Intracellular localisation of proteins to specific cellular areas by nanocapsule mediated delivery

Nanocapsules are promising carriers with great potential for intracellular protein transport. Although many studies have intended to improve cell uptake efficacy, there is an increasing interest in understanding of subcellular distribution of cargoes inside cells, which is essential for purposeful delivery of biomolecules into specific sites within cells. Herein, we interrogate the intracellular localisation of exogenous proteins, including fluorescein isothiocyanate (FITC)-labelled bovine serum albumin (BSA) and green fluorescent protein (GFP), mediated by specially designed nanocapsules. The results show that the designed nanocapsules can deliver the two types of fluorescent proteins into different cellular destinations (cytosol, nucleus or the whole cell), depending on the composition of nanocapsules. Meanwhile, several impact factors that influence the distribution of proteins in cells have also been investigated, and the results suggest that the localisation of capsule-mediated proteins in cells is strongly affected by the surface properties of nanocapsules, the types of stabilisers and proteins, and environmental temperatures. The rational control of intracellular localised delivery of exogenous proteins as we demonstrated in this study might open new avenues to obtain desired magnitude of drug effects for modulating cell activity.     

Nucleo-cytoplasmic transport of proteins as a target for therapeutic drugs

Recruitment of cytoplasmic signaling proteins into the nucleus is an essential step in the activation of gene expression in response to an extracellular signal. Nucleo-cytoplasmic transport of macromolecules is mediated by the transport receptors of an importin beta family. Post-translational modifications and masking/unmasking of specific signal sequences responsible for nuclear import and export are important for the coordinated control of the nucleo-cytoplasmic transport. Malfunctioning of the nucleo-cytoplasmic transport is profoundly involved in a number of diseases including cancer. Leptomycin B (LMB) is a Streptomyces metabolite that causes specific inhibition of the cell cycle of fission yeast and mammalian cells. The target molecule of LMB has been shown by genetic and biochemical analyses to be CRM1, a highly conserved protein in eukaryotes. CRM1 was shown to be a member of the importin beta family and a receptor for the nuclear export signal (NES) of proteins in both yeast and mammalian cells. LMB binds directly to CRM1, which results in dissociation of the NES from the nuclear export machinery containing CRM1. Thus, LMB serves as a potent tool for understanding the molecular mechanisms of nucleo-cytoplasmic transport of proteins and a potential therapeutic drug for diseases caused by mislocalization of regulatory proteins.     

Exploiting protein cationization techniques in future drug development

The development of a method for the efficient intracellular delivery of inherently non-permeable proteins is needed for manipulation of cellular phenotypes or the discovery of protein-based drugs. It has been demonstrated that proteins artificially cationized by chemical conjugation show efficient intracellular delivery via adsorptive-mediated endocytosis and then can exert their biological activity in cells. Studies have also revealed that cationic peptides known as cell-penetrating peptides (CPPs) provide a means to deliver molecules into mammalian cells. Although the internalization mechanisms remain controversial, it is now becoming clear that the main port of entry into cells by CPPs also involves adsorptive-mediated endocytosis rather than the direct penetration of the plasma membrane. As the mammalian cell membrane possesses an abundance of negatively charged glycoproteins and glycosphingolipids, cationization of proteins is a reasonable choice to endow them with the ability for intracellular delivery. Cationization of proteins is usually accompanied by drastic changes in protein properties, structure and biological activities. Recently developed sophisticated protein chemistry can minimize these side effects. Therefore, protein cationization techniques will hopefully prove to be powerful tools for innovative research and drug discovery. In this review, techniques for cationization of proteins and their intracellular delivery, as well as some of their potential therapeutic applications, are discussed.     

Biodegradable nanoparticles for cytosolic delivery of therapeutics

Many therapeutics require efficient cytosolic delivery either because the receptors for those drugs are located in the cytosol or their site of action is an intracellular organelle that requires transport through the cytosolic compartment. To achieve efficient cytosolic delivery of therapeutics, different nanomaterials have been developed that consider the diverse physicochemical nature of therapeutics (macromolecule to small molecule; water soluble to water insoluble) and various membrane associated and intracellular barriers that these systems need to overcome to efficiently deliver and retain therapeutics in the cytoplasmic compartment. Our interest is in investigating PLGA and PLA-based nanoparticles for intracellular delivery of drugs and genes. The present review discusses the various aspects of our studies and emphasizes the need for understanding of the molecular mechanisms of intracellular trafficking of nanoparticles in order to develop an efficient cytosolic delivery system.     

The (patho)physiological functions of the MRP family

The identification of certain members of the large superfamily of ATP binding cassette transport proteins such as MDR1 -P-glycoprotein and the multidrug resistance protein MRP1 as ATP-dependent drug efflux pumps has been a major contribution in our understanding of the multidrug resistance phenotype of cancer cells. Importantly, both transport proteins that exhibit only low structural homology have a very different substrate specificity but confer resistance to a similar spectrum of natural product chemotherapeutic drugs. In contrast to the drug transporter MDR1, MRP1 mainly transports anionic Phase II-conjugates. In addition MRP1-mediated drug resistance is highly dependent on high intracellular glutathione levels which may be linked to the apparent physiological involvement of MRP1 in glutathione-related cellular processes. This review summarizes the current knowledge about functional aspects of MRP1 and its five recently cloned homologues MRP2–MRP6 and discusses their substrate specificities and cellular localization with emphasis on drug resistance.     

Oligonucleotide delivery: a cellular prospective.

The use of oligonucleotides (ONs) for gene therapy of certain diseases has been discussed since the late 1970s. ONs are single stranded chains of nucleic acids that can hybridize with target nucleic acid sequences to inhibit specific proteins, and therefore allow selective treatment of various diseases. The use of ONs is limited due to their instability in biological tissues and difficulty in delivery to the intracellular compartments of the cell. Chemical analog approaches have been used to address the instability issue and delivery systems have been developed to increase cellular uptake of ONs. It is generally thought that ONs with or without a delivery system are transported into cells by endocytosis, and then accumulate within endosomes where they are significantly inactivated. The rate and extent of movement of ON from endosomes appears to be important in determining ON effects. Consequently, developing accessory compounds or delivery methods that enhance endosome to cytoplasm transfer may be vital to ON therapy. This review focuses on investigating mechanisms of various delivery approaches at the cellular/intracellular level that have demonstrated utility in increasing ON activity or cellular accumulation. The future prospects of ON delivery are also addressed.     

Liposomal drug delivery systems--clinical applications

Liposomes have been widely investigated since 1970 as drug carriers for improving the delivery of therapeutic agents to specific sites in the body. As a result, numerous improvements have been made, thus making this technology potentially useful for the treatment of certain diseases in the clinics. The success of liposomes as drug carriers has been reflected in a number of liposome-based formulations, which are commercially available or are currently undergoing clinical trials. The current pharmaceutical preparations of liposome-based therapeutic systems mainly result from our understanding of lipid-drug interactions and liposome disposition mechanisms. The insight gained from clinical use of liposome drug delivery systems can now be integrated to design liposomes that can be targeted on tissues, cells or intracellular compartments with or without expression of target recognition molecules on liposome membranes. This review is mainly focused on the diseases that have attracted most attention with respect to liposomal drug delivery and have therefore yielded most progress, namely cancer, antibacterial and antifungal disorders. In addition, increased gene transfer efficiencies could be obtained by appropriate selection of the gene transfer vector and mode of delivery.     

Intranasal drug delivery for brain targeting

Many drugs are not being effectively and efficiently delivered using conventional drug delivery approach to brain or central nervous system (CNS) due to its complexity. The brain and the central nervous system both have limited accessibility to blood compartment due to a number of barriers. Many advanced and effective approaches to brain delivery of drugs have emerged in recent years. Intranasal drug delivery is one of the focused delivery options for brain targeting, as the brain and nose compartments are connected to each other via the olfactory route and via peripheral circulation. Realization of nose to brain transport and the therapeutic viability of this route can be traced from the ancient times and has been investigated for rapid and effective transport in the last two decades. Various models have been designed and studied by scientists to establish the qualitative and quantitative transport through nasal mucosa to brain. The development of nasal drug products for brain targeting is still faced with enormous challenges. A better understanding in terms of properties of the drug candidate, nose to brain transport mechanism, and transport to and within the brain is of utmost importance. This review will discuss some pertinent issues to be considered and challenges to brain targeted intranasal drug delivery. A few marketed and investigational drug formulations will also be discussed.     

Strategies for in vivo siRNA delivery in cancer

A better understanding of the mechanisms involved in small interference RNA (siRNA) gene silencing opens new horizons for the development of the targeted therapy of malignant and benign diseases. As a research tool, siRNA has proven to be highly effective in silencing specific genes and modulating intracellular signaling pathways. However, systemic delivery of siRNA has been more problematic due to degradation and poor cellular uptake. In order to overcome these limitations, a variety of strategies are being developed including new delivery vehicles and chemical modifications. Here, we review potential approaches for the systemic delivery of siRNA for cancer treatment.     

Current status of polymeric gene delivery systems

Gene therapy provides great opportunities for treating diseases from genetic disorders, infections and cancer. To achieve successful gene therapy, development of proper gene delivery systems could be one of the most important factors. Several non-viral gene transfer methods have been developed to overcome the safety problems of their viral counterpart. Polymer-based non-viral gene carriers have been used due to their merits in safety including the avoidance of potential immunogenecity and toxicity, the possibility of repeated administration, and the ease of the establishment of good manufacturing practice (GMP). A wide range of polymeric vectors have been utilized to deliver therapeutic genes in vivo. The modification of polymeric vectors has also shown successful improvements in achieving target-specific delivery and in promoting intracellular gene transfer efficiency. Various systemic and cellular barriers, including serum proteins in blood stream, cell membrane, endosomal compartment and nuclear membrane, were successfully circumvented by designing polymer carriers having a smart molecular structure. This review explores the recent development of polymeric gene carriers and presents the future directions for the application of the polymer-based gene delivery systems in gene therapy.     

Orally active-targeted drug delivery systems for proteins and peptides

Introduction: In the past decade, extensive efforts have been devoted to designing ‘active targeted’ drug delivery systems (ATDDS) to improve oral absorption of proteins and peptides. Such ATDDS enhance cellular internalization and permeability of proteins and peptides via molecular recognition processes such as ligand–receptor or antigen?antibody interaction, and thus enhance drug absorption.

Areas covered: This review focuses on recent advances with orally ATDDS, including ligand–protein conjugates, recombinant ligand–protein fusion proteins and ligand-modified carriers. In addition to traditional intestinal active transport systems of substrates and their corresponding receptors, transporters and carriers, new targets such as intercellular adhesion molecule-1 and β-integrin are also discussed.

Expert opinion: ATDDS can improve oral absorption of proteins and peptides. However, currently, no clinical studies on ATDDS for proteins and peptides are underway, perhaps due to the complexity and limited knowledge of transport mechanisms. Therefore, more research is warranted to optimize ATDDS efficiency.     

Biodegradable nanoparticles for drug and gene delivery to cells and tissue

Biodegradable nanoparticles formulated from poly (D,L-lactide-co-glycolide) (PLGA) have been extensively investigated for sustained and targeted/localized delivery of different agents including plasmid DNA, proteins and peptides and low molecular weight compounds. Research about the mechanism of intracellular uptake of nanoparticles, their trafficking and sorting into different intracellular compartments, and the mechanism of enhanced therapeutic efficacy of nanoparticle-encapsulated agent at cellular level is more recent and is the primary focus of the review. Recent studies in our laboratory demonstrated rapid escape of PLGA nanoparticles from the endo-lysosomal compartment into cytosol following their uptake. Based on the above mechanism, various potential applications of nanoparticles for delivery of therapeutic agents to the cells and tissue are discussed.     

Intracellular delivery: exploiting viral membranotropic peptides

Recent advances in the understanding of cellular and molecular mechanisms of the pathogenesis of several diseases offer the possibility to address novel molecular targets for an improved diagnosis and therapy. In fact, in order to fulfill their function, macromolecular drugs, reporter molecules, and imaging agents often require to be delivered into specific intracellular compartments, usually the cytoplasm or the nucleus. From a medical perspective, biological membranes represent a critical hindrance due to their barrier-like behaviour not easily circumvented by many pharmacologically-active molecules. Therefore, identifying strategies for membrane translocation is essential. Several technologies have been designed to improve cellular uptake of therapeutic molecules, including cell-penetrating peptides (CPPs). These peptides, which are able to efficiently translocate macromolecules through the plasma membrane, have attracted a lot of attention, and new translocating peptides are continuously described. In this review, we will focus on the viral derived peptides, and in particular those derived by viral entry proteins that may be useful as delivery vehicles due to their intrinsic properties of inducing membrane perturbation.     

Misrouted cell surface receptors as a novel disease aetiology and potential therapeutic target: the case of hypogonadotropic hypogonadism due to gonadotropin-releasing hormone resistance

Molecules that are incorrectly folded or defectively assembled are recognised by cellular quality control mechanisms. This leads such conformationally abnormal molecules to intracellular retention and eventual degradation. A number of diseases caused by mutations that interfere with proper processing and intracellular trafficking of key cell surface proteins have been described. These include a particular variant of hypogonadotropic hypogonadism, which results from mislocalisation of the gonadotropin-releasing hormone (GnRH) receptor. It has been shown recently that membrane expression and function of misfolded GnRH receptor mutants can be rescued by a peptidomimetic antagonist of GnRH (IN3) that permeates into the cell and reaches the abnormally manufactured nascent receptor, stabilising a conformation compatible with cell-surface transport and reversing intracellular retention. This approach seems applicable for the development of defined therapeutic strategies for an array of diseases caused by incorrectly routed cell surface or secreted proteins.     

Intracellular targeting of polymer-bound drugs for cancer chemotherapy

Macromolecules have been traditionally employed as drug carriers due to their ability to selectively accumulate in malignant tissues compared to healthy tissues by either passive or active targeting, thus precluding undesirable side effects generated by free drug. The therapeutic activity proffered by such conjugates requires that the drug concentrate at its specific subcellular target such as the nucleus. Thus, the suitability of macromolecules as carriers also extends to their propensity to deliver the drug to a predetermined intracellular location. As binding a macromolecule to a drug facilitates cellular uptake by endocytosis, various approaches have been employed to either guide the drug to targets different from endosomal/lysosomal compartments by mediating vesicular escape, or to directly accomplish intracellular (cytoplasmic and nuclear) localization. This review discusses the utility of macromolecules in drug delivery and describes the numerous modalities (with a focus on cell-penetrating peptides) currently available for achieving effective intracellular drug delivery.     

Function and regulation of ATP-binding cassette transport proteins involved in hepatobiliary transport

Hepatobiliary transport of endogenous and exogenous compounds is mediated by the coordinated action of multiple transport systems present at the sinusoidal (basolateral) and canalicular (apical) membrane domains of hepatocytes. During the last few years many of these transporters have been cloned and functionally characterized. In addition, the molecular bases of several forms of cholestatic liver disease have been defined. Combined, this has greatly expanded our understanding of the normal physiology of bile formation, the pathophysiology of intrahepatic cholestasis, as well as of drug elimination and disposition processes. In this review recent advances, with respect to function and regulation of ATP binding cassette transport proteins expressed in liver, are summarized and discussed.     

Intracellular chloride channels: critical mediators of cell viability and potential targets for cancer therapy

The passage of ions to form and maintain electrochemical gradients is a key element for regulating cellular activities and is dependent on specific channel proteins or complexes. Certain ion channels have been the targets of pharmaceuticals that have had impact on a variety of cardiovascular and neurological diseases. Chloride channels regulate the movement of a major cellular anion, and in so doing they in part determine cell membrane potential, modify transepithelial transport, and maintain intracellular pH and cell volume. There are multiple families of chloride channel proteins, and respiratory, neuromuscular, and renal dysfunction may result from mutations in specific family members. Interest in chloride channels related to cancer first arose when the multidrug resistance protein (MDR/P-glycoprotein) was linked to volume-activated chloride channel activity in cancer cells from patients undergoing chemotherapy. More recently, CLC, CLIC, and CLCA intracellular chloride channels have been recognized for their contributions in modifying cell cycle, apoptosis, cell adhesion, and cell motility. Moreover, advances in structural biology and high-throughput screening provide a platform to identify chemical compounds that modulate the activities of intracellular chloride channels thereby influencing chloride ion transport and altering cell behavior. This review will focus on several chloride channel families that may contribute to the cancer phenotype and suggest how they may serve as novel targets for primary cancer therapy.     

Biodegradable nanoparticles for drug and gene delivery to cells and tissue

Biodegradable nanoparticles formulated from poly (d,l-lactide-co-glycolide) (PLGA) have been extensively investigated for sustained and targeted/localized delivery of different agents including plasmid DNA, proteins and peptides and low molecular weight compounds. Research about the mechanism of intracellular uptake of nanoparticles, their trafficking and sorting into different intracellular compartments, and the mechanism of enhanced therapeutic efficacy of nanoparticle-encapsulated agent at cellular level is more recent and is the primary focus of the review. Recent studies in our laboratory demonstrated rapid escape of PLGA nanoparticles from the endo-lysosomal compartment into cytosol following their uptake. Based on the above mechanism, various potential applications of nanoparticles for delivery of therapeutic agents to the cells and tissue are discussed.     

Mechanisms of TfR-mediated transcytosis and sorting in epithelial cells and applications toward drug delivery

Transferrin receptor has been an important protein for many of the advances made in understanding the intricacies of the intramolecular sorting pathways of endocytosed molecules. The unique internalization and recycling functions of transferrin receptor have also made it an attractive choice for drug targeting and delivery of large protein-based therapeutics and toxins. Recent advances in elucidating the role of the intracellular controllers of transferrin recycling and sorting, such as Rab proteins and their effectors, have led to enhancement of transferrin receptor as a drug delivery vehicle. This review focuses on the use of transferrin receptor as an agent for facilitating drug delivery and targeting, and the role that mechanisms of transferrin receptor sorting and transcytosis play in these events.     

Nuclear delivery of macromolecules: barriers and carriers

Recent evidence for efficient delivery of macromolecules, such as peptides and nucleic acids, from the cell exterior to the nucleus offers the interesting possibility of developing novel treatments directed at intranuclear targets. The findings should also stimulate the search for physiological ligands that utilize similar transport mechanisms to regulate pathobiological processes. Cytokines, growth factors and their receptors, as well as morphogens have all been shown to enter the nucleus to evoke biological responses in target cells. The rational design of intracellular drug delivery vehicles requires an increased understanding of the elaborate systems that mediate cellular communication and coordination with the extracellular environment without inflicting on the integrity of the cell. This review discusses some aspects of the carriers and barriers in macromolecular transport.