Approaches to the targeted intracellular delivery of photosensitizers in order to enhance their efficacy and cell specificity

The main physicochemical properties of photosensitizers used in the photodynamic therapy of cancer and their subcellular distribution after in vitro and in vivo administration were analyzed. It was shown that the effect of photosensitizers is realized at very short distances from the sites of their intracellular localization, and the sensitivities of different cellular compartments to the photocytotoxic action of photosensitizers are different. The necessity of intranuclear delivery of photosensitizers into the nuclei of target cells in order to enhance their efficacy and cell specificity was shown and the available approaches to the targeted delivery of photosensitizers were analyzed. The mechanisms of nucleocytoplasmic transport through the nuclear pore complex, which can be used for the delivery of photosensitizers inward the nucleus, are reviewed. Different modular transporters for photosensitizers comprising (i) a ligand module, which binds to an internalizable receptor overexpressed on the target cells, (ii) an intracellular localization signal, (iii) a carrier module, and (iv) an endosomolytic module were characterized. All these modules were shown to be fully functional within the chimeric polypeptide and the polypeptide as a whole. A significant enhancement of photocytotoxicity and cell specificity of photosensitizers delivered by these transporters were demonstrated. The transporters described represent a new generation of pharmaceuticals which can be widely used for targeted drug delivery.     

Targeted intracellular site-specific drug delivery: photosensitizer targeting to melanoma cell nuclei

A number of drugs are regarded as possessing local activity because their effects take place at an extremely short distance from their location site in the cell. The response of different cellular compartments to these effects is different. Such substances as photosensitizers (PSs), which are used in photodynamic cancer therapy, should be targeted to the cell compartments where their effect is the most pronounced. This study describes the construction and properties of the chimeric modular recombinant transporters (MRTs) expressed in Escherichia coli and used for PS targeting. These constructs include (1) the alpha-melanocyte-stimulating hormone as a ligand module, which is internalized by the target cells (mouse melanoma); (2) the optimized SV40 large T-antigen nuclear localization signal; (3) the hemoglobin-like protein from E. coli as a carrier module; (4) the endosomolytic module, the translocation domain of the diphtheria toxin. These MRTs were used for PS targeting to the mouse melanoma cell nuclei, the most PS-damaged intracellular compartment, which resulted in a PS photocytotoxic effect increase of several orders of magnitude. In our opinion, MRTs, which target locally active drugs into the desired cell compartment and thereby enhance the drug response, represent a new generation of the pharmacological agents.     

Targeted Intracellular Site-Specific Drug Delivery: Photosensitizer Targeting to Melanoma Cell Nuclei

A number of drugs are regarded as possessing local activity because their effects take place at an extremely short distance from their location site in the cell. The response of different cellular compartments to these effects is different. Such substances as photosensitizers (PSs), which are used in photodynamic cancer therapy, should be targeted to the cell compartments where their effect is the most pronounced. This study describes the construction and properties of the chimeric modular recombinant transporters (MRTs) expressed in Escherichia coli and used for PS targeting. These constructs include (1) the -melanocyte-stimulating hormone as a ligand module, which is internalized by the target cells (mouse melanoma); (2) the optimized SV40 large T-antigen nuclear localization signal; (3) the hemoglobin-like protein from E. coli as a carrier module; (4) the endosomolytic module, the translocation domain of the diphtheria toxin. These MRTs were used for PS targeting to the mouse melanoma cell nuclei, the most PS-damaged intracellular compartment, which resulted in a PS photocytotoxic effect increase of several orders of magnitude. In our opinion, MRTs, which target locally active drugs into the desired cell compartment and thereby enhance the drug response, represent a new generation of the pharmacological agents.     

Botulinum neurotoxin A and an engineered derivate targeted secretion inhibitor (TSI) A enter cells via different vesicular compartments

Botulinum neurotoxins (BoNTs) are highly potent multi-domain proteins, responsible for botulism in animals and humans. The modular structural organization of BoNTs has led to the development of novel engineered bio-therapeutic proteins called targeted secretion inhibitors (TSIs). We report here that botulinum neurotoxin A (BoNT/A) and a TSI/A in which the neuronal binding domain of BoNT/A has been substituted by an epidermal growth factor (EGF) ligand, named EGFR-targeted TSI/A, exploit different routes to gain entry in the same in vitro neuroblastoma cell system, SiMa cells. We found that the EGF ligand conferred the affinity to the EGFR-targeted TSI/A at the EGF receptor when compared to an untargeted TSI/A and also the ability to internalize into the cells and cleave its cytosolic target protein SNAP-25. Using high content analysis we found that both BoNT/A and the EGFR-targeted TSI/A enter the cell in a concentration-dependent manner and in compartments which are able to translocate the proteins into the cytosol within 4?h. The EGFR-targeted TSI/A internalized into a compartment which gave a punctate staining pattern by immunofluorescence and partially overlapped with structures positive for the early endosomal marker EAA1; whereas BoNT/A did not internalize into a punctate compartment but did so in an acidifying compartment consistent with local synaptic vesicle recycling. These findings show that the BoNT/A translocation domain, common to both BoNT/A and the EGFR-targeted TSI/A, is a versatile tool for cytosolic delivery from distinct intracellular vesicular compartments.     

Fabrication and Intracellular Delivery of Doxorubicin/Carbonate Apatite Nanocomposites: Effect on Growth Retardation of Established Colon Tumor

In continuing search for effective treatments of cancer, the emerging model aims at efficient intracellular delivery of therapeutics into tumor cells in order to increase the drug concentration. However, the implementation of this strategy suffers from inefficient cellular uptake and drug resistance. Therefore, pH-sensitive nanosystems have recently been developed to target slightly acidic extracellular pH environment of solid tumors. The pH targeting approach is regarded as a more general strategy than conventional specific tumor cell surface targeting approaches, because the acidic tumor microclimate is most common in solid tumors. When nanosystems are combined with triggered release mechanisms in endosomal or lysosomal acidic pH along with endosomolytic capability, the nanocarriers demonstrated to overcome multidrug resistance of various tumors. Here, novel pH sensitive carbonate apatite has been fabricated to efficiently deliver anticancer drug Doxorubicin (DOX) to cancer cells, by virtue of its pH sensitivity being quite unstable under an acidic condition in endosomes and the desirable size of the resulting apatite-DOX for efficient cellular uptake as revealed by scanning electron microscopy. Florescence microscopy and flow cytometry analyses demonstrated significant uptake of drug (92%) when complexed with apatite nanoparticles. In vitro chemosensitivity assay revealed that apatite-DOX nanoparticles executed high cytotoxicity in several human cancer cell lines compared to free drugs and consequently apatite-DOX-facilitated enhanced tumor inhibitory effect was observed in colorectal tumor model within BALB/cA nude mice, thereby shedding light on their potential applications in cancer therapy.     

Evidence for nonvectorial, retrograde transferrin trafficking in the early endosomes of HEp2 cells

We have previously characterized the trafficking of transferrin (Tf) through HEp2 human carcinoma cells (Ghosh, R. N., D. L. Gelman, and F. R. Maxfield, 1994. J. Cell Sci. 107:2177-2189). Early endosomes in these cells are comprised of both sorting endosomes and recycling compartments, which are distinct separate compartments. Endocytosed Tf initially appears in punctate sorting endosomes that also contain recently endocytosed LDL. After short loading pulses, Tf rapidly sorts from LDL with first-order kinetics (t1/2 approximately 2.5 min), and it enters the recycling compartment before leaving the cell (t1/2 approximately 7 min). Here, we report a second, slower rate for Tf to leave sorting endosomes after HEp2 cells were labeled to steady state with fluorescein Tf instead of the brief pulse used previously. We determined this rate using digital image analysis to measure the Tf content of sorting endosomes that also contained LDL. With an 11-min chase, the Tf in sorting endosomes was 24% of steady-state value. This was in excess of the amount expected (5% of steady state) from the rate of Tf exit after short filling pulses. The excess could not be accounted for by reinternalization of recycled cell surface Tf, implying that either some Tf was retained in sorting endosomes, or that Tf was delivered back to the sorting endosomes from the recycling compartment. The former is unlikely since nearly all sorting endosomes contain detectable Tf after an 11-min chase, even though more than one third of the sorting endosomes were formed during the chase time. Furthermore, while observing living cells by confocal microscopy, we saw vesicle movements that appeared to be fluorescent Tf returning from recycling compartments to sorting endosomes. The slow rate of exit after steady-state labeling was similar to the Tf exit rate from the cell, suggesting an equilibration of Tf throughout the early endosomal system by this retrograde pathway. This retrograde traffic may be important for delivering molecules from the recycling compartment, which is a long-lived organelle, to sorting endosomes, which are transient.     

Endosomal density shift is related to a decrease in fusion capacity.

Dinitrophenol (DNP)-beta-glucuronidase and mannosylated anti-DNP IgG, which are endocytosed by the mannose receptor and delivered to lysosomes, were previously developed as probes for examination of fusion between early endosomes in a cell-free system. In this study, these probes were found to be transported by intact cells to endocytic vesicles with heavy buoyant density at different rates, as determined by Percoll gradient fractionation of cell homogenates. There was a concomitant loss of in vitro fusion activity as the ligands moved to dense compartments. In monensin-treated cells, DNP-beta-glucuronidase was retained in a light compartment corresponding to intracellular vesicles capable of fusion in vitro. Pulse-chase studies using a DNP-derivatized transferrin-alkaline phosphatase conjugate showed that a recycling ligand was always found in light intracellular vesicles that were capable of fusion to early endosomes in vitro. In contrast to cell-free systems, intact cells sequentially labeled with DNP-beta-glucuronidase and then mannosylated anti-DNP IgG showed ligand mixing in both early and late endocytic compartments. Treatment with nocodazole or colchicine did not affect the rate of DNP-beta-glucuronidase transport to heavy vesicles in intact cells, however, the extent of ligand mixing in late endosomes was decreased by microtubule disruption. Using sequentially labeled cells split into two groups, we directly compared ligand mixing in vitro to mixing by intact cells. Fusion alone does not mediate increases in vesicle density, since DNP-beta-glucuronidase/anti-DNP IgG complexes formed in vitro were found in light vesicles, while intact cells showed immune complexes predominantly in heavy vesicles. These results suggest that the density shift is an initial step in targeting to lysosomes.     

HIV-1 buds and accumulates in "nonacidic" endosomes of macrophages

Macrophages represent viral reservoirs in HIV-1-infected patients and accumulate viral particles within an endosomal compartment where they remain infectious for long periods of time. To determine how HIV-1 survives in endocytic compartments that become highly acidic and proteolytic and to study the nature of these virus-containing compartments, we carried out an ultrastructural study on HIV-1-infected primary macrophages. The endosomal compartments contain newly formed virions rather than internalized ones. In contrast to endocytic compartments free of viral proteins within the same infected cells, the virus containing compartments do not acidify. The lack of acidification is associated with an inability to recruit the proton pump vacuolar ATPase into the viral assembly compartment. This may prevent its fusion with lysosomes, since acidification is required for the maturation of endosomes. Thus, HIV-1 has developed a strategy for survival within infected macrophages involving prevention of acidification within a devoted endocytic virus assembly compartment.     

Importin β-type nuclear transport receptors have distinct binding affinities for Ran-GTP

Cargos destined to enter or leave the cell nucleus are typically transported by receptors of the importin β family to pass the nuclear pore complex. The yeast Saccharomyces cerevisiae comprises 14 members of this protein family, which can be divided in importins and exportins. The Ran GTPase regulates the association and dissociation of receptors and cargos as well as the transport direction through the nuclear pore. All receptors bind to Ran exclusively in its GTP-bound state and this event is restricted to the nuclear compartment. We determined the Ran–GTP binding properties of all yeast transport receptors by biosensor measurements and observed that the affinity of importins for Ran–GTP differs significantly. The dissociation constants range from 230 pM to 270 nM, which is mostly based on a variability of the off-rate constants. The divergent affinity of importins for Ran–GTP suggests the existence of a novel mode of nucleocytoplasmic transport regulation. Furthermore, the cellular concentration of β-receptors and of other Ran-binding proteins was determined. We found that the number of β-receptors altogether about equals the amounts of yeast Ran, but Ran–GTP is not limiting in the nucleus. The implications of our results for nucleocytoplasmic transport mechanisms are discussed.     

Functional symmetry of endomembranes

In higher eukaryotic cells pleiomorphic compartments composed of vacuoles, tubules and vesicles move from the endoplasmic reticulum (ER) and the plasma membrane to the cell center, operating in early biosynthetic trafficking and endocytosis, respectively. Besides transporting cargo to the Golgi apparatus and lysosomes, a major task of these compartments is to promote extensive membrane recycling. The endocytic membrane system is traditionally divided into early (sorting) endosomes, late endosomes and the endocytic recycling compartment (ERC). Recent studies on the intermediate compartment (IC) between the ER and the Golgi apparatus suggest that it also consists of peripheral ("early") and centralized ("late") structures, as well as a third component, designated here as the biosynthetic recycling compartment (BRC). We propose that the ERC and the BRC exist as long-lived "mirror compartments" at the cell center that also share the ability to expand and become mobilized during cell activation. These considerations emphasize the functional symmetry of endomembrane compartments, which provides a basis for the membrane rearrangements taking place during cell division, polarization, and differentiation.     

Toxin entry: how bacterial proteins get into mammalian cells

Certain bacteria secrete protein toxins that catalytically modify and disrupt essential processes in mammalian cells, often leading to cell death. As the substrates modified by these toxins are located in the mammalian cell cytosol, a catalytically active toxin polypeptide must reach this compartment in order to act. The toxins bind to receptors on the surface of susceptible cells and enter them by endocytic uptake. Endocytosed toxins initially accumulate in endosomes, where some of these proteins take advantage of the acidic environment within these organelles to form, or contribute to the formation of, protein-conducting channels through which the catalytic polypeptide is able to translocate into the cytosol. Other toxins are unable to respond to low pH in this way and must undergo intracellular vesicular transport to reach a compartment where pre-existing protein-conducting channels occur and can be exploited for membrane translocation — the endoplasmic reticulum. In this way, cell entry by this second group of toxins demonstrates that the secretory pathway of mammalian cells is completely reversible.     

Cell wall pectins and xyloglucans are internalized into dividing root cells and accumulate within cell plates during cytokinesis

Recently, we have reported that cell wall pectins are internalized into apical meristem root cells. In cells exposed to the fungal metabolite brefeldin A, all secretory pathways were inhibited, while endocytic pathways remained intact, resulting in accumulation of internalized cell wall pectins within brefeldin A-induced compartments. Here we report that, in addition to the already published cell wall epitopes, rhamnogalacturonan I and xyloglucans also undergo large-scale internalization into dividing root cells. Interestingly, multilamellar endosomes were identified as compartments internalizing arabinan cell wall pectins reactive to the 6D7 antibody, while large vacuole-like endosomes internalized homogalacturonans reactive to the 2F4 antibody. As all endosomes belong topographically to the exocellular space, cell wall pectins deposited in these "cell wall islands", enclosed by the plasma-membrane-derived membrane, are ideally suited to act as temporary stores for rapid formation of cell wall and generation of new plasma membrane. In accordance with this notion, we report that all cell wall pectins and xyloglucans that internalize into endosomes are highly enriched within cytokinetic cell plates and accumulate within brefeldin A compartments. On the other hand, only small amounts of the pectins reactive to the JIM7 antibody, which are produced in the Golgi apparatus, localize to cell plates and they do not accumulate within brefeldin A compartments. In conclusion, meristematic root cells have developed pathways for internalization and recycling of cell wall molecules which are relevant for plant-specific cytokinesis.     

Tumor cell killing enabled by listeriolysin O-liposome-mediated delivery of the protein toxin gelonin

Gelonin is a type I plant toxin that has potential as an effective anti-tumor agent by virtue of its enzymatic capacity to inactivate ribosomes and arrest protein synthesis, thereby effectively limiting the growth of cancer cells. Being a hydrophilic macromolecule, however, gelonin has limited access to its target subcellular compartment, the cytosol; it is effectively plasma membrane-impermeant and subject to rapid degradation within endosomes and lysosomes upon cellular uptake as it lacks the membrane-translocating capability that is typically provided by a disulfide-linked B polypeptide found in the type II toxins (e.g. ricin). These inherent characteristics generate the need for the development of a specialized cytosolic delivery strategy for gelonin as an effective anti-tumor therapeutic agent. Here we describe an efficient means of delivering gelonin to the cytosol of B16 melanoma cells. Gelonin was co-encapsulated inside pH-sensitive liposomes with listeriolysin O, the pore-forming protein that mediates escape of the intracellular pathogen Listeria monocytogenes from the endosome into the cytosol. In in vitro experiments, co-encapsulated listeriolysin O enabled liposomal gelonin-mediated B16 cell killing with a gelonin IC50 of approximately 0.1 nM with an extreme efficiency requiring an incubation time of only 1 h. By contrast, cells treated with equivalent concentrations of unencapsulated gelonin or gelonin encapsulated alone in pH-sensitive liposomes exhibited no detectable cytotoxicity. Moreover, treatment by direct intratumor injection into subcutaneous solid tumors of B16 melanoma in a mouse model showed that pH-sensitive liposomes containing both listeriolysin O and gelonin were more effective than control formulations in curtailing tumor growth rates.     

Novel properties of the nucleolar targeting signal of human angiogenin

The polypeptide ligand angiogenin, a potent inducer of angiogenesis, localizes in the nucleus/nucleolus subsequent to endocytosis by relevant cell types. This study examines the kinetic properties of the nucleolar targeting signal (NTS) of angiogenin (IMRRRGL(35)) at the single cell level. We show that the NTS is sufficient to target green fluorescent protein (GFP), but not beta-galactosidase, to the nucleolus of rat hepatoma cells. Mutation of Arg(33) to Ala within the NTS abolishes targeting activity. Nuclear/nucleolar import conferred by the NTS of angiogenin is reduced by cytosolic factors as well as ATP and is independent of importins and Ran. The NTS also confers the ability to bind to nuclear/nucleolar components which is inhibited by ATP hydrolysis; nonhydrolysable GTP analogs prevent nuclear accumulation in the absence of an intact nuclear envelope through an apparent cytoplasmic retention mechanism. Since the lectin wheat germ agglutinin does not inhibit transport, we postulate a mechanism for angiogenin nuclear/nucleolar import involving passive diffusion of angiogenin through the nuclear pore and NTS-mediated nuclear/nucleolar retention, and with cytoplasmic retention modulating the process. This pathway is clearly distinct from that of conventional signal-mediated nuclear protein import.     

Control of compartment size by an EGF ligand from neighboring cells

Insect bodies are subdivided into anterior (A) and posterior (P) compartments: cohesive fields of distinct cell lineage and cell affinity . Like organs in many animal species, compartments can develop to normal sizes despite considerable variation in cell division . This implies that overall compartment dimensions are subject to genetic control, but the mechanisms are unknown. Here, studying Drosophila's embryonic segments, I show that P compartment dimensions depend on epidermal growth factor receptor (EGFR) signaling. I suggest the primary activating ligand is Spitz, emanating from neighboring A compartment cells. Spi/EGFR activity stimulates P compartment cell enlargement and survival, but evidence is presented that Spitz is secreted in limited amounts, so that increasing the number of cells within the P compartment causes the per-cell Spitz level to drop. This leads to compensatory apoptosis and cell-size reductions that preserve compartment dimensions. Conversely, I propose that lowering P compartment cell numbers enhances per-cell Spitz availability; this increases cell survival and cell size, again safeguarding compartment size. The results argue that the gauging of P compartment size is due, at least in part, to cells surviving and growing according to Spi availability. These data offer mechanistic insight into how diffusible molecules control organ size.     

Helicobacter pylori VacA cytotoxin: a probe for a clathrin-independent and Cdc42-dependent pinocytic pathway routed to late endosomes

The vacuolating cytotoxin VacA is a major virulence factor of Helicobacter pylori, a bacterium responsible for gastroduodenal ulcers and cancer. VacA associates with lipid rafts, is endocytosed, and reaches the late endocytic compartment where it induces vacuolation. We have investigated the endocytic and intracellular trafficking pathways used by VacA, in HeLa and gastric AGS cells. We report here that VacA was first bound to plasma-membrane domains localized above F-actin structures that were controlled by the Rac1 GTPase. VacA was subsequently pinocytosed by a clathrin-independent mechanism into cell peripheral early endocytic compartments lacking caveolin 1, the Rab5 effector early endosomes antigen-1 (EEA1) and transferrin. These compartments took up fluid-phase (as evidenced by the accumulation of fluorescent dextran) and glycosylphosphatidylinositol-anchored proteins (GPI-APs). VacA pinocytosis was controlled by Cdc42 and did not require cellular tyrosine kinases, dynamin 2, ADP-ribosylating factor 6, or RhoA GTPase activities. VacA was subsequently routed to EEA1-sorting endosomes and then sorted to late endosomes. During all these different endocytic steps, VacA was continuously associated with detergent resistant membrane domains. From these results we propose that VacA might be a valuable probe to study raft-associated molecules, pinocytosed by a clathrin-independent mechanism, and routed to the degradative compartment.     

Targeting of an Intestinal Apical Endosomal Protein to Endosomes in Nonpolarized Cells

Polarized cells such as epithelial cells and neurons have distinct endosomal compartments associated with different plasma membrane domains. The endosomes of the neuronal cell body and the basolateral cytoplasm of epithelial cells are thought to perform cellular “housekeeping” functions such as the uptake of nutrients and metabolites, while the endosomes in the apical cytoplasm or axons are thought to be specialized for the sorting and transcytosis of cell type–specific ligands and receptors. However, it is not known if nonpolarized cells such as fibroblasts contain a specialized endosomal compartment analogous to the specialized endosomes found in neurons and epithelia. We have expressed a protein that is normally found in the apical early endosomes of developing intestinal epithelial cells in normal rat kidney fibroblasts. This apical endosomal marker, called endotubin, is targeted to early endosomes in transfected fibroblasts, and is present in peripheral as well as perinuclear endosomes. The peripheral endosomes that contain endotubin appear to exclude transferrin, fluid phase markers, and the mannose-6-phosphate receptor, although in the perinuclear region colocalization of endotubin and these markers is present. In addition, endotubin positive structures do not tubulate in response to brefeldin A and instead redistribute to a diffuse perinuclear location. Since this endosomal compartment has many of the characteristics of an apical or axonal endosomal compartment, our results indicate that nonpolarized cells also contain a specialized early endosomal compartment.     

Association of the tyrosine phosphorylated epidermal growth factor receptor with a 55-kD tyrosine phosphorylated protein at the cell surface and in endosomes

After the intraportal injection of EGF, the EGF receptor (EGFR) is rapidly internalized into hepatic endosomes where it remains largely receptor bound (Lai et al., 1989. J. Cell Biol. 109:2751-2760). In the present study, we evaluated the phosphotyrosine content of EGFRs at the cell surface and in endosomes in order to assess the consequences of internalization. Quantitative estimates of specific radioactivity of the EGFR in these two compartments revealed that tyrosine phosphorylation of the EGFR was observed at the cell surface within 30 s of ligand administration. However, the EGFR was also highly phosphorylated in endosomes reaching levels of tyrosine phosphorylation significantly higher than those of the cell surface receptor at 5 and 15 min after EGF injection. A 55-kD tyrosine phosphorylated polypeptide (pyp55) was observed in association with the EGFR at the cell surface within 30 s of EGF injection. The protein was also found in association with the EGFR in endosomes as evidenced by coprecipitation studies using a mAb to the EGFR as well as by coelution with the EGR in gel permeation chromatography. Limited proteolysis of isolated endosomes indicated that the tyrosine phosphorylated domains of the EGFR and associated pyp55 were cytosolically oriented while internalized EGF was intraluminal. The identification of pyp55 in association with EGFR in both hepatic plasma membranes and endosomes may be relevant to EGFR function and/or trafficking of the EGFR.