectivity, enhanced photoproduction of reactive species, absorption at near-infrared spectrum, and drug-delivery strategies. Both experimental and clinical studies using first- and second-generation photosensitizers had pointed out the urgent need for developing improved photosensitizers for photodynamic applications and achieving better therapeutic outcomes. Bioconjugation and encapsulation with targeting moieties appeared as the main strategies for the new development of photosensitizers from their precursors. Factors influencing their cellular biodistribution and uptake are briefly discussed, as well as their roles as cancer diagnostic and therapeutic (theranostics) agents. The two-photon photodynamic approach using third-generation photosensitizers is present as an attempt in treating deeper tumors. Although significant advances had been made over the last decade, the development of next-generation photosensitizers is promising and should be conducted via chemo- and site-selective manner to facilitate PDT translation into further stages of treatment and cancer management.
Modular nanotransporters (MNTs) are multifunctional chimeric polypeptides for the multistep transport of locally acting cytotoxic agents into the nuclei of cancer target cells. MNTs consist of several polypeptide domains (functional modules) for the recognition of a cell-surface internalizable receptor, pH-dependent endosomal escape and subsequent transport into the nucleus through the nuclear pores. MNTs are a promising means for cancer treatment. As has been shown previously, all of the modules of MNTs retain their functionalities. Despite their importance, there is no structural information available about these chimeric polypeptides, which hampers the creation of new MNT variants. Here, a low-resolution 3D structure of an MNT is presented which was obtained by atomic force microscopy, transmission electron microscopy and small-angle X-ray scattering coupled to size-exclusion chromatography. The data suggest that the MNT can adopt two main conformations, but in both conformations the protein N- and C-termini are distanced and do not influence each other. The change in the MNT conformation during acidification of the medium was also studied. It was shown that the fraction of the elongated conformation increases upon acidification. The results of this work will be useful for the development of MNTs that are suitable for clinical trials and possible therapeutic applications.
The purpose of this study was to develop a method for exposing biological and chemical components to defined levels of (extracellular) oxidative stress, and to study their effects using conventional methods. Photodynamic therapy (PDT)-mimicking extracellular oxidative stress reactions (OSR); microbatch OSR (MBOSR) and microfluidic flow OSR (MFOSR) were carried out using colon carcinoma COLO320 cell line. An instrument was built and used in all the photosensitisation OSR. Several microfluidic chip configurations were designed, fabricated, studied for flow properties and used in the surface treatment reactions. Different types of photosensitisers were successfully covalently immobilised onto surfaces of the microfluidic channels, and their presence was verified using spectroscopic, colorimetric and photo-oxidation reactions. To conduct the MFOSR; syringe-, pressure-and peristaltic-pumping systems were tested to optimise the flow system and cell recovery was improved. Initially MFOSR with immobilised porphyrin were carried out. To determine the optimum OSR conditions, MBOSR were carried out having the photosensitiser, aluminium (III) phthalocyanine tetrasulfonate (AlPcS4) in solution, and the best set of photosensitisation effect inducing conditions were used in conducting the MFOSR with photosensitiser in solution. The treated samples were analysed to determine the levels of LDH released into supernatants, percentages of cells recovered, cell viability, and the modes of cellular disintegrations induced. MFOSR with immobilised photosensitiser showed high variations with no statistically significant differences, whereas MFOSR with AlPcS4 in solution showed significant induction of photosensitisation effects. It was identified from the MBOSR that the induction of photosensitisation effects significantly increased with the concentration of AlPcS4 or the light dose. The MBOSR and MFOSR with photosensitiser in solution were useful in the generation of extracellular oxidative stress environment. These types of reactions mainly induced cellular disintegration through necrosis relative to apoptosis. This system can possibly be adapted to study numerous diseases where oxidative stress is involved. 3
Fluorescence and Rayleigh light scattering in polymer systems based on the chlorine photosensitizer photoditazine (PD) for photodynamic therapy are studied upon excitation at wavelengths λex = 260, 280, 400, and 450 nm. Nontoxic water-soluble poly-N-vinylpyrrolidone (PVP) and a plant polysaccharide (sodium alginate (SA)) were used as polymers. The PD–SA double system is characterized by dependence of its characteristics on the synthesis conditions, while the PD–SA–PVP triple complex exhibits a positive combined effect of SA and PVP polymers on the PD molecule due to the formation of a local microenvironment of the active PD center with a higher polarity. It is shown that the introduction of tryptophan, which is used as a substrate in model photooxidation processes for testing the efficiency of photosensitizers in singlet-oxygen generation, does not considerably change the fluorescence parameters of the PD–SA–PVP complex.
Photodynamic therapy (PDT) is a clinically approved cancer treatment that uses light, oxygen and a photosen-sitizer to produce localized reactive oxygen species (ROS). Due to the short lifetime of ROS, the location of the photosensitizer in the cell is believed to be key de-terminant governing the outcome of PDT. To explore the effect of direct association between a photosensitizer and DNA a well know DNA-binding dye, DAPI, was con-verted into a photosensitizer. Br-DAPI – unlike native DAPI, upon irradiation produces ROS. We demonstrate that the ROS are only effective in inducing dsDNA breaks when Br-DAPI is bound to DNA. In cancer cells (A549) Br-DAPI causes rapid light dependent cell death. This work supports the design of photosensitizers which bind with high affinity to the DNA of target cells for ef-fective PDT.
Photodynamic Therapy (PDT) with methyl-aminolevulinate acid (MAL-PDT) is being used for the treatment of Basal cell carcinoma (BCC), but recurrences have been reported. In this work, we have evaluated resistance mechanisms to MAL-PDT developed by three BCC cell lines (ASZ, BSZ and CSZ), derived from mice on a ptch+/− background and with or without p53 expression, subjected to 10 cycles of PDT (10thG). The resistant populations showed mesenchymal-like structure and diminished proliferative capacity and size compared to the parental (P) cells. The resistance was dependent on the production of the endogenous photosensitiser protoporphyrin IX in the CSZ cell line and on its cellular localisation in ASZ and BSZ cells. Moreover, resistant cells expressing the p53 gene presented lower proliferation rate and increased expression levels of N-cadherin and Gsk3β (a component of the Wnt/β-catenin pathway) than P cells. In contrast, 10thG cells lacking the p53 gene showed lower levels of expression of Gsk3β in the cytoplasm and of E-cadherin and β-catenin in the membrane. In addition, resistant cells presented higher tumorigenic ability in immunosuppressed mice. Altogether, these results shed light on resistance mechanisms of BCC to PDT and may help to improve the use of this therapeutic approach.
2,4,6-trinitrophenol (PA) is a well-known aromatic compound, due to their higher explosive energy than TNT and their wide utilization when producing organic dyes, medicines and antiseptics. The following paper constructed a composite optical sensing system (denoted as Tb:[email protected]) for PA detection using [Zn8(ad)4(BPDC)6O·2 Me2NH2]·G, ad = adenine, BPDC = 4,4′-biphenyl dicarboxylic acid, G = DMF and water, as supporting matrix and two rare earth ions, Tb(III) and Eu(III), as dopant. Tb:[email protected] was carefully characterized by means of micromorphology, XRD, N2 adsorption and desorption. Detailed analysis on its photophysical performance indicated weak Tb emission and strong Eu emission, owing to the energy competing between two kinds of rare earth ions in Tb:[email protected] After meeting PA, such energy competing was blocked, leading to enhanced Tb emission and decreased Eu emission. Ratiometric fluorescent sensing for PA detection was thus constructed with good selectivity and low LOD (limit of detection) of 0.87 μM. Linear sensing response was achieved. Its practical sensing application and naked eye detection for PA were tentatively discussed as well.
Liposomes are microscopic lipid bubbles designed to entrap drugs. They have been used locally as well as systemically for targeting of drugs to specific organs or for prolonging drug effect. The encapsulation of drugs in liposomes has been shown to reduce the toxicity, provide solubility in plasma, and enhance permeability through tissue barriers. Some applications related to cancer and infectious diseases have reached clinical use, while others are currently in Phase I–III human clinical trials.
La thérapie photodynamique (PDT) repose sur l'utilisation d'une molécule photoactive telle que le Foscan® (m-THPC). Ce médicament de seconde génération combiné à la lumière et à l'oxygène, induit la mort cellulaire soit par nécrose soit par apoptose. La première étape de nos études consiste à détecter par MALDI-TOF/MS, dans des cellules HT29 intactes (acénocarcinome du colon humain), l'oxygène singulet produit par le Foscan® (Biolitec Pharma Ldt, Dublin, Irlande) ainsi que la distribution des protéines des cellules après PDT. Le MALDI-TOF/MS a été utilisé pour mettre en évidence l'ortho-benzoylbenzène(o-BB) produit de réaction entre l'oxygène singulet généré par le Foscan® pendant le traitement PDT et le 1.3-diphenylisobenzofurane (1,3-DPBF, sonde spécifique de l'oxygène singulet). Cette technique permet non seulement le suivi du comportement du photosensibilisateur in situ mais également la détection directe de l'oxygène singulet dans les cellules HT29 intactes. La seconde partie de nos travaux aborde le stress oxydatif induit au niveau des cellules HT29 après PDT. Pour ce faire, nous avons eu recours au gel 2D SDS-PAGE afin d'accéder à la distribution de protéines des HT29, une approche protéomique a ensuite été effectuée par spectrométrie de masse de MALDI-FT-ICR (9.4 T, Ion Spec Varian, Californie, USA). Grâce au logiciel Imagemaster 2D platinium, la visualisation de la sous expression de quelques protéines a été possible. L'empreinte protéique spécifique de ces protéines fut caractérisée par MALDI-FT-ICR/MS) et les premiers résultats indiqueraient que les protéines de la famille des disulfides isomérases seraient sollicitées lors des processus de PDT.
Depuis plusieurs années, les quadruplexes de guanine ou G4, structures particulières de l’ADN, suscitent un intérêt grandissant. Ces structures, largement étudiées in vitro, sont encore peu connues in vivo mais semblent jouer un rôle important dans la régulation de l’expression génétique. Elles ont rapidement été considérées comme des cibles thérapeutiques potentielles pour certaines maladies telles que le cancer. Le premier indice de leur existence dans les cellules n’a été obtenu qu’en 2013 par immunodétection sur des cellules fixées. Les recherches sont actuellement tournées vers le développement de nouveaux outils moléculaires qui permettraient la visualisation des G4 dans des cellules vivantes.C’est dans ce cadre que nous avons conçu et développé une série de complexes polyazaaromatiques de ruthéniumII à base de ligands plans étendus (heptacycle dpqp et octacycle dppqp). La combinaison des propriétés photophysiques des complexes de RuII associée à la présence d’un large plan étendu devant permettre l’interaction avec les G4, positionne ces molécules comme outils potentiels pour l’étude des G4 in vivo.La première partie de ce projet porte sur la synthèse de ces nouveaux complexes de ruthénium. Une méthode originale de "chimie sur complexe" a permis d'obtenir, entre autres, un complexe possédant le ligand dpqp, fonctionnalisé par une triple liaison. Il a également été possible, « par chimie sur complexe », de construire un cycle supplémentaire sur le ligand heptacyclique (dpqp) chélaté pour obtenir les complexes [Ru(L)2dppqp]2+. Les propriétés photophysiques des différents complexes ont été étudiées. Seuls deux complexes, [Ru(phen)2dpqp-Cl]2+ et [Ru(TAP)2dpqp-Cl]2+, présentent un comportement s’approchant de celui des complexes de référence; c’est à dire des rendements quantiques comparables à [Ru(bpy)3]2+ et des durées de vie de l’état excité de l’ordre de la centaine de nanosecondes. Les autres complexes sont non luminescents et l’hypothèse d’un quenching par transfert de proton à l’état excité a été avancée pour expliquer ce comportement.Les complexes ont aussi été évalués vis à vis de différentes structures oligonucléotidiques G4 et duplexes. Tous les complexes possèdent une affinité correcte envers les G4. Comme nous l'espérions, le complexe porteur du ligand octacyclique semble être particulièrement sélectif envers les G4 par rapport à l'ADN double brin. Il a aussi été montré que deux des complexes testés peuvent être utilisés comme sondes moléculaires "light-switch ON" pour les structures G4 en milieu cellulaire. Certains des complexes synthétisés sont donc d’excellents candidats en tant qu’outils moléculaires pour l’étude des G4 in vivo.
To inactivate methicillin-resistant Staphylococcus aureus (MRSA) with minimum damage to host cells and tissue, target-oriented photofunctional nanoparticles (TOPFNs) were fabricated and characterized. MRSA is a predominant infective pathogen even in hospital and non-hospital environments due to its ability to develop high levels of resistance to several classes of antibiotics through various pathways. To solve this major problem, photodynamic inactivation (PDI) method applies to treat antibiotic-resistant bacteria. PDI involves the photosensitizer (PS) and light with a specific wavelength to be able to apply for a non-invasive therapeutic procedure to treat pathogenic bacteria by inducing apoptosis or necrosis of microorganisms. However, most current PDI researches have suffered from the instability of PDI agents in the biological environment due to the lack of selectivity and low solubility of PDI agents, which leads to the low PDI efficiency. In this study, the TOPFNs were fabricated by an esterification reaction to introduce hematoporphyrin (HP) and MRSA antibody to the surface of Fe3O4 nanoparticles. The TOPFNs were designed as dispersible PDI agent in biological condition, which was effectively used for selectively capturing and killing of MRSA. The capture efficiency TOPFNs was compared with PFNs as a negative control. The results showed that the capture efficiency of TOPFNs and PFNs was 95.55% and 6.43% in MRSA and L-929 cell mixed condition, respectively. And TOPFNs have a selective killing ability for MRSA with minimum damage to L-929 cells. Furthermore, PDI effect of TOPFNs was evaluated on the mice in vivo condition in order to check the possibility of practical medical application.
La thérapie photodynamique (PDT) est basée sur l'action d'un photosensibilisateur qui en présence de la lumière et de l'oxygène induit la synthèse d'espèces réactives de l'oxygène, toxiques pour les cellules. La PDT a été utilisée pour le traitement et la prévention du cancer de la vessie. Les études ont été réalisées in vivo. La PDT a été développé en utilisant l'Hexvix® précurseur de la protoporphyrine IX (PpIX). Les paramètres biologiques liés à la PpIX et qui régissent l'efficacité du traitement photodynamique des tumeurs vésicales ont été déterminés en fonction de deux concentrations d'Hexvix® (8 et 16 mM) induisant des effets photodynamiques diamétralement opposés. L'efficacité du traitement photodynamique obtenue pour 8 mM d'Hexvix® est liée à la localisation mitochondriale de la PpIX induisant des dommages pro-apoptotiques. La PDT entraîne la diminution significative du pourcentage de l'implantation tumorale après la TUR et permet ainsi de prévenir les récidives du cancer de la vessie.
Pour améliorer la sélectivité cellulaire tumorale et/ou l'efficacité anti-tumorale de la thérapie photodynamique (PDT), des molécules actives (peptides, pseudopeptides, agent alkylant) ont été conjuguées à la molécule photo-activable. Dans une 1ère partie, l'effet vasculaire de la PDT a été favorisé par le ciblage de la vascularisation tumorale de phénotype angiogénique. Un photosensibilisateur de type chlorine a été couplé à un peptide ligand de neuropiline-1 (NRP-1) et a montré son efficacité anti-tumorale in vitro et in vivo en PDT. Cependant, la partie peptidique s'est avérée instable in vivo. Des pseudopeptides modifiés au niveau du site de clivage du peptide initial ont été synthétisés et testés in vitro, conjugués ou non à la chlorine. L'incorporation cellulaire de la chlorine a été améliorée après son couplage aux pseudopeptides. Une technique d'ARN interférence, visant à éteindre l'expression de NRP-1 dans les cellules MDA-MB-231, surexprimant NRP-1, a mis en évidence l'incorporation cellulaire récepteur-dépendante des conjugués et leur sélectivité cellulaire. Les pseudopeptides et conjugués se sont révélés affins pour NRP-1 et plus stables in vivo. Le ruthénium (Ru) s'affiche comme une molécule d'intérêt thérapeutique en oncologie. Dans une 2ème partie, nous avons appréhendé l'intérêt in vivo de coupler cet agent alkylant à des photosensibilisateurs de type porphyrine et chlorine. Les études d'efficacité in vivo sur un modèle de souris nude xénogreffées en ectopique avec des cellules de carcinome oral humain KB, ont été menées par une méthodologie de plans d'expérience. Deux conjugués [Ru(_6-p-PriC6H4Me)(5-(3-pyridyl)-10,15,20-triphenylporphyrin)Cl2, ou Rut1, et Ru4(_6-p-PriC6H4Me)4(5,10,15,20-tetra(3-pyridyl)porphyrin)Cl8, ou Rut4] efficaces in vitro ont été comparés in vivo. Le composé Rut4 est apparu comme le plus efficace. Des études de biodistribution par spectrofluorimétrie fibrée et de pharmacocinétique classique ont montré un intervalle drogue-lumière optimal de 24h. Néanmoins, malgré l'optimisation des modalités thérapeutiques, le couplage du Ru avec une molécule photo-activable (porphyrine ou chlorine) n'a pas conduit à une efficacité anti-tumorale significative in vivo
La thérapie pbotodynamique (PDT est une modalité de traitement des petites tumeurs localisées accessibles à la lumière. Son principe repose sur l'action conjuguée d'un photo sensibilisant, de la lumière et de l'oxygène. Une dosimétrie correcte est nécessaire pour assurer le traitement complet et des résultats reproductibles. Un élément clé de la dosimétrie à prendre en compte est le photoblanchiment, étant donné qu'il diminue la concentration du photosensibilisant au cours du traitement. Il est donc indispensabte d'appréhender les mécanismes du photoblanchiment pour maîtriser au mieux le traitement. Le rendement quantique de photoblanchiment de la m-THPBC en solution avec des protéines est de 5.7 X 10-4, les formes agrégées de photosensibilisants photoblanchissent plus lentement que les formes monomérisées. La m-THPBC se transforme sous l'effet de la lumière en m-THPC, ce rendement de phototransformation est influencé par l'état d'agrégation de la molécule. D'autres photoproduits tels que la m-THPBC di-bydroxylée et la m-THPC dibydroxylée ainsi que des dérivés dipyrines ont été observés. L'espèce responsable du photoblanchiment a été identifiée comme étant l'oxygène singulet. ln vitro, la LD50 de la mTHPBC est 0.7 J cm-2. Le photoblanchiment est 3 à 5 fois plus sensible que celui de la m-THPC. L'étude de la localisation intra-cellulaire des photosensibilisants a montré une différence, la m-THPC s'accumulant dans le réticulum endoplasmique alors que la m-THPBC se localise dans les mitochondries. ln vivo, la m-THPBC possède un photoblanchiment 4 à 10 fois plus élevé que la m-THPC, ainsi qu'une bonne accumulation tumorale. Ces résultats in vitro et in vivo sont particulièrement intéressants en terme de ratio thérapeutique, de sélectivité du traitement et de photosensibilité cutanée.
To address the issue of low cellular uptake of photosensitizers by cancer cells in photodynamic therapy (PDT), we designed a smart plasma membrane-activatable polymeric nanodrug by conjugating the photosensitizer protoporphyrin IX (PpIX) and polyethylene glycol (PEG) with glycol chitosan (GC). The as-prepared GC-PEG-PpIX can self-assemble into core-shell nanoparticles (NPs) in aqueous solution and the fluorescence of PpIX moieties in the inner core is highly quenched due to strong π–π stacking. Interestingly, when encountering plasma membranes, the GC-PEG-PpIX NPs can disassemble and stably attach to plasma membranes due to the membrane affinity of PpIX moieties, which effectively suppresses the self-quenching of PpIX, leading to significantly enhanced fluorescence and singlet oxygen (¹O2) production upon laser irradiation. The massively produced ¹O2 can compromise the integrity of the plasma membrane, enabling the influx of extracellular nanoagents into cells to promote cell death upon further laser irradiation. Through local injection, the membrane anchored GC-PEG-PpIX enables strong physical association with tumor cells and exhibits highly enhanced in vivo fluorescence at the tumor site. Besides, excellent tumor accumulation and prolonged tumor retention of GC-PEG-PpIX were realized after intravenous injection, which ensured its effective imaging-guided PDT.
The process by which viruses destabilize endosomal membranes in an acidification dependent manner has been mimicked with synthetic peptides that are able to disrupt liposomes, erythrocytes, or endosomes of cultured cells. Peptides containing the 20 amino-terminal amino acid sequence of influenza virus hemagglutinin as well as acidic derivatives showed erythrocyte lysis activity only when peptides were elongated by an amphipathic helix or by carboxyl-terminal dimerization. Interestingly, peptides consisting of the 23 amino-terminal amino acids of influenza virus hemagglutinin were also active in erythrocyte lysis. When peptides were in corporated into DNA complexes that utilize a receptor-mediated endocytosis pathway for uptake into cultured cells, either by ionic interaction with positively charged polylysine-DNA complexes or by a streptavidin-biotin bridge, a strong correlation between pH-specific erythrocyte disruption activity and gene transfer was observed. A high-level expression of luciferase or interleukin-2 was obtained with optimized gene transfer complexes in human melanoma cells and several cell lines.
Complexes containing plasmid DNA, transferrin-polylysine conjugates, and polylysine-conjugated peptides derived from the N-terminal sequence of the influenza virus hemagglutinin subunit HA-2 have been used for the transfer of luciferase or beta-galactosidase marker genes to K562 cells, HeLa cells, and BNL CL.2 hepatocytes. These DNA complexes mimic the entry of viruses into cells, as they contain functions for (i) the packaging of the nucleic acid with polylysine, (ii) the attachment to the cell and receptor-mediated endocytosis with transferrin as a ligand, and (iii) the release from endosomes by using membrane-disrupting influenza peptides. The presence of these influenza peptide conjugates in the DNA complexes renders the complexes active in membrane disruption in a liposome leakage assay and results in a substantial augmentation of the transferrin-polylysine-mediated gene transfer.
Photodynamic therapy involves administration of a tumorlocalizing photosensitizing agent, which may require metabolic synthesis
(i.e., a prodrug), followed by activation of the agent by light of a specific wavelength. This therapy results in a sequence
of photochemical and photobiologic processes that cause irreversible photodamage to tumor tissues. Results from preclinical
and clinical studies conducted worldwide over a 25-year period have established photodynamic therapy as a useful treatment
approach for some cancers. Since 1993, regulatory approval for photodynamic therapy involving use of a partially purified,
commercially available hematoporphyrin derivative compound (Photofrin®) in patients with early and advanced stage cancer of
the lung, digestive tract, and genitourinary tract has been obtained in Canada, The Netherlands, France, Germany, Japan, and
the United States. We have attempted to conduct and present a comprehensive review of this rapidly expanding field. Mechanisms
of subcellular and tumor localization of photosensitizing agents, as well as of molecular, cellular, and tumor responses associated
with photodynamic therapy, are discussed. Technical issues regarding light dosimetry are also considered. [J Natl Cancer Inst
1998;90:889-905]
Transfection of HC-11 murine epithelial mammary cells as well as murine and sheep mammary glands were carried out using insulin-containing constructs that deliver DNA by receptor-mediated endocytosis to receptor-expressing cells. In vivo transfection of mammary gland tissue with the luciferase gene was carried out by introducing the DNA constructs into the mammary ducts of both mice and sheep. The successful transfection of ewe mammary glands was demonstrated by the detection of luciferase activity in mammary gland biopsy material up to a month after a single administration of the construct. To test whether products of expression of transfected genes could be secreted into the milk in this system, the N-terminal secretory signal sequences of bovine beta-lactoglobulin or the entire coding sequence of human alpha-lactalbumin were fused to the N terminus of the luciferase gene. After transfection with the modified luciferases, both murine and sheep milk could be shown to contain luciferase activity, whereas mice, which had been transfected with the nonmodified luciferase gene, did not secrete any activity in the milk. This approach demonstrates for the first time the possibility of gene transfer in vivo into mammary gland epithelial cells using constructs delivering DNA via receptor-mediated endocytosis.
Plasmid DNA/glycosylated polylysine complexes were used to transfer in vitro a luciferase reporter gene into human hepatoma cells by a receptor-mediated endocytosis process. HepG2 cells which express
a galactose specific membrane lectin were efficiently and selectively transfected with pSV2Luc/lactosylated polylysine complexes
in a sugar dependent manner: I) HepG2 cells which do not express membrane lectin specific for mannose were quite poorly transfected
with pSV2Luc/mannosylated polylysine complexes, II) HeLa cells which do not express membrane lectin specific for galactose
were not transfected with pSV2Luc/lactosylated polylysine complexes. The transfection efficiency of HepG2 cells with pSV2Luc/lactosylated
polylysine complexes was greatly enhanced either in the presence of chloroquine or in the presence of a fusogenic peptide.
A 22-residue peptide derived from the influenza virus hemagglutinin HA2 N-terminal polypeptide that mimics the fusogenic activity
of the virus, was selected. In the presence of the fusogenic peptide, the luciferase activity in HepG2 cells was 10 fold larger
than that of cells transfected with pSV2Luc/lactosylated polylysine complexes in the presence of chloroquine.
The prognosis for patients with high-grade cerebral glioma is poor. Most treatment failures are due to local recurrence of tumor, indicating that a more aggressive local therapy could be beneficial. Adjuvant treatments such as porphyrin-sensitized photodynamic therapy (PDT) or boron neutron capture therapy (BNCT) have the potential to control local recurrence. The selective tumor uptake of a boronated porphyrin was studied in CBA mice bearing an implanted intracerebral glioma. Biopsy samples of tumor, normal brain, and blood were analyzed by a fluorometric assay following intraperitoneal and intravenous administration of boronated protoporphyrin (BOPP). This compound was selectively localized to tumor at ratios as high as 400:1 relative to normal brain. Confocal laser scanning microscopy of glioma cells in vitro and in vivo showed that BOPP was localized within mitochondria and excluded from the nucleus of these cells. This discrete subcellular localization was confirmed by density gradient ultracentrifugation after homogenization of mouse tumor biopsies. The selective discrete localization of these compounds within the tumor suggests that this compound may be used as a dual PDT/BNCT sensitizer.
NHIK 3025 cells were incubated with Photofrin II (PII) and/or tetra (3-hydroxyphenyl)porphyrin (3THPP) and exposed to light at either 400 or 420 nm, i.e. at the wavelengths of the maxima of the fluorescence excitation spectra of the two dyes. The kinetics of the photodegradation of the dyes were studied. When present separately in the cells the two dyes are photodegraded with a similar quantum yield. 3THPP is degraded 3-6 times more efficiently by light quanta absorbed by the fluorescent fraction of 3THPP than by light quanta absorbed by the fluorescent fraction of PII present in the same cells. The distance diffused by the reactive intermediate, supposedly mainly 1O2, causing the photodegradation was estimated to be on the order of 0.01-0.02 micron, which corresponds to a lifetime of 0.01-0.04 microsecond of the intermediate in the cells. PII has binding sites at proteins in the cells as shown by an energy transfer band in the fluorescence excitation spectrum at 290 nm. During light exposure this band decays faster than the Soret band of PII under the present conditions. Photoproducts (1O2 etc.) generated at one binding site contribute significantly in the destruction of remote binding sites.
Intracellular localization, intracellular translocation and photobleaching following non-lethal laser microirradiation of the fluorescing derivatives of sulfonated aluminum phthalocyanines (Al-PcSn, n = 1-4) in a human melanoma cell line (LOX) were studied by means of confocal laser scanning microscopy (LSM) and image processing. Use of confocal microscopy allowed 3-dimensional information to be obtained. Both Al-PcS1 and Al-PcS2 localized diffusely in the cytoplasm of the cells, while Al-PcS3 and Al-PcS4 exhibited a granular pattern in the extranuclear fraction of the cells. None of the Al-PcSn family was observed in the nuclei of the cells except that a small fraction of fluorescence was occasionally detected in nuclei of some cells treated with Al-PcS1 and Al-PcS2. Furthermore, exactly the same granular localization patterns and positions in the same cells were found after incubation initially with Al-PcS3 (or Al-PcS4) followed by acridine orange (AO) which emits red fluorescence from lysosomes of cells. Thus, the granular fluorescence of Al-PcS3 and Al-PcS4 is confined to the lysosomes of the LOX cells. Non-lethal laser exposure of cells incubated with high concentrations of the 2 dyes resulted in a translocation of the dyes from the lysosomes to the whole cytoplasm and an increase in total intracellular fluorescence intensity. Finally, a small fraction of the dyes localized into the nuclei of the cells. The laser exposure of cells incubated with low concentrations of the lysosomally localized dyes resulted in an increase in the intracellular fluorescence intensity with no translocation of the dyes. Under all conditions, high laser exposure resulted in a decrease in the total intracellular fluorescence intensity.
The yeast repressor alpha 2 is shown, by analysis of deletion-bearing alpha 2-beta-galactosidase hybrid proteins, to have two structurally distinct nuclear localization signals. The cellular location of hybrid proteins was determined by indirect immunofluorescence and optical sectioning of whole fixed yeast cells. The two nuclear localization signals are far apart in the alpha 2 primary structure and do not have any sequence homology. One signal is, as reported previously, within the aminoterminal 13 amino acids of alpha 2. Deletion of only this aminoterminal signal has no evident effect on nuclear localization. The second signal is in a central portion of alpha 2, within the alpha 2 homeodomain. Since this signal is within the amino terminus of the alpha 2 homeodomain, the homeodomain mediates nuclear localization in addition to, and independently of, DNA binding. Deletion of only this second signal results in inefficient localization and accumulation of mutant protein at discrete sites on the nuclear envelope assumed to be nuclear pores. We propose that the two signals in alpha 2 are functionally distinct and act at different steps in a localization pathway.
A photosensitizer conjugate, chlorin e6 (Ce6) covalently bound to 1-micron-diameter polystyrene microspheres, has been investigated in the photodynamic destruction of MGH-U1 human bladder carcinoma cells in vitro. The microspheres were taken up avidly by the carcinoma cells; confocal laser scanning fluorescence microscopy showed them to be localized in the cytoplasm, apparently within lysosomes, visualized by labeling with acridine orange. In contrast, fluorescence of unconjugated Ce6 was present within most cellular membranes. Use of Ce6-microsphere conjugates led to a 20-fold-higher mean intracellular concentration, compared with unconjugated Ce6. Cells incubated in the presence of Ce6-microsphere conjugates (0.43 microM equivalent) and subsequently irradiated at 659 nm with a dye laser pumped by an argon-ion laser showed dose-dependent phototoxicity, leading to total inhibition of colony formation at a radiant exposure of 5J/cm2; in contrast, cells incubated with either unconjugated Ce6 (0.43 microM) or unconjugated microspheres before laser irradiation were unaffected. Cells pretreated with Ce6-microsphere conjugates and irradiated in the presence of 90% 2H2O showed significantly increased phototoxicity, an effect consistent with an important role for excited-state singlet oxygen in the mechanism of injury. In solution, however, photosensitized generation of singlet oxygen with Ce6-microsphere conjugates was 9 times less efficient than with unconjugated Ce6. The markedly greater phototoxicity of Ce6-microsphere conjugates compared to unconjugated Ce6 was therefore a consequence of the high intracellular Ce6 concentration attained by phagocytosis of the conjugates and their particular sites of intracellular localization. Thus, these conjugates are an efficient system for the delivery of photosensitizing drugs to carcinoma cells.
The heparin-sensitive binding of 125I-labeled low-density lipoprotein (LDL) to homogenates from 18 different normal human tissues and some solid tumors was determined. The binding to adrenal and liver homogenates fulfilled criteria established for the binding of LDL to its receptor--namely, (i) saturability, (ii) sensitivity to proteolytic destruction, (iii) inhibition by EDTA, and (iv) heat sensitivity. When the binding of 125I-labeled LDL was assayed at a constant concentration (50 micrograms/ml), the adrenal gland and the ovary had the highest binding of normal tissues. The highest binding per g of tissue overall was obtained in homogenates of a gastric carcinoma and a parotid adenoma. When the weights of the parenchymatous organs were considered, the major amount of LDL receptors was contained in the liver. To study the possible regulation of hepatic LDL-receptor expression, 11 patients were pretreated with cholestyramine (8 g twice a day for 3 weeks). Increased binding activity (+105%, P less than 0.001) was obtained in homogenates from liver biopsies from the cholestyramine-treated patients as compared with 12 untreated controls. It is concluded that the liver is the most important organ for LDL catabolism in humans and that the receptor activity in this organ can be regulated upon pharmacologic intervention. Further studies are needed to confirm the possibility that certain solid tumors can exhibit high numbers of LDL receptors.
A synthetic, amphipathic 30-amino acid peptide with the major repeat unit Glu-Ala-Leu-Ala (GALA) was designed to mimic the behavior of the fusogenic sequences of viral fusion proteins. GALA is a water-soluble peptide with an aperiodic conformation at neutral pH and becomes an amphipathic α-helix as the pH is lowered to 5.0 where it interacts with bilayers. Fluorescence energy transfer measurements indicated that GALA induced lipid mixing between phosphatidylcholine small unilamellar vesicles but not large unilamellar vesicles. This lipid mixing occurred only at pH 5.0 and not at neutral pH. Concomitant with lipid mixing, the vesicles increased in diameter from 500 to 750 to 1000 Å as measured by dynamic light scattering and internal volume determination. GALA induced leakage of small molecules (M(r) 450) at pH 5.0 was too rapid to permit detection of contents mixing. However, retention of larger molecules (M(r) 4100) under the same conditions suyggests that vesicle fusion is occurring. For a 100/1 lipid/peptide ratio all vesicles fused just once, whereas for a 50/1 ratio higher order fusion products formed. A mass action model gives good simulation of the kinetics of increase in fluorescence intensity and yields rate constants of aggregation and fusion. As the lipid to peptide ratio decreases from 100/1 to 50/1 both rate constants of aggregation and fusion increase, indicating that GALA is a genuine inducer of vesicle fusion. The presence of divalent cations which can alter GALAs conformation at pH 7.5 had little effect on its lipid mixing activity. These peptides did not have any lipid mixing activity nor did they induce an increase in vesicle size. Together, these results indicate that fusion of phosphatidylcholine small unilamellar vesicles induced by GALA requires both a peptide length greater than 16 amino acids as well as a defined topology of the hydrophobic residues.
A monoclonal antibody that reacts with proteins in the nuclear pore complex of rat liver (Snow, C. M., A. Senior, and L. Gerace. 1987. J. Cell Biol. 104:1143-1156) has been shown to cross react with similar components in Xenopus oocytes, as determined by immunofluorescence microscopy and immunoblotting. We have microinjected the antibody into oocytes to study the possible role of these polypeptides in nucleocytoplasmic transport. The antibody inhibits import of a large nuclear protein, nucleoplasmin, in a time- and concentration-dependent manner. It also inhibits export of 5S ribosomal RNA and mature tRNA, but has no effect on transcription or intranuclear tRNA processing. The antibody does not affect the rate of diffusion into the nucleus of two small proteins, myoglobin and ovalbumin, indicating that antibody binding does not result in occlusion of the channel for diffusion. This suggests that inhibition of protein and RNA transport occurs by binding of the antibody at or near components of the pore that participate in mediated transport.
The binding, internalization, subcellular trafficking and in vitro cytotoxicity of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer–anti-cancer drug–OV-TL16 antibody (Ab) conjugates in the ovarian carcinoma OVCAR-3 cell line have been investigated. Adriamycin (ADR) and meso chlorin e6 mono(N-2-aminoethylamide) (Mce6) photosensitizer were used as anti-cancer drugs. Targeted (Ab-containing) conjugates were compared with non-targeted HPMA copolymer–drug conjugates and with free drugs. Targeted conjugates were taken up rapidly by cells and detected within lysosomes by confocal fluorescence microscopy. The ADR attached to polymer chains via a degradable GFLG spacer was released from the conjugate, diffused via the lysosomal membrane into the cytoplasm and ultimately accumulated in the cell nuclei. In contrast, conjugates containing ADR bound via the GG spacer accumulated in the lysosomes, but no fluorescence could be detected in the cell nuclei. Binding the drugs to a non-targeted HPMA copolymer decreased their cytotoxicity in vitro. The IC50 dose increased from 2 μM for free ADR to 150 μM for P(GFLG)–ADR (P is the HPMA copolymer backbone) and from 0.34 μM for free Mce6 (with light) to 290 μM for P–(GG)–Mce6. However, attachment of OV-TL16 Abs rendered HPMA copolymer–drug conjugates biorecognizable by OVCAR-3 cells and markedly increased their cytotoxicity. The IC50 doses were 4.4 and 0.38 μM for the targeted conjugates P(GFLG)–ADR–Ab and P(GG)–Mce6–Ab (with light), respectively. Biorecognition was shown to be specific by inhibition experiments with free Ab. The findings indicate the potential of these conjugates as effective agents in the treatment of ovarian cancer. Int. J. Cancer 75:600–608, 1998. © 1998 Wiley-Liss, Inc.
A photosensitizer conjugate, chlorin eâ (Ceâ) covalently bound to 1-μm-diameter polystyrene microspheres, has been investigated in the photodynamic destruction of MGH-U1 human bladder carcinoma cells in vitro. The microspheres were taken up avidly by the carcinoma cells; confocal laser scanning fluorescence microscopy showed them to be localized in the cytoplasm, apparently within lysosomes, visualized by labeling with acridine orange. Use of Ceâ-microsphere conjugates led to a 20-fold-higher mean intracellular concentration, compared with unconjugated Ceâ. Cells incubated in the presence of Ceâ-microsphere conjugated (0.43 μM equivalent) and subsequently irradiated at 659 nm with a dye laser pumped by an argon-ion laser showed dose-dependent phytotoxicity, leading to total inhibition of colony formation at a radiant exposure of 5 J/cm². Cells pretreated with Ceâ-microsphere conjugates and irradiated in the presence of 90% ²HâO showed significantly increased phototoxicity, and effect consistent with an important role for excited-state singlet oxygen in the mechanism of injury. The markedly greater phototoxicity of Ceâ-microsphere conjugates compared to unconjugated Ceâ was therefore a consequence of the high intracellular Ceâ concentration attained by phagocytosis of the conjugates and their particular sites of intracellular localization. Thus, these conjugates are an efficient system for the delivery of photosensitizing drugs to carcinoma cells.
Confocal fluorescence microscopy, using a newly constructed laser line-scanning confocal microscope, was applied to an investigation of the early stages of photoinduced destruction of V79–4Chinese hamster fibroblasts using aluminum and zinc phthalocyanines as photosensitize. Results obtained in this work show that aluminum and zinc phthalocyanines, once internalized, localize in perinuclear sites that are disrupted upon light exposure resulting in fluorescence redistribution. The combination of laser-line scanning with charge-coupled device detection used in the confocal microscope developed in this work can enable rapid high-resolution sequential imaging, which is ideal for studying photoinduced intracellular fluorescence dynamics.
The development of second-generation photosensitizers to improve photodynamic therapy (PDT) is an area of extensive research, Three such compounds that have been synthesized in our group are polysubstituted Zn(II) phthalocyanines that differ in their overall net charge (one cationic, one anionic and one neutral). The aim of this study was to characterize the drugs in terms of their uptake, cell killing efficacy and subcellular localization in RIF-1 cells in vitro and to identify any possible structure/function relationships, The results show that the relative uptakes and cell killing efficacy of each of the drugs follows the order cationic much greater than neutral > anionic. For the anionic and cationic drugs the initial subcellular localization was in the lysosomes as determined by fluorescence microscopy. The neutral phthalocyanine demonstrated a more diffuse localization characteristic of membrane staining with some involvement of the Golgi apparatus in the perinuclear area. Following light exposure the drugs rapidly relocalized to different sites within the cell in a manner that was apparently charge dependent and this relocalization was accompanied by an increase in the fluorescence associated with the drugs. This indicates that the primary sites of localization of these photosensitizers may not be as important as their secondary sites in producing cell killing during PDT, especially as the fluorescence intensity increases on relocalization.
We have evaluated the potential usefulness of radiolabelled [DTPA0,Tyr3]octreotide and [DOTA0,Tyr3]octreotide as radiopharmaceuticals for somatostatin receptor–targeted scintigraphy and radiotherapy. In vitro somatostatin receptor binding and in vivo metabolism in rats of the compounds were investigated in comparison with [111In-DTPA0] octreotide. Comparing different peptide–chelator constructs, [DTPA0,Tyr3]octreotide and [DOTA0, Tyr3]octreotide were found to have a higher affinity than [DTPA0]octreotide for subtype 2 somatostatin receptors (sst2) in mouse AtT20 pituitary tumour cell membranes (all IC50 values obtained were in the low nanomolar range). In vivo studies in CA20948 tumor-bearing Lewis rats revealed a significantly higher uptake of both 111In-labelled [DOTA0,Tyr3]octreotide and [DTPA0,Tyr3]octreotide in sst2-expressing tissues than after injection of [111In-DTPA0]octreotide, showing that substitution of Tyr for Phe at position 3 in octreotide results in an increased affinity for its receptor and in a higher target tissue uptake. Uptake of 111In-labelled [DTPA0]octreotide, [DTPA0,Tyr3]octreotide and [DOTA0,Tyr3]octreotide in pituitary, pancreas, adrenals and tumour was decreased to less than 7% of control by pre-treatment with 0.5 mg unlabelled octreotide/rat, indicating specific binding to sst2. Comparing different radionuclides, [90Y-DOTA0,Tyr3]octreotide had the highest uptake in sst2-positive organs, followed by the [111In-DOTA0,Tyr3]octreotide, whereas [DOTA0, 125I-Try3]octreotide uptake was low compared to that of the other radiopharmaceuticals, when measured 24 hr after injection. Renal uptake of 111In-labelled [DTPA0]octreotide, [DTPA0, Tyr3]octreotide and [DOTA0,Tyr3]octreotide was reduced over 50% by an i.v. injection of 400 mg/kg d-lysine, whereas radioactivity in blood, pancreas and adrenals was not affected. Int. J. Cancer 75:406–411, 1998. © 1998 Wiley-Liss, Inc.
We evaluated the following '"In-labeled somatostatin (SS) analogues (diethylenetriaminepentaacetic acid, Oil'A: tetraazacyclododecanetet- raacetic acid, DOTA): (DTPA°loctreotide, |DTPA",Tyr'loctreotide, (DTPA°,D-Tyr'loctreotide. |DTPA°,Tyr')octreotate (Thr(ol) in octreotide replaced with Thr|, and (DOTA°,Tyr'loctreotide, in vitro and in vivo. In vitro, all compounds .showed high and specific binding to SS recep tors in mouse pituitary AtT20 tumor cell membranes, and K '.,,,s were in the nanomolar range. Furthermore, all compounds showed specific inter- nalization in rat pancreatic tumor cells; uptake of |'"ln- DTPA",Tyr*)octreotate was the highest of the compounds tested, and that of |l"ln-DTPA0,l)-Tyr'loctreotide was the lowest. Biodistribution exper iments in rats showed that, 4, 24, and 48 h after injection of |'"ln- DTPA°,Tyr')octreotide, |mIn-DTPA0.Tyr3loctreotate, and ('"In- DOTA°,Tyr'loctreotide, radioactivity in the octreotide-binding, receptor- expressing tissues and tumor-to-blood ratios were significantly higher than those after injection of ("'In-DTPA")octreotide. Uptake of l'"ln- DTPA°,Tyr')octreotate in the target organs was also, in vivo, the highest of the radiolabeled peptides tested, whereas that of ('"in-DTPA",!)- Tyr'loctreotide was the lowest. Uptake of |"'ln-DTPA",Tyr*loctreotide, ('"ln-DTPA",TyrJloctreotate, and ('"ln-DOTA0,TyrJloctreotide in tar get tissues was blocked by >90% by 0.5 mg of unlabeled octreotide, indicating specific binding to the octreotide receptors. Blockade of ('"In- DTPA°,D-Tyr'loctreotide was >70%. In conclusion, radiolabeled (DTPA°,Tyr
Experiments with human hepatoma PLC/PRF/5 cells and human embryo skin fibroblasts involving the use of three different tests (colony formation, Trypan blue exclusion, labeled thymidine incorporation) have demonstrated a significantly higher photosensitizing activity of chlorin e6 conjugates with internalizable ligands as compared to that of chlorin e6 itself. Receptor-mediated internalization of chlorin e6 conjugates ensures a greater photosensitization of cells than binding of those conjugates to cell surface receptors. The suitability of such conjugates that permit the delivery of a photosensitizer to sensitive intracellular targets is discussed.
Breaking a Yang–Mills symmetry at very high energy could lead to macroscopic structures in the vacuum called ‘strings’, which may be the cause of galaxy formation and clustering. Dissipative gravitational interactions of string loops can produce unusual astrophysical side effects. Such scenarios generally predict a stochastic background of gravitational radiation which should be observable using the millisecond pulsar 1937 + 21. Gravitational radiation recoil accelerates loops, but dynamical friction on ordinary matter tends to slow them down so they may be able to accrete matter. Internal oscillations of loops produce a time-varying shear perturbation which may lead to an observable heating of stellar or gaseous systems in their immediate neighbourhood.
Nuclear sites of action have long been recognized for steroid and thyroid hormones. Evidence accumulated over the past decade suggests that polypeptide hormones and growth factors may also have nuclear sites of action.
The role of membrane receptors is regarded as being to transduce the signal represented by ligand binding from the external cell surface across the membrane into the cell. Signals are subsequently conveyed from the cytoplasm to the nucleus through a combination of second-messenger molecules, kinase/phosphorylation cascades, and transcription factor (TF) translocation to effect changes in gene expression. Mounting evidence suggests that through direct targeting to the nucleus, polypeptide ligands and their receptors may have an important additional signaling role. Ligands such as those of the platelet-derived and fibroblast growth factor classes, as well as cytokines such as interferon-γ and interleukins-1 and -5, have been found to localize in the nucleus through the action of nuclear localization sequences (NLSs). Where tested, these NLSs appear to be essential for full signaling activity and may be responsible for cotranslocating receptors to the nucleus in complexes with their ligands. The implication is that, subsequent to endocytosis at the membrane, particular polypeptide ligands or their receptors, or both, may translocate to the nucleus to participate directly in gene regulation. BioEssays20:400–411, 1998.© 1998 John Wiley & Sons Inc.
We have evaluated the potential usefulness of radiolabelled [DTPA0,Tyr3]octreotide and [DOTA0,Tyr3]octreotide as radiopharmaceuticals for somatostatin receptor–targeted scintigraphy and radiotherapy. In vitro somatostatin receptor binding and in vivo metabolism in rats of the compounds were investigated in comparison with [111In-DTPA0] octreotide. Comparing different peptide–chelator constructs, [DTPA0,Tyr3]octreotide and [DOTA0, Tyr3]octreotide were found to have a higher affinity than [DTPA0]octreotide for subtype 2 somatostatin receptors (sst2) in mouse AtT20 pituitary tumour cell membranes (all IC50 values obtained were in the low nanomolar range). In vivo studies in CA20948 tumor-bearing Lewis rats revealed a significantly higher uptake of both 111In-labelled [DOTA0,Tyr3]octreotide and [DTPA0,Tyr3]octreotide in sst2-expressing tissues than after injection of [111In-DTPA0]octreotide, showing that substitution of Tyr for Phe at position 3 in octreotide results in an increased affinity for its receptor and in a higher target tissue uptake. Uptake of 111In-labelled [DTPA0]octreotide, [DTPA0,Tyr3]octreotide and [DOTA0,Tyr3]octreotide in pituitary, pancreas, adrenals and tumour was decreased to less than 7% of control by pre-treatment with 0.5 mg unlabelled octreotide/rat, indicating specific binding to sst2. Comparing different radionuclides, [90Y-DOTA0,Tyr3]octreotide had the highest uptake in sst2-positive organs, followed by the [111In-DOTA0,Tyr3]octreotide, whereas [DOTA0, 125I-Try3]octreotide uptake was low compared to that of the other radiopharmaceuticals, when measured 24 hr after injection. Renal uptake of 111In-labelled [DTPA0]octreotide, [DTPA0, Tyr3]octreotide and [DOTA0,Tyr3]octreotide was reduced over 50% by an i.v. injection of 400 mg/kg d-lysine, whereas radioactivity in blood, pancreas and adrenals was not affected. Int. J. Cancer 75:406–411, 1998. © 1998 Wiley-Liss, Inc.
Recent progress indicates that there are multiple pathways of nucleocytoplasmic transport which involve specific targeting sequences, such as nuclear localization sequences (NLSs), and cytosolic receptor molecules of the importin/karyopherin superfamily which recognise and dock the NLS-containing proteins at the nuclear pore. This first step of nuclear import/export is of central importance, with the affinity of the importin-targeting sequence interaction a critical parameter in determining transport efficiency. Different importins possess distinct NLS-binding specificities, which allows the system to be modulated through differential expression of the importins themselves, as well as through competition between different importins for the same protein, and between different proteins for the same importin. The targeting sequence-importin interaction can also be influenced directly by phosphorylation increasing the affinity of the interaction with importins or by targeting sequence masking through phosphorylation or specific protein binding. Targeting sequence recognition thus appears to represent a key control point in the regulation of nuclear transport. BioEssays 22:532–544, 2000. © 2000 John Wiley & Sons, Inc.
The interaction of native and cationized bovine serum albumin-gold complex with confluent monolayers of sheep brain microvascular endothelial cells was investigated. These cells were used as anin vitro model of the blood-brain barrier. After exposure to both complexes for 5, 15, 30, and 60 min, the endothelial cells were washed with phosphate-buffered saline, fixed, and processed for electron microscopy. The results obtained suggest that (a) contrary to previous observationsin vivo, cultured endothelial cells were able to transfer native bovine serum albumin-gold complex particles from apical to basolateral surfaces presumably via a fluid-phase mechanism; (b) the most conspicuous difference in the interaction of both albumin-gold complexes consisted in a significantly greater adsorption of catonized than of native bovine serum albumin-gold complex to the apical surface of the endothelial cells; (c) the further fate of the endocytosed complexes was similar, that is, their major part was internalized into endosomes, multivesicular bodies and lysosomes whereas a smaller fraction was apparently transferred to the basolateral plasma membrane where presumably they were exocytosed into the subendothelial space.
These observations provide new ultrastructural evidence that both native and cationized albumin are endocytosed and eventually transported by a non-specific, fluid-phase mechanism, and also support the results of earlier quantitative studies indicating the absence of albumin receptors in brain endothelia.
The fluorescence emission of hydrophilic tetrasulphonated aluminium phthalocyanine (AlPcS4) and hydrophobic zinc phthalocyanine (ZnPc), bound to the membrane of liposomes, was investigated in vivo in an appropriate tumour model of the rat bladder and in RR 1022 epithelial cells of the rat. The sensitizers were administered systemically to the rats and photodynamic therapy (PDT) was performed 24 h later. During PDT treatment, the fluorescence was measured every 30 s. The fluorescence was excited with 633 nm light from an HeNe laser and the fluorescence spectra were detected with an optical multichannel analyser system. PDT was performed for both sensitizers using 672 nm light from an Ar+ dye laser.The fluorescence changes during PDT were significantly different for the two phthalocyanines. For AlPcS4, an initial fluorescence intensity increase, followed by subsequent photobleaching, was observed. In contrast, ZnPc fluorescence showed an exponential decrease and no increase at the start of treatment. Tumour necrosis 24 h after PDT was significant only for ZnPc.RR 1022 cells incubated for 24 h with AlPcS4 revealed a granular fluorescence pattern, whereas ZnPc was localized diffusely in the cytoplasm of the cells. In agreement with the in vivo measurements, subcellular relocalization and a fluorescence intensity increase were detected exclusively in the case of AlPcS4. Morphological changes at this time were significant only for ZnPc. The subcellular localization and fluorescence kinetics were obtained using a confocal laser scanning microscope.
Meso-tetra(4-sulphonatophenyl)porphyrin (TPPS4) taken up by cells is mainly localized in lysosomes as previously shown by fluorescence microscopical and fluorescence spectroscopical investigations. In the present study the intracellular fluorescence behaviour and the intracellular amount of this dye at various growth periods of cells were examined. For cells irradiated in the growth phase a relocalization of TPPS4 from the lysosomes into the cytoplasm and finally into the nucleus was observed. In contrast, for cells irradiated in the stationary phase no redistribution could be detected and therefore no evidence for severe damage of the lysosomal membranes and subsequently for the release of lytical enzymes is given. In both cases lethal damage of the cells was achieved as examined using the trypan blue exclusion test. This indicates that damage of the lysosomes is less important in the photodynamic inactivation of cells sensitized by TPPS4.
A soluble construct consisting of a plasmid carrying the gene of the SV40 large T-antigen and an insulin—poly-l-lysine conjugate is able to selectively transfect PLC/PRF/5 human hepatoma cells which possess insulin receptors. Transfection can be efficiently competed by excess free insulin. To examine intracellular transport of the construct, it was fluorescently labeled and its accumulation on and in cells visualized by video-enhanced microscopy and quantitative confocal laser scanning microscopy. After 2 h at 37 °C, the labeled construct was found predominantly in intracellular acidic compartments, with a substantial portion of fluorescence localized both near and in the cell nucleus. Binding, endocytosis, and nuclear localization of the labeled conjugate could all be competed by excess free insulin, thus indicating that entry of the conjugate into cells was specifically mediated by the insulin receptor.
Photodynamic therapy (PDT) has successfully been used to induce vascular occlusion via endothelial damage and subsequent thrombosis. To increase the selective of this method for neovascularizations, characteristics in the ultrastructure of the proliferative vessel wall allow physiological vessels to be spared and predominantly neovascularizations to be occluded: (a) Due to the disturbance of the blood-retina barrier, free dye molecules accumulate within the vascular wall. Using a dye with prolonged retention, such as phthalocyanine (CASPc), it is possible to thrombose neovascularizations 24 h post injection while leaving the physiological vasculature of the anterior segment of the rabbit eye unaffected. (b) Proliferating endothelial cells express high numbers of low-density lipoprotein (LDL) receptors. Chlorin e6 (Ce6), a potent photosensitizer, is covalently bound to LDL. Intravascularly, ce6-LDL complexes selectively label neovascular walls. Since ce6-LDL is incorporated intracellular into enzymatically active lysosomes, photothrombosis is effectively achieved at low drug and light doses in vivo. In addition, the induced damage is spatially confined to the inner vascular lining. We conclude that carrier-mediated PDT may offer a new and sensitive approach for selective treatment of intraocular neovascularizations.
Experiments with human hepatoma PLC/PRF/5 cells involving the use of two different tests (colony formation and Trypan blue exclusion) have demonstrated a significantly higher photosensitizing activity of chlorin e6 conjugates with bovine serum albumin (BSA) and internalizable ligand insulin as compared to that of chlorin e6 itself. Receptor-mediated internalization of insulin-BSA-chlorin e6 conjugates ensures greater photosensitization of cells than the binding of those conjugates to cell surface receptors. The suitability of such conjugates permitting the delivery of a photosensitizer to most sensitive cell structures is discussed.
Zn-phthalocyanine (ZnPc) and Sn-etiopurpurin (SnET2) incorporated in unilamellar liposomes or solubilized in a Cremophor-EL emulsion have been incubated in vitro with rabbit plasma or intravenously administered to rabbits. Ultracentrifugation and chromatographic analysis of the plasma showed that ZnPc and SnET2 are mainly released to lipoproteins; within the lipoprotein family, both dyes are preferentially bound by low-density (LDL) and high-density (HDL) lipoproteins. The amount of dye bound with these two lipoprotein classes was related to their relative concentration in the plasma; in most cases a larger amount of photosensitizer was bound to HDL as compared to LDL on a protein concentration basis.
Native bovine serum albumin (BSA) was endocytosed and degraded at a steady rate by resident peritoneal murine macrophages with barely detectable amounts remaining within the cells. Radical-damaged BSA was endocytosed and degraded up to 2.5-fold more rapidly than native BSA, but some radical-damaged BSA accumulated within the cells in a time-dependent manner. The extent of accumulation increased in parallel with that of radical damage. Thus, some radical-damaged BSA was processed less efficiently than native BSA. Such inefficient catabolism of radical-damaged proteins may contribute to certain diseases such as atherosclerosis.
The interaction of native and cationized bovine serum albumin-gold complex with confluent monolayers of goat [corrected] brain microvascular endothelial cells was investigated. These cells were used as an in vitro model of the blood-brain barrier. After exposure to both complexes for 5, 15, 30, and 60 min, the endothelial cells were washed with phosphate-buffered saline, fixed, and processed for electron microscopy. The results obtained suggest that (a) contrary to previous observations in vivo, cultured endothelial cells were able to transfer native bovine serum albumin-gold complex particles from apical to basolateral surfaces presumably via a fluid-phase mechanism; (b) the most conspicuous difference in the interaction of both albumin-gold complexes consisted in a significantly greater adsorption of cationized than of native bovine serum albumin-gold complex to the apical surface of the endothelial cells; (c) the further fate of the endocytosed complexes was similar, that is, their major part was internalized into endosomes, multivesicular bodies and lysosomes whereas a smaller fraction was apparently transferred to the basolateral plasma membrane where presumably they were exocytosed into the subendothelial space. These observations provide new ultrastructural evidence that both native and cationized albumin are endocytosed and eventually transported by a non-specific, fluid-phase mechanism, and also support the results of earlier quantitative studies indicating the absence of albumin receptors in brain endothelia.
The patterns of in vitro intracellular and in vivo intratumoral localization of Photofrin II (P-II) and aluminum phthalocyanine tetrasulfonate (AlPCS4) in human melanoma LOX were studied by means of computer-enhanced video fluorescence microscopy (CEVFM). The hydrophobic drug P-II localized diffusely in the perinuclear fraction of the cytoplasm of the LOX cells cultivated in vitro. Light exposure did not result in any observable change in the localization pattern. The hydrophilic dye AlPCS4 was distributed as granular and grain patterns in the cytoplasm before light exposure, in exactly the same pattern as that of acridine orange incubated in the same cells, which is known to emit red fluorescence from lysosomes, thus indicating that AlPCS4 was also primarily localized in the lysosomes of the LOX cells. After light exposure the distribution of the intracellular AlPCS4 fluorescence was altered and the intensity increased. In vivo, P-II had a combined cellular localization pattern (i.e. a strongly cytoplasmic membrane-localizing pattern and a weakly intracellular distribution pattern) and an extracellular distribution pattern in the tumor tissue, while the AlPCS4 fluorescence was seen mainly in the stroma of the tumor. The total fluorescence intensity of P-II and AlPCS4 in the LOX tumor tissue at different times after injection was quantitatively determined by means of CEVFM.
Cells acquire cholesterol via de novo synthesis and receptor-mediated uptake of low-density lipoprotein (LDL), the major cholesterol-carrying lipoprotein in blood. Human leukemic cells and certain tumor tissues display elevated receptor-mediated uptake of LDL as compared with the corresponding normal cells or tissues. LDL has therefore been proposed as a potential carrier for chemotherapeutic agents. Various methods have been employed to incorporate antineoplastic lipophilic drugs into LDL, and the resultant drug-LDL complexes have been shown to be cytotoxic toward tumor cells in vitro. Initial experiments with tumor-bearing animals suggest that LDL may be a promising carrier for drugs in the treatment of malignant diseases.
Point mutagenesis of the nuclear targeting sequence of nucleoplasmin has identified two interdependent basic domains. These are separated by 10 intervening "spacer" amino acids that tolerate point mutations and some insertions. Amino acids in both basic domains are required for nuclear targeting, and the transport defect of a mutation in one domain is amplified by a simultaneous mutation in the other. Therefore, these basic domains are interdependent. A strikingly similar motif of two clusters of basic residues is seen in the nuclear targeting sequence of Xenopus N1. It is also conserved in the related nucleolar protein NO38. Several other short sequences known to be necessary for nuclear targeting fall within a similar motif.
Spatial and temporal alterations in carbon tetrachloride-induced oxidative stress were studied in perfused hepatic microcirculatory units of the rat by digital imaging microscopic fluorography using dichlorofluorescin diacetate, a hydroperoxide-sensitive fluorogenic probe. The surface of the liver loaded with dichlorofluorescin was microscopically visualized, and the fluorescence of dichlorofluorescin (DCF), a highly fluorescent molecule generated by hydroperoxide-mediated oxidation of dichlorofluorescin, was digitally processed. After completing the experiments, fluorescein-labeled albumin was injected into the perfusate to confirm the state of sinusoidal perfusion and the topography of hepatic microangioarchitecture in the area studied. During transportal infusion of carbon tetrachloride, DCF fluorescence was observed predominantly in the pericentral area and was attenuated by pretreatment with SKF-525A, suggesting the involvement of cytochrome P-450. After peaking at the maximum level, the pericentral DCF fluorescence gradually decreased in parallel to the loss of viability, implicating the causal role of intracellular hydroperoxide formation in hepatocellular injury. In retrogradely perfused liver, in which intralobular O2 gradient was reversed, no significant activation of DCF fluorescence was observed among hepatic lobules. It is therefore conceivable that the zonal heterogeneity of carbon tetrachloride-induced lipid peroxidation may depend on at least two factors, sinusoidal oxygenation and the intralobular distribution of cytochrome P-450. Furthermore, development of the current technique will provide a useful method to investigate the microtopography of oxidative stress in organ microcirculatory units.
Human cervix carcinoma cells of the line NHIK 3025 were incubated for 18 h with sulfonated meso-tetraphenylporphines (TPPSn where n = 1, 2a, 2o or 4) followed by 1 h in sensitizer-free medium and then exposed to light. The fluorescing fraction of TPPS4, TPPS2o and TPPS2a has recently been shown to be located intracellularly in extracellular granules which are intracellularly localized in a similar pattern as acridine orange-stained granules, assumed to be endosomes and lysosomes (Berg, K., A. Western, J. Bommer and J. Moan. Photochem. Photobiol. 52, 481-487). Light exposure induced a relocalization of TPPS4 from its granular pattern to mainly the nuclear area while TPPS2o and TPPS2a relocalized mainly to cytoplasmic areas. After the light-induced relocalization TPPS4 became less efficient in sensitizing photoinactivation of cells as measured per fluorescing cellbound TPPS4 molecules while TPPS2a and TPPS2o became more efficient. These changes were independent of the extracellular concentration of TPPSn applied to the cells, except for cells incubated with 75 micrograms/mL TPPS4. These cells became more sensitive to light after a light exposure inactivating 20% of the cells. This increased photosensitivity seems to be related to a 2-2.5 fold increase in the amount of fluorescing cellbound TPPS4 induced by the first light exposure.
In the course of an attempt to identify genes that encode Escherichia coli dihydropteridine reductase (DHPR) activities, a chromosomal DNA fragment that directs synthesis of two soluble polypeptides of Mr 44000 and 46000 was isolated. These proteins were partially purified and were identified by determination of their N-terminal amino acid sequences. The larger was serine hydroxymethyltransferase, encoded by the glyA gene, while the smaller was the previously described product of an unnamed gene closely linked to glyA, and transcribed in the opposite direction. Soluble extracts of E. coli cells that overproduced the 44 kDa protein had elevated DHPR activity, and were yellow in colour. Their visible absorption spectra were indicative of a CO-binding b-type haemoprotein that is high-spin in the reduced state. The sequence of the N-terminal 139 residues of the protein, deduced from the complete nucleotide sequence of the gene, had extensive homology to almost all of Vitreoscilla haemoglobin. We conclude that E. coli produces a soluble haemoglobin-like protein, the product of the hmp gene (for haemoprotein). Although the protein has DHPR activity, it is distinct from the previously purified E. coli DHPR.
The cellular uptake, relative fluorescence quantum yields and photosensitizing efficiencies of meso-tetraphenylporphines sulfonated to different degrees (TPPSn) have been investigated using the human carcinoma cell line NHIK 3025. The efficiencies of these dyes in photoinactivation of cells were highly dependent on the number of sulfonate groups on the derivatives. These differences in phototoxicity were primarily due to different abilities to be taken up by cells, but were also dependent upon the cellular localization of the dyes. TPPS1 and TPPS2a were more efficiently taken up by the cells than TPPS2o and TPPS4. Plasma membrane associated TPPS4 was less efficient in cell inactivation per quantum of fluorescence emitted than intracellularly located dye. This was also to some extent the case for TPPS1 but not for TPPS2a and TPPS2o. The results presented here indicate that TPPS2a and TPPS1 are the most promising of the TPPSns for possible future use in photodynamic therapy.
The mechanism controlling insulin accumulation in nuclei of H35 hepatoma cells was investigated by incubating intact cells with 125I-labeled insulin in the presence or absence of agents that perturb different intracellular sites involved in the processing of ligand-receptor complexes. Purified nuclei were isolated, and nuclear-associated 125I-insulin was determined. Insulin accumulation in the nuclei was time and temperature dependent. Nuclear accumulation was linear and insulin-concentration dependent between 5 and 50 ng insulin/ml. However, pharmacological concentrations of insulin increased the amount of insulin translocated to the nucleus to a far greater extent than it increased total cell-associated insulin. Chloroquine, an acidotrophic agent, increased total cell-associated and intracellular insulin but had no effect on nuclear accumulation. The monovalent ionophores monensin and nigericin inhibited nuclear accumulation of insulin at low concentrations (0.5-5.0 microM) without affecting total insulin binding or intracellular accumulation. At 10 or 25 microM, monensin and nigericin also acted as acidotrophic agents and increased total insulin binding and intracellular accumulation but inhibited nuclear accumulation by a maximum of 50%. Low concentrations of monensin and nigericin were additive; maximal concentrations were not. A 23187 and valinomycin did not affect insulin binding or intracellular and nuclear accumulation of insulin. Neither depletion of ATP by sodium azide, 2,4-dinitrophenol, sodium cyanide, or oligomycin nor disruption of cytoskeletal elements by cytochalasin D or colchicine had any effect on nuclear accumulation of insulin.(ABSTRACT TRUNCATED AT 250 WORDS)
By means of laser scanning fluorescence microscopy the intratumoral localization patterns of several photosensitizers in LOX tumors in nude mice were studied. Lipophilic dyes such as P-II (Photofrin II), 3-THPP tetra(3-hydroxyphenyl)porphin, TPPS1 (tetraphenylporphine monosulfonate), TPPS2a (tetraphenylporphine disulfonates with the sulfonate groups on adjacent rings), A1PCS1 (aluminium phthalocyanine monosulfonate) and A1PCS2 (aluminium phthalocyanine disulfonates) localized mainly in tumor cells. The fluorescence intensity of these dyes increased from 4 h to 48 h post-injection and the fluorescence was still observable 120 h post-injection. The more hydrophilic dyes such as TPPS2o (tetraphenylporphine disulfonates with the sulfonates groups on opposite rings), TPPS3 (tetraphenylporphine trisulfoantes), TPPS4 (tetraphenylporphine tetrasulfonates), A1PCS3 (aluminium phthalocyanine trisulfonates) and A1PCS4 (aluminium phthalocyanine tetrasulfonates) localized mainly extracellularly in the tumorous stroma. The fluorescence intensity of these dyes decreased from 4 h to 48 h post-injection. 120 h post-injection no significant fluorescence of these dyes could be seen in the tumors. The data are discussed in relation to what is known about the in vivo photosensitizing efficiency of some of the dyes.
To minimize the side effect of porphyrin photosensitizers and to reduce their active concentration, chlorine e6 was conjugated with concanavalin A. Photodynamic action of chlorine e6 and concanavalin A-chlorine e6 conjugate has been studied in human skin embryonic fibroblasts. The conjugate appeared to be 5 times more effective as compared to chlorine e6 due to concanavalin A-chlorine e6 endocytosis into intracellular compartments.
We have studied the uptake of 125I-labelled low-density lipoprotein (LDL) by seven experimental murine tumours in vivo. Four tumours (Lewis Lung carcinoma, B-16, MS-2 and Colon 26) showed a higher relative uptake of lipoprotein as compared to the liver, two (L-1210 and P-388) had a very low lipoprotein uptake, while lipoprotein uptake by tumour M5 was similar to that of the liver. The data was confirmed by tracing tissue uptake of lipoproteins using [14C]sucrose-labeled LDL. These in vivo findings correlated well with the in vitro specific binding of 125I-beta-VLDL to membranes prepared from tumours, thus suggesting that the expression of the LDL receptor in the tumours is related to the in vivo uptake of lipoprotein. Further analysis of the LDL receptor by ligand blotting showed that the tumor receptor has several of the liver LDL receptor characteristics (including apparent Mr, sensitivity to proteinases, and Ca2+ requirement of lipoprotein binding). In summary, our data show that experimental murine tumours express the LDL receptor and suggest that the high relative in vivo uptake of LDL is determined by the elevated LDL-receptor expression in the tumours.
The mechanisms of cellular uptake, subcellular localization, and cellular retention kinetics of the photosensitizers photofrin II (PfII), mono-L-aspartyl chlorin e6 (MACE), and chloro-aluminum sulfonated phthalocyanine (CASPc) are reported in this paper. Each photosensitizer's cellular uptake mechanism was determined by preferentially inhibiting endocytosis by chilling cells to 2 degrees C, while allowing diffusion across the membrane. Subcellular localization was studied by computer-enhanced low-light level video fluorescence microscopy, while flow cytometry was used to determine uptake and retention kinetics. The results indicate that PfII enters the cell primarily by diffusion across the membrane, whereas MACE and CASPc enter the cell through endocytosis.