Targeting intracellular targets

Many therapeutic agents have intracellular compartments as their site of action. Targeted delivery of these agents to their specific intracellular targets could result in enhanced therapeutic efficacy and reduced toxicity. Various carriers have been shown useful in targeted delivery of different classes of therapeutic agents. Among these carriers, biodegradable nanoparticles formulated from biocompatible polymers poly(D,L-lactide-co-glycolide) (PLGA) and polylactide (PLA) have shown the potential for sustained intracellular delivery of different therapeutic agents. In this review, we discuss different intracellular targets, barriers to intracellular delivery, mechanism and pathways of intracellular delivery, and various carriers and approaches that have been investigated for intracellular drug delivery.     

Nanovehicular intracellular delivery systems

This article provides an overview of principles and barriers relevant to intracellular drug and gene transport, accumulation and retention (collectively called as drug delivery) by means of nanovehicles (NV). The aim is to deliver a cargo to a particular intracellular site, if possible, to exert a local action. Some of the principles discussed in this article apply to noncolloidal drugs that are not permeable to the plasma membrane or to the blood-brain barrier. NV are defined as a wide range of nanosized particles leading to colloidal objects which are capable of entering cells and tissues and delivering a cargo intracelullarly. Different localization and targeting means are discussed. Limited discussion on pharmacokinetics and pharmacodynamics is also presented. NVs are contrasted to micro-delivery and current nanotechnologies which are already in commercial use. Newer developments in NV technologies are outlined and future applications are stressed. We also briefly review the existing modeling tools and approaches to quantitatively describe the behavior of targeted NV within the vascular and tumor compartments, an area of particular importance. While we list "elementary" phenomena related to different level of complexity of delivery to cancer, we also stress importance of multi-scale modeling and bottom-up systems biology approach.     

Modulators of intracellular calcium

The importance of calcium in excitation-contraction coupling in both cardiac and vascular smooth muscle has resulted in an intense research interest into the intracellular regulation of this ion. Selective foci for the modulation of intracellular calcium include the interaction of calcium with the contractile protein apparatus, sites of calcium release and sequestration, and pathways for the extrusion of calcium into the extracellular space. Research efforts directed towards elucidating these phenomena have met with varied degrees of success. The presence of different calcium regulatory systems for contractile protein function, i.e., troponin in cardiac and calmodulin-myosin light chain kinase in vascular, provides an attractive rationale for the design of selective compounds. The inherent difficulty in studying intracellular release and sequestration presently presently precludes examining the physiological implications of specific inhibition of these phenomena. However, the apparent absence of a sodium-dependent calcium extrusion pathway in vascular tissue may lead to the design of novel cardiotonics. It is anticipated that further clarification of the similarities and differences in the calcium cycle between these tissues will result in the development of tissue-selective therapeutic agents.     

Sensing intracellular pathogens-NOD-like receptors

The innate immune system uses different molecules that sense pathogen associated molecular patterns. These include Toll-like receptors (TLRs), RIG-1-like receptors (RLRs) and the NOD-like receptors (NLRs). The NLRs, consisting of more than 20 related family members, are present in the cytosol and recognize intracellular ligands. Members of the NLR can be grouped into molecules that contain either a CARD or a Pyrin motif. The NOD proteins mediate NF-kappaB activation, whereas Pyrin molecules such as NALP3 regulate IL-1beta and IL-18 production. In this review, we will discuss the role of NLRs in pattern recognition of microbial components and their role in health and disease.     

Pharmacology of intracellular signalling pathways

This article provides a brief and somewhat personalized review of the dramatic developments that have occurred over the last 45 years in our understanding of intracellular signalling pathways associated with G-protein-coupled receptor activation. Signalling via cyclic AMP, the phosphoinositides and Ca(2+) is emphasized and these systems have already been revealed as new pharmacological targets. The therapeutic benefits of most of such targets are, however, yet to be realized, but it is certain that the discipline of pharmacology needs to widen its boundaries to meet these challenges in the future.     

Vaccines against intracellular bacterial pathogens

There is a long history of remarkable success in developing vaccines against bacteria that are extracellular pathogens. In general, the development of vaccines against intracellular bacterial pathogens has proven to be more challenging. Typically, such vaccines need to induce a range of immune responses, including antibody, CD4(+) and CD8(+) T cell responses. These responses can be induced by live attenuated vaccines, but eliciting these responses with non-living vaccines has proven to be difficult. The difficulties appear to be related partly to the problems associated with the identification of protective antigens and partly with the difficulties associated with inducing CD8(+) T cell responses.     

HAP1 and intracellular trafficking

Huntington's disease is caused by a polyglutamine expansion in the protein huntingtin. Several studies suggest that huntingtin and its associated protein HAP1 participate in intracellular trafficking and that polyglutamine expansion affects vesicular transport. A study now provides new evidence that HAP1 is also involved in the endocytosis of membrane receptors. These studies offer insight into the normal function of HAP1 and its involvement in Huntington's disease.     

灵芝多糖对小鼠T细胞胞浆游离Ca2+浓度和胞内pH的影响

目的通过考察灵芝多糖(GLP)对免疫细胞信号转导过程的影响,探讨GLP免疫调节作用机制。方法采用激光扫描共聚焦显微镜(LSCM)技术,动态监测GLP均一体组分GLB7对小鼠T细胞胞浆游离Ca     

灵芝多糖对小鼠T细胞胞浆游离Ca~(2+)浓度和胞内pH的影响

目的通过考察灵芝多糖（ＧＬＰ）对免疫细胞信号转导过程的影响,探讨ＧＬＰ免疫调节作用机制。方法采用激光扫描共聚焦显微镜（ＬＳＣＭ）技术,动态监测ＧＬＰ均一体组分ＧＬＢ７对小鼠Ｔ细胞胞浆游离Ｃａ２＋浓度（［Ｃａ２＋）和胞内 ｐＨ（［ｐＨ］ｉ）的影响。结果 ＧＬＢ７（２０ｍｇ·Ｌ－１）引起小鼠 Ｔ细胞中［Ｃａ２＋］;和［ｐＨ］;明显升高,１ｍｉｎ即分别升高至３３４．７０％±１６,４％（ｎ＝３）、１７１．６％ ± １０．４％（ｎ＝３）,之后一直分别维持在该平台期,至 １０ ｍｉｎ仍维持高峰水平。加入ＥＣＴＡ和 ｖｅｒａｐａｍｉｌ处理后, １０ ｍｉｎ ＧＬＢ７ 引起 Ｔ细胞［Ｃａ２＋］;和［ｐＨ］;分别升高为 ２０２．４％± １３．６％（ｎ＝３）。１４０．２％±７．８％（ｎ＝３）,与正常对照组比较差异有显著性（Ｐ＜０．０1）。以 ＥＧＴＡ和 ｖｅｒａｐａｍｉｌ处理后,再分别以 ｈｅｐ－ｅｒｉｎ和 ｐrocaine处理细胞 １０ ｍｉｎ,之后以 ＧＬＢ７刺激Ｔ细胞,１０ｍｉｎ［Ｃａ２＋］ｉ值分别为１５１．５％±９．４％（ｎ＝３）、１４３．２％± ８．１ ％（ ｎ＝ ３）,与 ＥＧＴＡ和 ｖｅｒａｐａｍｉｌ处理组相比差异有显著性（ Ｐ＜ ０．０１）。同时以     

Alterations in intracellular calcium during sepsis

Tissue injury and/or infection produce significant alterations in intracellular calcium ion regulation. These alterations in cellular calcium has recently been studied following both short term and long term septic model which uses two types of gram-negative bacteria frequently encountered human abdominal sepsis. Changes in calcium flux as well as functional disturbances has been observed in the major organs, specially in skeletal muscle. The changes in calcium flux in different organs were studied using 45Ca exchange, 19F NMR study or by using calcium-fluorescence probes. Calcium-channel blockers attenuate the increased effects of calcium flux. Further anti-cytokines may be useful to prevent septic injury in tissues.     

线粒体:新的细胞内药物作用靶点

线粒体除了作为细胞内能量生成的关键细胞器 ,还与细胞凋亡、自由基生成、脂质代谢等重要生化过程密切相关。近年来随着对线粒体结构和功能的深入研究 ,发现线粒体是多类药物的细胞内作用靶点 ,与相关药物之间存在广泛而复杂的相互作用。研究线粒体与相关药物之间的相互作用对于明确药物作用机制、研发新药和避免药物毒副作用等方面具有重要意义。该文就线粒体的靶点特性及与药物之间的相互作用作一简要介绍     

Cell-penetrating peptides with intracellular organelle targeting

Introduction: One of the major limiting steps in order to have an effective drug is the passage through one or more cell membranes to reach its site of action. To reach the action-site, the specific macromolecules are required to be delivered specifically to the cell compartment/organelle in their (pre)active form.

Areas covered: In this review, we will discuss cell-penetrating peptides (CPPs) developed in the last decade to transport small RNA/DNA, plasmids, antibodies, and nanoparticles into specific sites of the cell. The article describes CPPs in complex with cargo molecules that target specific intracellular organelles and their potential for pharmacological or clinical use.

Expert opinion: Organelle targeting is the ultimate goal to ensure selective delivery to the site of action in the cells. CPP technologies represent an important strategy to address drug delivery to specific intracellular compartments by covalent conjugation to targeting sequences, potentially enabling strategies to combat genomic diseases as well as infections, cancer, neurodegenerative and hereditary diseases. They have proven to be successful in delivering various therapeutic agents into cells however, further in vivo experiments and clinical trials are required to demonstrate the efficacy of this technology.     

Cardiac alternans and intracellular calcium cycling

Cardiac alternans refers to a condition in which there is a periodic beat‐to‐beat oscillation in electrical activity and the strength of cardiac muscle contraction at a constant heart rate. Clinically, cardiac alternans occurs in settings that are typical for cardiac arrhythmias and has been causally linked to these conditions. At the cellular level, alternans is defined as beat‐to‐beat alternations in contraction amplitude (mechanical alternans), action potential duration (APD; electrical or APD alternans) and Ca²⁺ transient amplitude (Ca²⁺ alternans). The cause of alternans is multifactorial; however, alternans always originate from disturbances of the bidirectional coupling between membrane voltage (V_m) and intracellular calcium ([Ca²⁺]_i). Bidirectional coupling refers to the fact that, in cardiac cells, V_m depolarization and the generation of action potentials cause the elevation of [Ca²⁺]_i that is required for contraction (a process referred to as excitation–contraction coupling); conversely, changes of [Ca²⁺]_i control V_m because important membrane currents are Ca²⁺ dependent. Evidence is mounting that alternans is ultimately caused by disturbances of cellular Ca²⁺ signalling. Herein we review how two key factors of cardiac cellular Ca²⁺ cycling, namely the release of Ca²⁺ from internal stores and the capability of clearing the cytosol from Ca²⁺ after each beat, determine the conditions under which alternans occurs. The contributions from key Ca²⁺‐handling proteins (i.e. surface membrane channels, ion pumps and transporters and internal Ca²⁺ release channels) are discussed.     

Synthetic nanocarriers for intracellular protein delivery

Introducing exogenous proteins intracellularly presents tremendous chances in scientific research and clinical applications. The effectiveness of this method, however, has been limited by lack of efficient ways to achieve intracellular protein delivery and poor stability of the delivered proteins. Over the years, a variety of nanomaterials have been explored as intracellular protein delivery vectors, including liposomes, polymers, gold nanoparticles, mesoporous silica particles, and carbon nanotubes. Nanomaterials stand out in various protein delivery systems due to various advantages, such as efficient intracellular delivery, long circulation time, and passive tumor targeting. Additionally, chemistry behind these nanomaterials provides readily engineered materials, enabling versatile designs of delivery agents. Intracellular delivery mediated by such nanocarriers achieved varying degrees of success. Different problems associated with these nanocarriers, however, still hamper their real-world applications. Developing new delivery methods or vectors remains essential but challenging. This review surveys the current developments in protein delivery based on synthetic nanocarriers, including liposomes, polymers and inorganic nanocarriers; Prospects for future development of protein delivery nanocarriers are also provided.     

Prodrug-based intracellular delivery of anticancer agents

There are numerous anticancer agents based on a prodrug approach. However, no attempt has been made to review the ample available literature with a specific focus on the altered cell uptake pathways enabled by the conjugation and on the intracellular drug-release mechanisms. This article focuses on the cellular interactions of a broad selection of parenterally administered anticancer prodrugs based on synthetic polymers, proteins or lipids. The report also aims to highlight the prodrug design issues, which are key points to obtain an efficient intracellular drug delivery. The chemical basis of these molecular concepts is put into perspective with the uptake and intracellular activation mechanisms, the in vitro and in vivo proofs of concepts and the clinical results. Several active targeting strategies and stimuli-responsive architectures are discussed throughout the article.     

Caveolae and intracellular trafficking of cholesterol

Caveolae, free cholesterol (FC)-rich microdomains of the plasma membrane, are both a terminus for the intracellular transit of newly synthesized and recycling cellular FC, and a site for FC efflux to the extracellular medium. The same domains play key roles as locations for the assembly of signaling complexes and for the endocytosis of selected ligands. Caveolin, the major structural protein of caveolae, plays a regulatory role in growth, the cell cycle, and cell adhesion. Each of these functions is FC-dependent. Caveolae appear to act as both sensors and regulators of cellular FC content, and in this way mediate an array of membrane-dependent cell functions.     

Histamine increases vascular tone and intracellular calcium level using both intracellular and extracellular calcium in porcine coronary arteries

Effects of histamine on the tone and intracellular calcium level (Ca2+i) in porcine coronary arteries were simultaneously investigated by use of the fura-2 microscopic fluorometric method. Histamine (10(-6)-10(-4) M) induced concentration-dependent increases in tone and Ca2+i, but these responses were not sustained. Histamine induced a larger contraction than did KCl with a similar increase in Ca2+i. Depletion of the caffeine-sensitive Ca2+ store with ryanodine (3 x 10(-5) M) and repetitive applications of caffeine (2.5 x 10(-2) M) scarcely affected contractile and Ca2+i responses to histamine. In Ca2(+)-free medium or in the presence of verapamil (10(-6) M), histamine produced a briefer increase in Ca2+i and a smaller contraction than in normal medium. When histamine or caffeine was repetitively applied in Ca2(+)-free medium, the first application produced an increase in Ca2+i but the second application produced no increase. Although caffeine increased Ca2+i after repetitive histamine applications, histamine failed to increase Ca2+i after repetitive caffeine applications in Ca2(+)-free medium. These results indicate that vascular contraction induced by histamine may involve the following mechanisms: an increase in Ca2+ influx through Ca2+ channels, release of Ca2+ from the intracellular Ca2+ store which has an interaction with the caffeine-sensitive Ca2+ store, and sensitization of contractile proteins to Ca2+.     

Antibody-onconase conjugates: cytotoxicity and intracellular routing

Onconase, a member of the pancreatic ribonuclease A superfamily, is currently in Phase III clinical trials for treatment of unresectable malignant mesothelioma. The anticancer effect of onconase may relate to its intracellular target, a non-coding RNA. Some non-coding RNAs are aberrantly expressed in cancer cells. This discovery is creating new interest in drugs that target RNA. Conjugating onconase to agents that recognize tumor associated molecules further increases its potency and specificity. Analysis of onconase activity when directed to two different internalizing and one non-internalizing receptor reveals that the ideal targeting agents would rapidly enter lysosomal compartments before onconase escaped to the cytosol. Antibody-onconase conjugates are effective in preclinical models, cause little non-specific toxicities in mice and have favorable formulation properties. Understanding the reason for their potency coupled with understanding novel RNA-based mechanisms of tumor cell death will lead to improved variations of targeted onconase.