Protein release from collagen matrices

The effective delivery of protein drugs is an important research subject in the field of pharmacology, and to prolong the effect of protein drugs, many studies are being conducted to control the release of proteins from various carrier materials. Collagen is one of the most useful candidates for this purpose, and many studies have been reported; pharmaceutical formulations containing collagen in gel, film and sponge form are used to incorporate low-molecular-weight compounds such as antibiotics and carcinostatics, and the release of these compounds is controlled by the concentration of the gel as well as the shape and degree of crosslinking of the matrix. However, it is still difficult to retain protein drugs in the collagen. In this article, we report on the controlled release of protein drugs using collagen which exhibits good biocompatibility as a carrier, focusing on a new drug delivery system, the Minipellet, which we have developed.     

Development of intracellular drug delivery system using fusogenic liposomes

Drug delivery system (DDS) research has contributed greatly toward improving chemotherapy efficacy and reducing its adverse effects through the development of approaches to optimize pharmacokinetics, such as controlled release and targeting. On the other hand, the remarkable progress of this latest life science research has altered the concept of what constitutes medical supplies. A change in this concept would allow for the consideration of medical materials that use not only conventional low molecular-weight organic compounds, but also biomacromolecules, including nucleic acids and proteins, that constitute living organisms. Although these biomacromolecular drugs are expected to demonstrate excellent efficacy based on their intrinsic bioactivity, they quickly degrade when administered in vivo and only a limited number have therefore been developed into medicines. In addition, most biomacromolecular drugs are ineffective until they are delivered to particular cells within a tissue or to particular organelles within a cell. To develop effective biomacromolecular medicines, it is necessary to introduce a DDS that is capable of ensuring internal stability as well as precise control of internal and intracellular dynamics, and to establish a new fundamental technology for DDS that can accommodate the material properties and mechanisms of action of the biomacromolecular drugs. In this context, this review introduces our approach to the design and creation of "Intracellular DDS" using fusogenic liposomes for application to gene therapy and tumor peptide vaccines. We suggest that this technology is very important for controlling the intracellular pharmacokinetics of biomacromolecular drugs.     

Promotion of optimized protein therapy by bioconjugation as a polymeric DDS

In recent years, clinical applications of recombinantly produced bioactive proteins such as cytokines have attracted attention. However, since these recombinant proteins are rather unstable in vivo, their clinical use as therapeutic agents requires frequent administration at a high dosage. This regimen disrupts homeostasis and results in severe side effects. To overcome these problems, bioactive proteins have been conjugated with water-soluble synthetic (WSS) polymeric carriers. Chemical modification of a protein with a WSS polymeric carrier (bioconjugation) regulates tissue distribution, resulting in a selective increase in its desirable therapeutic effects and a decrease in undesirable side effects. Among several drug delivery system (DDS) technologies, bioconjugation has been recognized as one of the most efficient methods for improving therapeutic potency of proteins. However, for further enhancement of the therapeutic potency and safety of conjugated bioactive proteins, more precise regulation of the in vivo behavior of each protein is necessary for selective expression of its therapeutic effect. Therefore, alternative WSS polymeric modifiers in which new functions such as targeting and controlled release of drugs can be added are required for further development of bioconjugated drugs. Recently, we have synthesized a novel polymeric drug carrier, poly(vinylpyrrolidone-co-dimethyl maleic anhydride) [PVD], which was a powerful candidate drug carrier for cancer therapy. In this review, we introduce useful information that enabled us to design polymeric drug carriers and their application for protein therapy.     

Drug and gene delivery by "bubble liposomes" and ultrasound

Gene therapy has a potentiality for treatment of cancer and diseases from genomic defects. It is important to select a vector which has good potency in terms of gene transduction efficiency, and is safe and easy to apply. Many researchers have attempted to develop an effective gene delivery carrier. Recently, it was reported that microbubbles, which are ultrasound (US) contrast agents, improved the transfection efficiency by cavitation with US exposure. However, microbubbles had problems with stability and targeting ability. To solve these problems, we focused on liposomes that had many advantages such as being stable and safe in vivo and easily modifying targeting ligand. We succeeded in preparing the liposomes ("Bubble liposomes" (BLs)) entrapping perfluoropropane gas which was utilized for contrast enhancement in ultrasonography. In this study, we assessed the feasibility of BLs as gene delivery carrier utilized cavitation by US exposure. BLs could deliver plasmid DNA to various cell types in vitro by combining with US without cytotoxicity. To evaluate the ability of BLs to in vivo gene delivery, we attempted to deliver plasmid DNA into the femoral artery. The gene expression at this artery treated with BLs and US combination was higher than with US only, BLs without US or Lipofectamine 2000. This result suggested that Bubble liposomes could quickly deliver plasmid DNA into the artery even under conditions of short contact time between BLs and the endothelial cells and the existence of the bloodstream and serum. These results suggested that BLs might be a non-invasive and effective carrier for gene delivery.     

Cyclodextrin-based controlled drug release system

Because of their bioadaptability and multi-functional characteristics, cyclodextrins (CDs) are capable of alleviating the undesirable properties of drug molecules in various routes of administration through the formation of inclusion complexes. This article outlines the current application of natural and chemically modified CDs in the design of advanced dosage forms. In an oral drug delivery system (DDS), the hydrophilic and ionizable CDs can serve as potent drug carriers in the immediate release- and delayed release-formulations, respectively, while the release rate of water-soluble drugs can be retarded by hydrophobic CDs. Since CDs are able to extend the function of pharmaceutical additives, the combination of molecular encapsulation with other carrier materials will become effective and a valuable tool in the improvement of drug formulation. Moreover, the most desirable attribute for the drug carrier is its ability to deliver a drug to a targeted site; conjugates of a drug with CDs can be a versatile means of constructing a new class of colon-targeting prodrugs. On the basis of this knowledge, the advantages and limitations of CDs in DDS are addressed.     

A new gene delivery system based on controlled release technology

The recent rapid development of molecular biology together with the steady progress of genome projects has given us some essential and revolutionary informations of gene to elucidate all the biological phenomena at the molecular level. Under these circumstances, gene transfection has become a fundamental technology indispensable to the basic research of medicine and biology. On the other hand, the technology of gene transfection is also important for gene therapy of several diseases. Some human gene therapies have been performed with a plasmid DNA alone or virus vectors but are clinically limited by the poor gene expression of plasmid DNA and the adverse effects of virus itself, such as immunogenicity and toxicity or the possible mutagenesis of cells transfected. Therefore, several non-viral vectors of synthetic materials have been explored to enhance the transfection efficiency of gene into mammalian cells both in vitro and in vivo. In this paper, the researches about non-viral vectors and recent research trials about the controlled release of plasmid DNA are briefly reviewed to emphasize the significance of gene delivery technology in basic biology and medicine as well as clinical medicine. A new system of gene release based on biodegradable hydrogel is introduced.     

Development of site specific gene delivery system with sonoporation

In gene therapy, it is important to develop an effective and safe gene delivery system. Especially, from the viewpoint of reducing side effects, gene delivery into a specific site is essential. We previously, developed liposomal bubbles (Bubble liposomes) containing perfluoropropane. Bubble liposomes were useful as ultrasound enhanced gene delivery tools in vitro and in vivo. In this review, we introduced the characteristics of Bubble liposomes as ultrasound imaging agents and ultrasound enhanced gene delivery tools. Bubble liposomes worked as ultrasound imaging agents in cardiosonography. In addition, their combination with ultrasound exposure was able to deliver plasmid DNA in the femoral artery. The gene expression was only observed at the site of ultrasound exposure. Moreover, the gene delivery by Bubble liposomes and ultrasound exposure was more efficient than that by conventional lipofection method using Lipofectamine 2000. Therefore, it was suggested that Bubble liposomes might be a new class of tools for site specific gene delivery.     

Three-dimensional printing in pharmaceutics: promises and problems

Three-dimensional printing (3DP) is a rapid prototyping (RP) technology. Prototyping involves constructing specific layers that uses powder processing and liquid binding materials. Reports in the literature have highlighted the many advantages of the 3DP system over other processes in enhancing pharmaceutical applications, these include new methods in design, development, manufacture, and commercialization of various types of solid dosage forms. For example, 3DP technology is flexible in that it can be used in applications linked to linear drug delivery systems (DDS), colon-targeted DDS, oral fast disintegrating DDS, floating DDS, time controlled, and pulse release DDS as well as dosage form with multiphase release properties and implantable DDS. In addition 3DP can also provide solutions for resolving difficulties relating to the delivery of poorly water-soluble drugs, peptides and proteins, preparation of DDS for high toxic and potent drugs and controlled-release of multidrugs in a single dosage forms. Due to its flexible and highly reproducible manufacturing process, 3DP has some advantages over conventional compressing and other RP technologies in fabricating solid DDS. This enables 3DP to be further developed for use in pharmaceutics applications. However, there are some problems that limit the further applications of the system, such as the selections of suitable excipients and the pharmacotechnical properties of 3DP products. Further developments are therefore needed to overcome these issues where 3DP systems can be successfully combined with conventional pharmaceutics. Here we present an overview and the potential 3DP in the development of new drug delivery systems.     

How controlled release technology can aid gene delivery

Introduction: Many types of gene delivery systems have been developed to enhance the level of gene expression. Controlled release technology is a feasible gene delivery system which enables genes to extend the expression duration by maintaining and releasing them at the injection site in a controlled manner. This technology can reduce the adverse effects by the bolus dose administration and avoid the repeated administration. Biodegradable biomaterials are useful as materials for the controlled release-based gene delivery technology and various biodegradable biomaterials have been developed.

Areas covered: Controlled release-based gene delivery plays a critical role in a conventional gene therapy and genetic engineering. In the gene therapy, the therapeutic gene is released from biodegradable biomaterial matrices around the tissue to be treated. On the other hand, the intracellular controlled release of gene from the sub-micro-sized matrices is required for genetic engineering. Genetic engineering is feasible for cell transplantation as well as research of stem cells biology and medicine.

Expert opinion: DNA hydrogel containing a sequence of therapeutic gene and the exosome including the individual specific nucleic acids may become candidates for controlled release carriers. Technologies to deliver genes to cell aggregates will play an important role in the promotion of regenerative research and therapy.     

基于水凝胶的核酸药物负载策略的研究进展

基因治疗在癌症以及遗传疾病的治疗中具有广阔的应用前景,基因治疗的关键在于如何实现将核酸药物精准递送至靶部位。近年来,研究人员致力于将核酸药物负载于水凝胶中,以实现全身或局部的基因递送。水凝胶系统由于其良好的生物相容性、高效的核酸药物负载能力和局部定位控制释放等优势,为核酸药物的递送提供了有效的工具,在实体瘤和再生医学领域具有巨大的潜力。本文综述了近年来水凝胶系统作为核酸药物载体的研究,并重点探讨基于水凝胶的核酸药物负载策略,以期为基于水凝胶的核酸药物递送系统的研究提供参考。     

外泌体作为药物载体在中枢神经系统疾病中的研究进展

外泌体作为细胞间通讯的重要信使，在正常和病理条件下通过转运蛋白质、核酸等物质参与多种生物学功能的调控过程。治疗药物难以跨越血脑屏障（BBB）致使脑内药物浓度过低，无法达到预期治疗效果一直是中枢神经系统（CNS）疾病的治疗瓶颈。考虑到外泌体具有良好的生物相容性、较好的渗透性、能够穿越血脑屏障、天然的稳定性以及低免疫原性和毒性等优点，研究者们开始将其作为新型药物递送系统（DDS）来提升药物在脑内的生物利用度，为临床治疗CNS疾病提供一种新兴治疗策略。本文就外泌体DDS治疗CNS疾病的研究进展进行综述，概述了目前外泌体的载药方式和作为脑部递送载体的特性，重点介绍了外泌体作为核酸、蛋白质和化学药物的递送载体在CNS疾病中的治疗应用。     

Nanoparticles as drug delivery systems

Controlled drug delivery systems (DDS) have several advantages compared to the traditional forms of drugs. A drug is transported to the place of action, hence, its influence on vital tissues and undesirable side effects can be minimized. Accumulation of therapeutic compounds in the target site increases and, consequently, the required doses of drugs are lower. This modern form of therapy is especially important when there is a discrepancy between the dose or the concentration of a drug and its therapeutic results or toxic effects. Cell-specific targeting can be accomplished by attaching drugs to specially designed carriers. Various nanostructures, including liposomes, polymers, dendrimers, silicon or carbon materials, and magnetic nanoparticles, have been tested as carriers in drug delivery systems. In this review, the aforementioned nanocarriers and their connections with drugs are analyzed. Special attention is paid to the functionalization of magnetic nanoparticles as carriers in DDS. Then, the advantages and disadvantages of using magnetic nanoparticles as DDS are discussed.     

Galactosylated chitosan-g-PEI/DNA complexes-loaded poly(organophosphazene) hydrogel as a hepatocyte targeting gene delivery system

Hydrogels are widely used in drug delivery systems because they can control the release and thereby enhance the efficiency of locally delivered bioactive molecules such as therapeutic drugs, proteins, or genes. For gene delivery, localized release of plasmid DNA or polymer/DNA complexes can transfect cells and produce sustained protein production. We tested the galactosylated chitosan-graft-polyethylenimine (GC-g-PEI)/DNA complexes-loaded poly(organophosphazene) thermosensitive biodegradable hydrogel as a hepatocyte targeting gene delivery system. The poly(organophosphazene) hydrogel loaded with GC-g-PEI/DNA complexes showed low cytotoxicity and higher transfection efficiency than PEI/DNA complexes, as well as good hepatocyte specificity in vitro and in vivo. Our results indicate that poly(organophosphazene) hydrogels loaded with GC-g-PEI/DNA complexes may be a safe and efficient hepatocyte targeting gene delivery system.     

Preparation of poly-lactic acid microspheres containing the angiogenesis inhibitor TNP-470 with medium-chain triglyceride and the in vitro evaluation of release profiles.

TNP-470 (AGM-1470, 6-0-(N-chloroacetylcarbamoyl)-fumagillol), a derivative of fumagillin, is a promising angiogenesis inhibitor. However, as TNP-470 is very unstable in in vitro and in vivo, it has been difficult to verify its pharmacological efficacy in the clinical medicine. The preparation of a drug delivery system (DDS) in a microsphere form was studied for the stable inclusion and controlled release of TNP-470. Medium-chain triglyceride (MCTG) as an effective stabilizer and poly-lactic acid (PLA) as a biodegradable carrier were used for this purpose. The release of TNP-470 from the MCTG containing DDS continued for approximately 2 weeks, while the release of TNP-470 from the one without MCTG stopped after only 5 days. It was proved that TNP-470 could be released much more stable for much longer period from the MCTG containing DDS compared to the one without DDS.     

Non-methylated CpG motif packaged into fusogenic liposomes enhance antigen-specific immunity in mice

DNA rich in non-methylated CG motifs (CpGs) enhances induction of immune responses against co-administered antigen encoding genes. CpGs are therefore among the promising adjuvants known to date. However, naked plasmid DNA, even which contains CpG motifs, are taken up by antigen presenting cells via the endocytosis pathway. Endocytosed DNAs are thus degraded and their gene expression levels are inefficient. In this context, an effective plasmid delivery carrier is required for DNA vaccine development. We show in the present study that packaging plasmids containing CpGs into fusogenic liposomes (FL) derived from conventional liposomes and Sendai virus-derived active accessory proteins is an attractive method for enhancing the efficacy of a DNA vaccine. These CpG-enhanced plasmids (possessing 16 CpG repeats) that were packaged into FL, enhanced ovalbumin (OVA)-specific T cell proliferation and cytotoxic T cell activity after immunization. In fact, vaccination with CpG enhanced plasmid-loaded FL induced effective prophylactic effects compared with 13 repeats CpG containing plasmid in a tumor challenge experiment. Thus, the development of a CpG-enhanced DNA-FL genetic immunization system represents a promising tool for developing candidate vaccines against some of the more difficult infectious, parasitic, and oncologic disease targets.     

Development of novel technology of DDS for gene therapy

In the near future, not only "systemic pharmacokinetics" but also "intracellular pharmacokinetics" seems to be important in Drug Delivery System (DDS) research for gene therapy. Beyond the basic philosophy of DDS of "delivering the optimal amounts of drugs to a target site", it is now necessary to "express the gene (as a drug) efficiently in a target cell for a required period" in gene therapy. To achieve these objectives, vectors for introducing the gene into the target cell are being improved, and techniques to efficiently express the transgene and to regulate the transgene expression are being developed. DDS is expected to play a large part in achieving this goal. Here, we review a novel DDS technology to satisfy these criteria.     

New aerosol drug delivery systems for the treatment of immune-mediated pulmonary diseases

In the lung, unchecked immune responses mediated predominantly by T-lymphocytes and concurrent inflammation can lead to the development of different pathological conditions such as parenchymal disease, interstitial fibrosis, hypersensitivity pneumonitis, bronchiolitis obliterans and bronchiolar asthma. Targeted modulation of uncontrolled T-cell activation and inhibition of cytokine production within different pulmonary compartments is the challenge for the development of novel methods for immunotherapeutic intervention. Utilization of aerosol technology for pulmonary drug delivery represents new potential opportunities for therapeutic application for such immune-mediated pulmonary diseases. For targeted aerosol pulmonary drug delivery, continuous-flow jet nebulizers have several advantages over metered dose or dry powder inhalers since they are the simplest and most effective for aerosol droplet deposition into the peripheral lung tissues. At the present, the major limitations for targeted pulmonary immunosuppression through effective utilization of nebulizer technology has been the conspicuous lack of suitable formulations. The development of liposomal formulations compatible with aerosol delivery with jet nebulizers has expanded the potential for more effective utilization with an array of potent and effective immunosuppressive drugs. For pulmonary therapy, the utilization of liposomes for aerosol delivery has many potential advantages, including universal carrier suitability for most lipophilic drugs, aqueous compatibility, sustained pulmonary release or depot and intracellular delivery. Drug liposomes may also prevent local irritation in the lung, and increase potency with reduced systemic toxicity. Successful utilization of potent immunosuppressive drugs, like cyclosporin, tacrolimus (FK-506), rapamycin, mycophenolate and budesonide, in a variety of immunopathological conditions for other indications demonstrates their potential efficacy for the treatment of many different immune-mediated pulmonary diseases. The route of delivery to the pulmonary tissues can potentially limit adverse effects and markedly affect localized immunosuppressive activity in the lung. Combination of liposomal formulations with topical aerosol delivery to the central and peripheral lung tissues has expanded potential for more effective utilization with these lipophilic immunosuppressive (and antiinflammatory) drugs. Synergistic combinations can also be developed for localized and sustained delivery of therapeutic drug concentrations within the lung to provide multisite immunosuppression. Drug liposome aerosol technology represents one readily available approach for more effective therapeutic intervention in the lung using cyclosporin, FK-506, rapamycin, mycophenolate, budesonide and other lipophilic drugs.     

Key issues in non-viral gene delivery

The future of non-viral gene therapy depends on a detailed understanding of the barriers to delivery of polynucleotides. These include physicomechanical barriers, which limit the design of delivery devices, physicochemical barriers that influence self-assembly of colloidal particulate formulations, and biological barriers that compromise delivery of the DNA to its target site. It is important that realistic delivery strategies are adopted for early clinical trials in non-viral gene therapy. In the longer term, it should be possible to improve the efficiency of gene delivery by learning from the attributes which viruses have evolved; attributes that enable translocation of viral components across biological membranes. Assembly of stable, organized virus-like particles will require a higher level of control than current practice. Here, we summarize present knowledge of the biodistribution and cellular interactions of gene delivery systems and consider how improvements in gene delivery will be accomplished in the future.