内质网靶向递药系统用于抗肿瘤免疫治疗的研究进展

内质网作为真核细胞内功能多样的细胞器,不仅是蛋白质合成与修饰的关键场所,也是胞内信号转导的重要位点,影响着细胞的功能发挥、存活及凋亡。内质网稳态的失衡会引起内质网应激,这与肿瘤的发生与发展密不可分。在一些药物(蒽醌类及奥沙利铂等)的作用下,过度的内质网应激则会介导肿瘤细胞的免疫原性细胞死亡并激发抗肿瘤免疫反应,在治疗后期形成长期免疫记忆。但是,这些药物往往存在靶向性差等问题,在肿瘤细胞内质网的蓄积量极低,限制了其临床疗效。因此,内质网靶向药物的研究将为更高效更精准的抗肿瘤免疫治疗提供参考。本文就内质网与肿瘤免疫的关系,以及近年来基于内质网靶向的抗肿瘤免疫治疗的策略进行综述,并对内质网靶向策略存在的问题及未来发展方向进行总结与展望。     

Periocular routes for retinal drug delivery

Despite numerous scientific efforts, delivery of therapeutic amounts of a drug to the retina remains a challenge. This challenge is compounded if chronic therapy is desired. The inability or inefficiency of topical and systemic routes for retinal delivery of existing drugs is now widely accepted. Although the intravitreal route offers high local concentrations in the vitreous and, hence, retina, these advantages are offset by side effects, such as cataracts, endophthalmitis and retinal detachment, following repeated intravitreal injections, or intravitreal placement of sustained-release implants. As discussed in this review, periocular routes, including subconjunctival, sub-tenon, retrobulbar, peribulbar and posterior juxtascleral routes, potentially offer a more promising alternative for enhanced drug delivery to the retina compared with topical and systemic routes. Periocular routes exploit the permeability of sclera for retinal drug delivery, and they are particularly useful for administering sustained-release systems of potent drugs. This review discusses the various periocular routes with respect to their anatomical location, pharmacokinetics, safety and mechanisms of drug delivery. In the coming years, several innovations in absorption enhancement, drug delivery systems and drug administration devices are anticipated for improving retinal drug delivery via periocular routes.     

Periocular routes for retinal drug delivery

Despite numerous scientific efforts, delivery of therapeutic amounts of a drug to the retina remains a challenge. This challenge is compounded if chronic therapy is desired. The inability or inefficiency of topical and systemic routes for retinal delivery of existing drugs is now widely accepted. Although the intravitreal route offers high local concentrations in the vitreous and, hence, retina, these advantages are offset by side effects, such as cataracts, endophthalmitis and retinal detachment, following repeated intravitreal injections, or intravitreal placement of sustained-release implants. As discussed in this review, periocular routes, including subconjunctival, sub-tenon, retrobulbar, peribulbar and posterior juxtascleral routes, potentially offer a more promising alternative for enhanced drug delivery to the retina compared with topical and systemic routes. Periocular routes exploit the permeability of sclera for retinal drug delivery, and they are particularly useful for administering sustained-release systems of potent drugs. This review discusses the various periocular routes with respect to their anatomical location, pharmacokinetics, safety and mechanisms of drug delivery. In the coming years, several innovations in absorption enhancement, drug delivery systems and drug administration devices are anticipated for improving retinal drug delivery via periocular routes.     

Polyplexes traffic through caveolae to the Golgi and endoplasmic reticulum en route to the nucleus

The cellular machinery involved in the internalization of nonviral gene carriers and their subsequent trafficking to the nucleus directly impacts their therapeutic efficiency. Hence, identifying key endocytic pathways and organelles that contribute to the successful transfer of polyplexes to the nucleus generates new opportunities for improving carrier design. Previously, we showed that histone H3 tail peptides encoding a sequence known to participate in chromatin activation exhibit synergistic gene delivery activity with poly(ethylenimine) (PEI). Polyplexes containing H3 and PEI exhibited a reduced dependence on endocytic pathways that trafficked to lysosomes, and had enhanced sensitivity to an inhibitor associated with retrograde trafficking through the Golgi apparatus. Thus, we sought to determine whether caveolar uptake and transport through the Golgi and/or endoplasmic reticulum (ER) preceded nuclear delivery. By the use of a panel of chemical endocytic inhibitors, we determined that H3 polyplexes utilized caveolar pathways to a greater degree than PEI polyplexes. Caveolae-mediated endocytosis was found to be a productive route for gene expression by the H3/PEI-pDNA polyplexes, consistent with previous studies of polymer-mediated gene delivery. Additionally, the polyplexes substantially colocalized within the ER after only 5 min of incubation, and utilized retrograde Golgi-to-ER pathways at levels similar to pathogens known to traffic by these routes during infection. The results of this study have expanded our understanding of how caveolar polyplexes are trafficked to cell nuclei, and provide new evidence for the role of Golgi-ER pathways in transfection. These findings suggest new design criteria and opportunities to stragetically target nonviral gene delivery vehicles.     

Capsaicin-induced apoptosis is regulated by endoplasmic reticulum stress- and calpain-mediated mitochondrial cell death pathways

Capsaicin, a pungent compound found in hot chili peppers, induces apoptotic cell death in various cell lines, however, the precise apoptosis signaling pathway is unknown. Here, we investigated capsaicin-induced apoptotic signaling in the human breast cell line MCF10A and found that it involves both endoplasmic reticulum (ER) stress and calpain activation. Capsaicin inhibited growth in a dose-dependent manner and induced apoptotic nuclear changes in MCF10A cells. Capsaicin also induced degradation of tumor suppressor p53; this effect was enhanced by the ER stressor tunicamycin. The proteasome inhibitor MG132 completely blocked capsaicin-induced p53 degradation and enhanced apoptotic cell death. Capsaicin treatment triggered ER stress by increasing levels of IRE1, GADD153/Chop, GRP78/Bip, and activated caspase-4. It led to an increase in cytosolic Ca²⁺, calpain activation, loss of the mitochondrial transmembrane potential, release of mitochondrial cytochrome c, and caspase-9 and -7 activation. Furthermore, capsaicin-induced the mitochondrial apoptotic pathway through calpain-mediated Bid translocation to the mitochondria and nuclear translocation of apoptosis-inducing factor (AIF). Capsaicin-induced caspase-9, Bid cleavage, and AIF translocation were blocked by calpeptin, and BAPTA and calpeptin attenuated calpain activation and Bid cleavage. Thus, both ER stress- and mitochondria-mediated death pathways are involved in capsaicin-induced apoptosis.     

Distinct endoplasmic reticulum signaling pathways regulate apoptotic and necrotic cell death following iodoacetamide treatment.

Environmental stress induces the synthesis of glucose-regulated proteins (Grps) in the endoplasmic reticulum (ER) and heat shock proteins (Hsps) in the cytoplasm. Iodoacetamide (IDAM), a prototypical alkyating agent, induces both Grp and Hsp synthesis in renal epithelial cells and causes necrosis which is prevented by prior activation of the ER stress response (pre-ER stress) [Liu, H., et al. (1997) J. Biol. Chem. 272, 21751-21759]. In this study, we examined the biochemical pathways leading to IDAM-induced apoptosis and investigated the role of the ER stress response in apoptotic cell death. The antioxidant N,N'-diphenyl-p-phenylenediamine (DPPD) prevented necrosis after IDAM treatment, but the cells went on to die with hallmarks of apoptosis, i.e., cell detachment, caspase-3 activation, cleavage of poly(ADP-ribose)polymerase (PARP), and DNA-ladder formation, all of which were blocked by the general caspase inhibitor zVAD. As with IDAM-induced necrosis, dithiothreitol protected against apoptosis, but cell permeable calcium chelators did not, suggesting that distinct biochemical pathways mediate these two forms of cell death. Pre-ER stress, but not heat shock, prevented IDAM-induced apoptosis. pkASgrp78 cells are deficient in Grp78 induction due to expression of a grp78 antisense RNA and are more sensitive to necrosis. However, these cells were resistant to IDAM-induced apoptosis and had increased basal levels of Grp94 and a KDEL-containing protein of about 50 kDa. Thus, the expression of grp78 antisense perturbs ER functions and activates expression of other ER stress genes accounting for the resistance to apoptosis. Taken together, the data describe functionally distinct signaling pathways through which the ER regulates apoptosis and necrosis caused by chemical toxicants.     

Gateways and tools for drug delivery: endocytic pathways and the cellular dynamics of cell penetrating peptides

A major goal in drug delivery is to be able to design a macromolecular entity that utilises an endocytic pathway to deliver a bioactive payload into a malfunctioning cell. However, the effectiveness of this approach may be constrained by insufficient information regarding the fate of the delivery vector within the confines of the endo-lysosomal network. Successful drug delivery through this mechanism is therefore dependent on an equal high level of understanding of the specific endocytic pathways that are inherent in the target cell and the traffic and fate of the macromolecule within endocytic organelles. Cell penetrating peptides (CPPs) are promising candidate vectors for delivering macromolecules, however, there is little consensus regarding their exact mechanism of uptake. This review highlights the numerous endocytic pathways and sorting mechanisms that may deliver CPPs to a number of cellular destinations. Our use of non-adherent leukaemia cell lines to study the cellular dynamics of CPPs HIV-TAT and octaarginine is also discussed.     

Endothelial endocytic pathways: gates for vascular drug delivery

Vascular endothelium plays strategic roles in many drug delivery paradigms, both as an important therapeutic target itself and as a barrier for reaching tissues beyond the vascular wall. Diverse means are being developed to improve vascular drug delivery including stealth liposomes and polymer carriers. Affinity carriers including antibodies or peptides that specifically bind to endothelial surface determinants, either constitutive or pathological, enhance targeting of drugs to endothelial cells (EC) in diverse vascular areas. In many cases, binding to endothelial surface determinants facilitates internalization of the drug/carrier complex. There are several main endocytic pathways in EC, including clathrin- and caveoli-mediated endocytosis, phagocytosis and macropinocytosis (these two are less characteristic of generic EC) and the recently described Cell Adhesion Molecule (CAM)-mediated endocytosis. The latter may be of interest for intracellular drug delivery to EC involved in inflammation or thrombosis. The metabolism and effects of internalized drugs largely depend on the routes of intracellular trafficking, which may lead to degrading lysosomal compartments or other organelles, recycling to the plasma membrane or transcytosis to the basal surface of endothelium. The latter route, characteristic of caveoli-mediated endocytosis, may serve for trans-endothelial drug delivery. Paracellular trafficking, which can be enhanced under pathological conditions or by auxiliary agents, represents an alternative for transcytosis. Endothelial surface determinants involved in endocytosis, mechanisms of the latter and trafficking pathways, as well as specific characteristics of EC in different vascular areas, are discussed in detail in the context of modern paradigms of vascular drug delivery.     

内质网应激与肿瘤细胞耐药的相关机制

内质网是真核细胞内一种重要的细胞器,当细胞低氧、糖类供应不足或有化学药物处理时均可引发内质网应激(endoplasmic reticulum stress,ERS)。目前研究表明,内质网应激可激活多条通路,其中以未折叠蛋白反应(unfolded protein response,UPR)通路研究最为广泛。UPR通路激活后,可参与调控肿瘤细胞耐药。其中,参与的机制涉及DNA损伤修复、凋亡抑制、自噬等。     

Noninvasive routes of proteins and peptides drug delivery

Recent advances in the field of pharmaceutical biotechnology have led to the formulation of many protein and peptide-based drugs for therapeutic and clinical application. The route of administration has a significant impact on the therapeutic outcome of a drug. The needle and syringe is a well established choice of protein and peptide delivery which has some drawback related to patient and to formulation such as pain, cost, sterility etc. Thus, the noninvasive routes which were of minor importance as parts of drug delivery in the past have assumed added importance in protein and peptide drug delivery and these include nasal, ophthalmic, buccal, vaginal, transdermal and pulmonary routes. The pharmaceutical scientists have some approaches to develop the formulations for protein and peptide delivery by noninvasive routes. But, due to the physiochemical instability and enzymatic barrier of proteins and peptides there are several hurdle to develop suitable formulation. So there is need of penetration enhancers, enzyme inhibitors and suitable vehicles for noninvasive delivery to increase the bioavailability. In this review, the aim is to focus on the approaches to formulation of protein and peptide based drug administration by noninvasive route.     

基于内质网应激的抗肿瘤药物研发

在缺氧、钙代谢紊乱、氧化应激等条件下,细胞内质网功能受到干扰,未折叠或错误折叠蛋白积聚,将产生内质网应激(endoplasmic reticulum stress,ERS)效应。早期ERS可促使肿瘤细胞存活,但长时间或严重的ERS可诱导肿瘤细胞产生凋亡等效应,这使得基于ERS的抗肿瘤药物研发成为可能。该文主要介绍当前基于ERS的抗肿瘤药物研发概况,总结具有诱导ERS效应的天然产物,并在此基础上对基于ERS的抗肿瘤药物研发进行分析,以期为相关领域的发展提供参考。     

Current oral and non-oral routes of antiepileptic drug delivery

Antiepileptic drugs are commonly given orally for chronic treatment of epilepsy. The treatment of epilepsy requires administration of medications for both acute and chronic treatment using multiple types of formulations. Parenteral routes are used when the oral route is unavailable or a rapid clinical response is required. Lorazepam and midazolam can be administered by the buccal, sublingual or intranasal routes. Consensus documents recommend rectal diazepam, buccal midazolam or intranasal midazolam for the out-of-hospital treatment of early status epilepticus. In the United States, diazepam is the only FDA approved rectal formulation. With the lack of parenteral, buccal or intranasal formulations for many of the antiepileptic drugs, the use of the rectal route of delivery to treat acute seizures or to maintain therapeutic concentrations is suitable for many, but not all antiepileptic medications. There is a significant need for new non-oral formulations of the antiepileptic drugs when oral administration is not possible.     

Tributyltin induces apoptotic signaling in hepatocytes through pathways involving the endoplasmic reticulum and mitochondria

Tri-n-butyltin is a widespread environmental toxicant, which accumulates in the liver. This study investigates whether tri-n-butyltin induces pro-apoptotic signaling in rat liver hepatocytes through pathways involving the endoplasmic reticulum and mitochondria. Tri-n-butyltin activated the endoplasmic reticulum pathway of apoptosis, which was demonstrated by the activation of the protease calpain, its translocation to the plasma membrane, followed by cleavage of the calpain substrates, cytoskeletal protein vinculin, and caspase-12. Caspase-12 is localized to the cytoplasmic side of the endoplasmic reticulum and is involved in apoptosis mediated by the endoplasmic reticulum. Tri-n-butyltin also caused translocation of the pro-apoptotic proteins Bax and Bad from the cytosol to mitochondria, as well as changes in mitochondrial membrane permeability, events which can activate the mitochondrial death pathway. Tri-n-butyltin induced downstream apoptotic events in rat hepatocytes at the nuclear level, detected by chromatin condensation and by confocal microscopy using acridine orange. We investigated whether the tri-n-butyltin-induced pro-apoptotic events in hepatocytes could be linked to perturbation of intracellular calcium homeostasis, using confocal microscopy. Tri-n-butyltin caused changes in intracellular calcium distribution, which were similar to those induced by thapsigargin. Calcium was released from a subcellular compartment, which is likely to be the endoplasmic reticulum, into the cytosol. Cytosolic acidification, which is known to trigger apoptosis, also occurred and involved the Cl(-)/HCO(3)(-) exchanger. Pro-apoptotic events in hepatocytes were inhibited by the calcium chelator, Bapta-AM, and by a calpain inhibitor, which suggests that changes in intracellular calcium homeostasis are involved in tri-n-butyltin-induced apoptotic signaling in rat hepatocytes.     

Ricin: the endoplasmic reticulum connection.

Ricin is a potent, plant-derived, ribosome inactivating protein. To target ribosomes in the mammalian cytosol, ricin must firstly negotiate the endomembrane system of the cell to reach the endoplasmic reticulum. Here, the toxin is reduced and the catalytic A chain is recognised by ER components that facilitate its membrane translocation to the cytosol. To be toxic, ricin A chain must then avoid degradation, a conundrum made more tricky in that ubiquitination and proteasomal degradation are normally tightly coupled to the translocation process. This mini-review summarises current understanding of these events.     

Microparticulate delivery systems: potential drug/vaccine carriers via mucosal routes

There is currently considerable research interest in the use of microparticles as carriers for poorly bioavailable drugs and vaccines via mucosal (particularly oral) routes. A variety of therapeutic moieties, including peptides and proteins, have shown enhanced oral uptake when entrapped within various types of microparticulate system constructs, and this approach has also been used successfully for the oral, nasal and rectal delivery of a variety of vaccines. As drug-delivery technology becomes more sophisticated and our understanding of the uptake and immunological mechanisms of mucosal sites becomes more defined, many further microparticulate constructs are emerging and demonstrating their potential.     

Scaffold: a novel carrier for cell and drug delivery

Scaffolds are implants or injects, which are used to deliver cells, drugs, and genes into the body. Different forms of polymeric scaffolds for cell/drug delivery are available: (1) a typical three-dimensional porous matrix, (2) a nanofibrous matrix, (3) a thermosensitive sol-gel transition hydrogel, and (4) a porous microsphere. A scaffold provides a suitable substrate for cell attachment, cell proliferation, differentiated function, and cell migration. Scaffold matrices can be used to achieve drug delivery with high loading and efficiency to specific sites. Biomaterials used for fabrication of scaffold may be natural polymers such as alginate, proteins, collagens, gelatin, fibrins, and albumin, or synthetic polymers such as polyvinyl alcohol and polyglycolide. Bioceramics such as hydroxyapatites and tricalcium phosphates also are used. Techniques used for fabrication of a scaffold include particulate leaching, freeze-drying, supercritical fluid technology, thermally induced phase separation, rapid prototyping, powder compaction, sol-gel, and melt moulding. These techniques allow the preparation of porous structures with regular porosity. Scaffold are used successfully in various fields of tissue engineering such as bone formation, periodontal regeneration, repair of nasal and auricular malformations, cartilage development, as artificial corneas, as heart valves, in tendon repair ,in ligament replacement, and in tumors. They also are used in joint pain inflammation, diabetes, heart disease, osteochondrogenesis, and wound dressings. Their application of late has extended to delivery of drugs and genetic materials, including plasmid DNA, at a controlled rate over a long period of time. In addition, the incorporation of drugs (i.e., inflammatory inhibitors and/or antibiotics) into scaffolds may be used to prevent infection after surgery and other disease for longer duration. Scaffold also can be used to provide adequate signals (e.g., through the use of adhesion peptides and growth factors) to the cells, to induce and maintain them in their desired differentiation stage, and to maintain their survival and growth. The present review gives a detailed account of the need for the development of scaffolds along with the materials used and techniques adopted to manufacture scaffolds for tissue engineering and for prolonged drug delivery.     

Archaeosomes: an excellent carrier for drug and cell delivery

Archaeosomes as liposomes made with one or more ether lipids that are unique to the domain of Archaeobacteria, found in Archaea constitute a novel family of liposome. Achaean-type lipids consist of archaeol (diether) and/or caldarchaeol (tetraether) core structures. Archaeosomes can be produced using standard procedures (hydrated film submitted to sonication, extrusion and detergent dialysis) at any temperature in the physiological range or lower, therefore making it possible to encapsulate thermally stable compounds. Various physiological as well as environmental factors affect its stability. Archaeosomes are widely used as drug delivery systems for cancer vaccines, Chagas disease, proteins and peptides, gene delivery, antigen delivery and delivery of natural antioxidant compounds. In this review article, our major aim was to explore the applications of this new carrier system in pharmaceutical field.     

Transplacental drug delivery: gene and virus delivery to the trophoblast

The development of successful strategies for delivering genes to the placenta may provide new opportunities for modifying trophoblast function in order to learn more about trophoblast physiology and to offer novel therapeutic options for complications of pregnancy that result from placental dysfunction. Replication-deficient recombinant viral vectors are useful vehicles for introducing genes into cells in vitro and in vivo. Recombinant adenovirus and herpes simplex virus vectors are unable to efficiently infect and transduce terminally differentiated trophoblastic cells. However, recombinant adeno-associated virus vectors transduce terminally differentiated trophoblastic cells, and a Sindbis virus construct efficiently transduces and destroys trophoblastic cancer cells. We describe the features that make particular viral vectors attractive for gene transfer to trophoblastic cells.