The use of neural stem cells in cancer gene therapy: predicting the path to the clinic

Gene therapy is a novel means of anticancer treatment that has led to preliminary positive results in the preclinical setting, as well as in clinical trials; however, successful clinical application of this approach has been hampered by the inability of gene delivery systems to target tumors and to deliver a therapeutic payload to disseminated tumor foci efficiently. Along with viral vector systems, various mammalian cells with tropism for tumor cells have been considered as vehicles for delivery of anticancer therapeutics. The discovery of the inherent tumor-tropic properties of neural stem cells (NSCs) has provided a unique opportunity to develop targeted therapies that use NSCs as a vehicle to track invasive tumor cells and deliver anticancer agents selectively to diseased areas. Many in vivo and in vitro studies have demonstrated that the targeted migration of NSCs to infiltrative brain tumors, including malignant glioma, provides a potential therapeutic approach. In this review, the development of NSCs as targeted carriers for anticancer gene therapy is discussed, and barriers in the path to the clinic, as well as approaches to overcoming such barriers are presented.     

Advanced molecular design of biopolymers for transmucosal and intracellular delivery of chemotherapeutic agents and biological therapeutics

Hydrogels have been instrumental in the development of polymeric systems for controlled release of therapeutic agents. These materials are attractive for transmucosal and intracellular drug delivery because of their facile synthesis, inherent biocompatibility, tunable physicochemical properties, and capacity to respond to various physiological stimuli. In this contribution, we outline a multifaceted hydrogel-based approach for expanding the range of therapeutics in oral formulations from classical small-molecule drugs to include proteins, chemotherapeutics, and nucleic acids. Through judicious material selection and careful design of copolymer composition and molecular architecture, we can engineer systems capable of responding to distinct physiological cues, with tunable physicochemical properties that are optimized to load, protect, and deliver valuable macromolecular payloads to their intended site of action. These hydrogel carriers, including complexation hydrogels, tethered hydrogels, interpenetrating networks, nanoscale hydrogels, and hydrogels with decorated structures are investigated for their ability to respond to changes in pH, to load and release insulin and fluorescein, and remain non-toxic to Caco-2 cells. Our results suggest these novel hydrogel networks have great potential for controlled delivery of proteins, chemotherapeutics, and nucleic acids.     

Cell vehicle targeting strategies

Use of cells as therapeutic carriers has increased in the past few years and has developed as a distinct concept and delivery method. Cell-based vehicles are particularly attractive for delivery of biotherapeutic agents that are difficult to synthesize, have reduced half-lives, limited tissue penetrance or are rapidly inactivated upon direct in vivo introduction. Initial studies using cell-based approaches served to identify some of the key factors for the success of this type of therapeutic delivery. These factors include the efficiency of cell loading with a therapeutic payload, the means of cell loading and the nature of therapeutics that cells can carry. However, one important aspect of cell-based delivery yet to be fully investigated is the process of actual delivery of the cell payload in vivo. In this regard, the potential ability of cell carriers to provide site-specific or targeted delivery of therapeutics deserves special attention. The present review focuses on a variety of targeting approaches that may be utilized to improve cell-based therapeutic delivery strategies. The different aspects of targeting that can be applied to cell vehicles will be discussed, including physical methods for directing cell distribution, intrinsic cell-mediated homing mechanisms and the feasibility of engineering cells with novel targeting mechanisms. Development of cell targeting strategies will further advance cell vehicle applications, broaden the applicability of this delivery approach and potentiate therapeutic outcomes.     

药物缓释载体材料在医药领域中的研究及应用

背景：药物缓释就是将小分子药物与高分子载体以物理或化学方法结合,在体内通过扩散、渗透等控制方式,将小分子药物以适当的浓度持续地释放出来,从而达到充分发挥药物功效的目的。目的：总结药物缓释载体材料特征及其在医药领域中的应用。方法：以＂药物缓释、载体材料、生物降解、壳聚糖、聚乳酸、海藻酸钠＂为中文关键词,以＂Drug delivery,carrier material,biodegradable,chitosan,polylactic acid,sodium alginate＂为英文关键词,采用计算机检索中国期刊全文数据库、PubMed数据库（1993-01/2010-11）相关文章。纳入高分子生物材料-药物缓释载体等相关的文章,排除重复研究或Meta分析类文章,共入选31篇文章进入结果分析。结果与结论：壳聚糖和聚乳酸是当前在药物缓释体系中应用较多的材料,它是将小分子药物与高分子载体以物理或化学方法结合,以适当的浓度持续地释放出来,从而达到充分发挥药物功效的目的,较单一生物材料具有显著优越性,具有更好的生物相容性和生物可降解性。目前很多研究仍处于实验阶段,还有一些问题有待于解决,如制剂质量方法不成熟,剂量较难控制,成本较高等。     

Smart polymeric carriers for enhanced intracellular delivery of therapeutic macromolecules

Limited cytoplasmic delivery of enzyme-susceptible drugs remains a significant challenge facing the development of protein and nucleic acid therapies that act in intracellular compartments. Researchers have examined several approaches, including fusogenic proteins and protein transduction domains, to enhance the intracellular delivery of the therapeutic cargo. This review summarises efforts to develop 'smart' pH-sensitive and membrane-destabilising polymers that can shuttle therapeutic peptide, protein and nucleic acid molecules past the endosomal membrane into the cytoplasm of targeted cells. Several classes of 'smart' non-degradable polymeric carriers have been developed that have proved effective both in vitro and in vivo in enhancing the cytoplasmic delivery of a variety of therapeutic molecules.     

A dual-ligand approach for enhancing targeting selectivity of therapeutic nanocarriers.

Conjugation of ligands to nano-scale drug carriers targeting over-expressed cell surface receptors is a promising approach for delivery of therapeutic agents to tumor cells. However, most commonly utilized ligands are directed at receptors expressed not only on target cells but also on other cells in the body, leading to unintended uptake in these off-target cells. In this study, a novel, dual-ligand approach is reported, which targets tumor cells while sparing off-target cells by exploiting the fact that tumor cells typically over-express multiple types of surface receptors. This approach was tested in the human KB cell line, which over-expresses both folate receptor (FR) and the epidermal growth factor receptor (EGFR). Liposomal nanocarriers loaded with doxorubicin and bearing controlled numbers of both folic acid and a monoclonal antibody against the EGFR were designed. Cytotoxicity was used to determine targeting selectivity of the designed carriers in vitro by utilizing KB cells expressing both FR and EGFR and off-target control cells in which one or both receptors were blocked. The data demonstrates that nanocarriers can be designed to achieve toxicity only when all targeted receptors are available, providing an approach to improve selectivity over current single-ligand approaches.     

Fibronectin conformational changes induced by adsorption to liposomes.

One of the major drawbacks of drug delivery techniques that utilize liposomes as carriers is that they are often cleared from the body before they can deliver their therapeutic cargo. It is well known that serum proteins can adsorb to these drug delivery vehicles and influence their uptake by phagocytic cells. For this reason, protein adsorption to liposomes has been extensively quantified, and strategies have been developed to minimize protein adsorption to improve drug delivery. However, the conformation of proteins on surfaces can play an even greater role in controlling cell behavior than the quantity of adsorbed protein. We have therefore used fluorescence resonance energy transfer (FRET) to measure changes in the structure of fibronectin (Fn)--a key serum protein involved in phagocytosis--upon interaction with phosphatidylcholine (PC) liposomes. Our experiments reveal that fibronectin opens up from its inactive, compact conformation upon interaction with gel phase PC liposomes. We also used FRET to estimate a physiologically relevant dissociation constant, KD=1.1 nM, for the interaction. Conformational changes in serum proteins may result in the exposure of otherwise concealed recognition sites and therefore influence the interaction of liposomes with phagocytic cells.     

Dual-targeted polyplexes: One step towards a synthetic virus for cancer gene therapy

Incorporating ligands into nano-scale carriers for specific delivery of therapeutic nucleic acids to tumor sites is a promising approach in anti-cancer strategies. Current artificial vector systems however still suffer from efficient and specific delivery, compared to their natural counterparts and addressed receptor types rarely are exclusively expressed on target cells. In this study synthetic dual receptor targeted polyplexes were developed, mimicking biphasic cell entry characteristics of natural viruses to increase efficiency and specificity by a dual-receptor internalization mechanism. For engineering the synthetic dual targeted vector system, the transferrin targeting peptide B6 was evaluated for the first time in the context of PEGylated PEI based polyplexes. As a second ligand, arginine-glycine-aspartic acid (RGD) containing peptide was incorporated for simultaneous integrin targeting.Cellular association, cellular uptake, transfection efficiency and accordant competition experiments displayed specificity of both ligands for each targeted receptor in two prostate cancer cell lines. A clear synergy of dual targeting over the combination of single-targeted polyplexes was found, suggesting that the dual targeting strategy is one step towards safe vectors for therapeutic approaches.     

Erythrocyte-mediated delivery of drugs,peptides and modified oligonucleotides

An important determinant for the success of every new therapy is the ability to deliver the molecules of interest to the target cells or organ. This selective delivery is even more complex when the therapeutic agents are peptides, modified oligonucleotides or genes. In this paper we summarize the possibility of using autologous erythrocytes for the delivery and targeting of new and conventional therapeutics. In fact, a number of macromolecules can be encapsulated by different procedures into human erythrocytes. These modified cells can then be re-infused into the same or a compatible recipient where they can circulate for several weeks. However, drug-loaded erythrocytes can also be modified to be selectively recognized by tissue macrophages. These phagocyte cells recognize the modified drug-loaded erythrocytes which are able to release their content into the macrophage. The feasibility and safety of the use of erythrocytes as drug delivery systems was evaluated in 10 cystic fibrosis patients, where a sustained release of corticosteroids from dexamethasone 21-phosphate-loaded erythrocytes was obtained. In vitro human erythrocytes were found to be able to deliver ubiquitin analogues and modified oligonucleotides to macrophages. Thus, drug-loaded erythrocytes are safe and useful carriers of new and conventional therapeutics and can be advantageous delivery systems for new clinical applications where proteins and oligonucleotides are therapeutic agents.     

The coiled coils in the design of protein-based constructs: hybrid hydrogels and epitope displays.

Recombinant DNA technology provides a powerful tool for producing protein-based biomaterials. Genetically engineered coiled coils have been used as a structural module for the construction of a variety of bio-based systems useful in drug delivery studies. Two of such approaches developed in the authors' laboratory were described here. One approach was to assemble hybrid hydrogels from coiled coil protein domains and synthetic polymers. Preliminary results showed that temperature-sensitive volume transition of the hybrid hydrogels could be triggered by the thermal unfolding of the engineered coiled coil protein domains. The other approach, discussed in detail, was to construct an epitope display model system based on a coiled coil stem loop peptide self-assembled on a solid substrate. This model construct displayed a constrained nonapeptide sequence, which was found to mediate specific binding with immunocompetent cells bearing complementary surface receptors. These novel approaches will likely find important applications in the rational design of more effective drug delivery systems.     

Advances in molecular biology. Potential impact on diagnosis and treatment of disorders of the thyroid

Advances in molecular biology research continue to have a major impact on clinical medicine. These advances have provided a means to produce proteins previously available in limited supply and allow for the production of novel proteins that are improved agonists or else antagonists with greater specificity for therapeutic targets. Newer drug delivery systems should facilitate delivery of these proteins. By combining the capabilities to produce drug targets in acceptable quantities with improved methods for determining the three-dimensional structures of these targets, novel organic therapeutic molecules that act on these targets will be designed. Gene transfer therapy using genes that express important proteins or that encode "antisense" RNAs that inhibit the translation of specific mRNAs will soon become a reality. The use of RFLPs and PCR methodologies promises increased means to diagnose specific genetic diseases and infections. Most importantly, molecular biology is helping to understand the mechanisms of disease such that novel diagnostic and therapeutic approaches can be described. These advances are providing an understanding of the mechanisms involved in cancers of the thyroid gland. They have already led to an enhanced understanding of (1) the growth factors that control proliferation of the thyroid gland, (2) the potential steps in thyroid nodule and neoplasia development, (3) particular mutations that may occur as thyroid cancers develop, (4) oncogenes that are expressed in thyroid cancers, and (5) the genetic defects that are responsible for thyroid gland malignancies in the multiple endocrine neoplasia (MEN) syndromes. With the latter, the RFLP technology has already provided an enhanced means to diagnose the disorder. With further progress, enhanced means for diagnosis and treatment should emerge. Molecular biology techniques are contributing to an increased understanding of the mechanisms of development of autoimmune thyroid disease as with Graves' disease and Hashimoto's thyroiditis. The potential role of infections, histocompatibility antigens, thyroid gland and extraglandular antigens, immune modulators, subpopulations of T-cells such as suppressor and helper cells, other cells involved in immune responses, and a combination of influences of several different functions on the thyroid gland are being defined. This knowledge should soon suggest improved means for diagnosis and treatment. Understanding of the function of the thyroid hormone receptors should have clinical importance. This knowledge suggests a means to develop thyroid hormone antagonists that may be used to more rapidly ameliorate the effects of hyperthyroidism and could be useful in nonthyroidal disorders such as cardiac arrhythmias.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pulmonary delivery of insulin by liposomal carriers.

Growing attention has been given to the potential of a pulmonary route as a non-invasive administration for systemic delivery of therapeutic agents (mainly peptides and proteins). The lungs provide a large absorptive surface area, extremely thin absorptive mucosal membrane, and good blood supply. The non-invasive nature of this pathway makes it especially valuable for the delivery of large molecular protein. However, pulmonary delivery of peptides and proteins is complicated by the complexity of the anatomic structure of the human respiratory system and the effect of disposition exerted by the respiration process. In this study, novel nebulizer-compatible liposomal carrier for aerosol pulmonary drug delivery of insulin was developed and characterized. Experimental results showed that insulin could be efficiently encapsulated into liposomes by preformed vesicles and detergent dialyzing method. The optimal encapsulation efficiency was achieved when 40% ethanol was used. The particle size of liposomal aerosols from ultrasonic nebulizer approximated to 1 mum. Insulin was stable in the liposomal solution. Animal studies showed that plasma glucose level was effectively reduced when liposomal insulin was delivered by inhalation route of using aerosolized insulin-encapsulated liposomes. Including fluorescent probe (phosphatidylethanolamine-rhodamine) into liposome, we found that the liposomal carriers were effectively and homogeneously distributed in the lung aveolar. Liposome-mediated pulmonary drug delivery promotes an increase in drug retention-time in the lungs, and more importantly, a reduction in extrapulmonary side-effects which invariably results in enhanced therapeutic efficacies.     

Nanoscale polymer carriers to deliver chemotherapeutic agents to tumours

Nanoscale polymer carriers have the potential to enhance the therapeutic efficacy of antitumour drugs as they can regulate their release, improve their stability and prolong circulation time by protecting the drug from elimination by phagocytic cells or premature degradation. Moreover, nanoscale polymeric carriers are capable of accumulating in tumour cells and tissues due to enhanced permeability and retention effect or by active targeting bearing ligands designed to recognise overexpressed tumour-associated antigens. The diversity in the polymer structures being studied as drug carriers in cancer therapy allows an optimal solution for a particular drug to be provided regarding its delivery and efficacy, and thus the patient's quality of life. This review is focused on the different types of nanoscale polymer carriers used for the delivery of chemotherapeutic agents and on the factors that affect their cellular uptake and trafficking.     

Electroporation-induced siRNA precipitation obscures the efficiency of siRNA loading into extracellular vesicles

Extracellular vesicles (EVs) are specialised endogenous carriers of proteins and nucleic acids and are involved in intercellular communication. EVs are therefore proposed as candidate drug delivery systems for the delivery of nucleic acids and other macromolecules. However, the preparation of EV-based drug delivery systems is hampered by the lack of techniques to load the vesicles with nucleic acids. In this work we have now characterised in detail the use of an electroporation method for this purpose. When EVs were electroporated with fluorescently labelled siRNA, siRNA retention was comparable with previously published results (20–25% based on fluorescence spectroscopy and fluorescence fluctuation spectroscopy), and electroporation with unlabelled siRNA resulted in significant siRNA retention in the EV pellet as measured by RT-PCR. Remarkably, when siRNA was electroporated in the absence of EVs, a similar or even greater siRNA retention was measured. Nanoparticle tracking analysis and confocal microscopy showed extensive formation of insoluble siRNA aggregates after electroporation, which could be dramatically reduced by addition of EDTA. Other strategies to reduce aggregate formation, including the use of cuvettes with conductive polymer electrodes and the use of an acidic citrate electroporation buffer, resulted in a more efficient reduction of siRNA precipitation than EDTA. However, under these conditions, siRNA retention was below 0.05% and no significant differences in siRNA retention could be measured between samples electroporated in the presence or absence of EVs. Our results show that electroporation of EVs with siRNA is accompanied by extensive siRNA aggregate formation, which may cause overestimation of the amount of siRNA actually loaded into EVs. Moreover, our data clearly illustrate that electroporation is far less efficient than previously described, and highlight the necessity for alternative methods to prepare siRNA-loaded EVs.     

Emerging technologies in the delivery of erythropoietin for therapeutics

Deciphering the function of proteins and their roles in signaling pathways is one of the main goals of biomedical research, especially from the perspective of uncovering pathways that may ultimately be exploited for therapeutic benefit. Over the last half century, a greatly expanded understanding of the biology of the glycoprotein hormone erythropoietin (Epo) has emerged from regulator of the circulating erythrocyte mass to a widely used therapeutic agent. Originally viewed as the renal hormone responsible for erythropoiesis, recent in vivo studies in animal models and clinical trials demonstrate that many other tissues locally produce Epo independent of its effects on red blood cell mass. Thus, not only its hematopoietic activity but also the recently discovered nonerythropoietic actions in addition to new drug delivery systems are being thoroughly investigated in order to fulfill the specific Epo release requirements for each therapeutic approach. The present review focuses on updating the information previously provided by similar reviews and recent experimental approaches are presented to describe the advances in Epo drug delivery achieved in the last few years and future perspectives. © 2009 Wiley Periodicals, Inc. Med Res Rev, 31, No. 2, 284–309, 2011     

Antitubercular inhaled therapy: opportunities, progress and challenges

Pulmonary tuberculosis remains the commonest form of this disease and the development of methods for delivering antitubercular drugs directly to the lungs via the respiratory route is a rational therapeutic goal. The obvious advantages of inhaled therapy include direct drug delivery to the diseased organ, targeting to alveolar macrophages harbouring the mycobacteria, reduced risk of systemic toxicity and improved patient compliance. Research efforts have demonstrated the feasibility of various drug delivery systems employing liposomes, polymeric microparticles and nanoparticles to serve as inhalable antitubercular drug carriers. In particular, nanoparticles have emerged as a remarkably useful tool for this purpose. While some researchers have preferred dry powder inhalers, others have emphasized nebulization. Beginning with the respiratory delivery of a single antitubercular drug, it is now possible to deliver multiple drugs simultaneously with a greater therapeutic efficacy. More experience and expertise have been observed with synthetic polymers, nevertheless, the possibility of using natural polymers for inhaled therapy has yet to be explored. Several key issues such as patient education, cost of treatment, stability and large scale production of drug formulations, etc. need to be addressed before antitubercular inhaled therapy finds its way from theory to clinical reality.     

Development of polymeric nanoparticles-based vaccine

Traditional vaccines are mainly composed of attenuated or heat-inactivated viruses. These vaccines often generate many unwanted side effects. Subunit protein and peptide vaccines are generally very safe vaccines with well-defined components. However, proteins and peptides are often poorly immunogenic and thus require the use of adjuvants to induce adequate immunity. Therefore, particulate adjuvants have been widely investigated in vaccine delivery systems. In particular, polymeric nanoparticles with entrapped antigens represent an exciting approach to control the release of vaccine antigens and optimize the desired immune response via selective targeting of antigen to professional antigen presenting cells. It is expected that biodegradable polymeric nanoparticles have great potential as carriers for systemic and mucosal vaccine delivery systems.     

Erythrocytes as Carriers for Drug Delivery in Blood Transfusion and Beyond

Red blood cells (RBCs) are innate carriers that can also be engineered to improve the pharmacokinetics and pharmacodynamics of many drugs, particularly biotherapeutics. Successful loading of drugs, both internally and on the external surface of RBCs, has been demonstrated for many drugs including anti-inflammatory, antimicrobial, and antithrombotic agents. Methods for internal loading of drugs within RBCs are now entering clinical use. Although internal loading can result in membrane disruption that may compromise biocompatibility, surface loading using either affinity or chemical ligands offers a diverse set of approaches for the production of RBC drug carriers. A wide range of surface determinants is potentially available for this approach, although there remains a need to characterize the effects of coupling agents to these surface proteins. Somewhat surprisingly, recent data also suggest that red cell–mediated delivery may confer tolerogenic immune effects. Questions remaining before widespread application of these technologies include determining the optimal loading protocol, source of RBCs, and production logistics, as well as addressing regulatory hurdles. Red blood cell drug carriers, after many decades of progress, are now poised to enter the clinic and broaden the potential application of RBCs in blood transfusion.     

Application of nanoparticle technology in the treatment of Systemic lupus erythematous

Systemic lupus erythematous (SLE) is a chronic heterogeneous multisystem autoimmune disease characterized by loss of tolerance to self-antigens and production of numerous autoantibodies. Current therapeutic approaches used to treat inflammatory autoimmune diseases are associated with debilitating side effects. In spite of significant advances in therapeutic options and increased understanding of the pathogenesis, novel therapeutic approaches are needed for treatment of SLE. Nanoparticle-based delivery systems that selectively deliver drugs to inflamed tissue or specific cell have the potential to improve drug delivery. This article reviews recent nanotechnology-based therapeutics strategies that are being developed for the treatment of SLE.