Fc-fusion proteins and FcRn: structural insights for longer-lasting and more effective therapeutics

Nearly 350 IgG-based therapeutics are approved for clinical use or are under development for many diseases lacking adequate treatment options. These include molecularly engineered biologicals comprising the IgG Fc-domain fused to various effector molecules (so-called Fc-fusion proteins) that confer the advantages of IgG, including binding to the neonatal Fc receptor (FcRn) to facilitate in vivo stability, and the therapeutic benefit of the specific effector functions. Advances in IgG structure-function relationships and an understanding of FcRn biology have provided therapeutic opportunities for previously unapproachable diseases. This article discusses approved Fc-fusion therapeutics, novel Fc-fusion proteins and FcRn-dependent delivery approaches in development, and how engineering of the FcRn–Fc interaction can generate longer-lasting and more effective therapeutics.     

Small interfering RNA delivery into the liver by cationic cholesterol derivative-based liposomes

Purpose: Previously, we reported that the cationic liposomes composed of a cationic cholesterol derivative, cholesteryl (2-((2-hydroxyethyl)amino)ethyl)carbamate (OH-C-Chol) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (termed LP-C), could deliver small interfering RNAs (siRNAs) with high transfection efficiency into tumor cells. In this study, to develop a liposomal vector for siRNA delivery in vivo, we prepared the poly(ethyleneglycol) (PEG)-modified cationic liposomes (LP-C-PEG) and evaluated their transfection efficiency in vitro and in vivo.

Materials and methods: We prepared LP-C-PEG/siRNA complexes (LP-C-PEG lipoplexes) formed in water or 50?mM NaCl solution, and evaluated their siRNA biodistribution and gene silencing effect in mice after intravenous injection.

Results: LP-C-PEG lipoplexes strongly exhibited in vitro gene silencing effects in human breast tumor MCF-7 cells as well as LP-C lipoplexes. In particular, formation of LP-C and LP-C-PEG lipoplexes in the NaCl solution increased the cellular association. When LP-C-PEG lipoplexes with Cy5.5-labeled siRNA formed in water or NaCl solution were injected into mice, accumulation of the siRNA was observed in the liver. Furthermore, injection of LP-C-PEG lipoplexes with ApoB siRNA could suppress ApoB mRNA levels in the liver and reduce very-low-density lipoprotein/low-density lipoprotein levels in serum compared with that after Cont siRNA transfection, although the presence of NaCl solution in forming the lipoplexes did not affect gene silencing effects in vivo.

Conclusions: LP-C-PEG may have potential as a gene vector for siRNA delivery to the liver.     

ZEBRA cell‐penetrating peptide as an efficient delivery system in Candida albicans

There is increasing interest in drug delivery systems, such as nanoparticles, liposomes, and cell‐penetrating peptides, for the development of new antimicrobial treatments. In this study, we investigated the transduction capacity of a carrier peptide derived from the Epstein–Barr virus ZEBRA protein in the pathogenic fungus Candida albicans. ZEBRA‐minimal domain (MD) was able to cross the cell wall and cell membrane, delivering eGFP to the cytoplasm. Uptake into up to 70% of the cells was observed within two hours, without toxicity. This new delivery system could be used in C. albicans as a carrier for different biological molecules including peptides, proteins, and nucleic acids. Thereby, in antifungal therapy, MD may carry promising bioactive fungal inhibitors that otherwise penetrate poorly into the cells. Furthermore, MD will be of interest for deciphering molecular pathways involving cell‐cycle control in yeast or signaling pathways. Short interfering peptides could be internalized using MD, providing new tools for the inhibition of metabolic or signaling cascades essential for the growth and virulence of C. albicans, such as yeast‐to‐hyphae transition, cell wall remodeling, stress signaling and antifungal resistance. These findings create new possibilities for the internalization of cargo molecules, with applications for both treatment and functional analyses.     

Transtactin: a universal transmembrane delivery system for Strep‐tag II‐fused cargos

The delivery of molecules into cells poses a critical problem that has to be solved for the development of diagnostic tools and therapeutic agents acting on intracellular targets. Cargos which by themselves cannot penetrate cellular membranes due to their biophysical properties can achieve cell membrane permeability by fusion to protein transduction domains (PTDs). Here, we engineered a universal delivery system based on PTD‐fused Strep‐Tactin, which we named Transtactin. Biochemical characterization of Transtactin variants bearing different PTDs indicated high thermal stabilities and robust secondary structures. Internalization studies demonstrated that Transtactins facilitated simple and safe transport of Strep‐tag II‐linked small molecules, peptides and multicomponent complexes, or biotinylated proteins into cultured human cells. Transtactin‐introduced cargos were functionally active, as shown for horseradish peroxidase serving as a model protein. Our results demonstrate that Transtactin provides a universal and efficient delivery system for Strep‐tag II‐fused cargos.     

SiRNA based strategies for inhibition of apoptotic pathways in vivo—analytical and therapeutic implications

Apoptosis is a genetically controlled mechanism of cell death which is important for embryogenesis, metamorphosis, tissue homeostasis and tumor regression of multicellular organisms. In normal cells as well as in transformed cells signals released from the cytoplasm and/or the cell membrane can trigger the activation of caspases which in turn cleave many cellular substrates, leading to the characteristic morphology of apoptosis.Systematic analysis and dissection of apoptotic pathways was obtained by the use of knock out or transgenic organisms, expressing dominant active or negative proteins.The use of antisense oligonucleotides (ASO) for analysis of apoptotic pathways was commonly restricted to cultured cells, because of the low efficacy of ASO in in vivo experiments. In order to investigate the contribution of specific apoptotic pathways in the onset and maintenance of disease in vivo experiments are needed. This approach allows the analysis of apoptotic pathways within their physiological/pathophysiological environment. The combination of recent advances in in vivo gene delivery with siRNA technique for efficient gene silencing provides new, unique possibilities to study apoptotic pathways thereby evaluating new molecular therapeutic strategies in vivo. In this minireview we will focus on the use of RNA interference for analytical and therapeutical suppression of apoptotic pathways in vivo with special consideration of the liver.     

Exosomes: Nanoparticulate tools for RNA interference and drug delivery

Exosomes are naturally occurring extracellular vesicles released by most mammalian cells in all body fluids. Exosomes are known as key mediators in cell‐cell communication and facilitate the transfer of genetic and biochemical information between distant cells. Structurally, exosomes are composed of lipids, proteins, and also several types of RNAs which enable these vesicles to serve as important disease biomarkers. Moreover, exosomes have emerged as novel drug and gene delivery tools owing to their multiple advantages over conventional delivery systems. Recently, increasing attention has been focused on exosomes for the delivery of drugs, including therapeutic recombinant proteins, to various target tissues. Exosomes are also promising vehicles for the delivery of microRNAs and small interfering RNAs, which is usually hampered by rapid degradation of these RNAs, as well as inefficient tissue specificity of currently available delivery strategies. This review highlights the most recent accomplishments and trends in the use of exosomes for the delivery of drugs and therapeutic RNA molecules.

     

Delivery of small interfering RNA with a synthetic collagen poly(Pro‐Hyp‐Gly) for gene silencing in vitro and in vivo

Silencing gene expression by small interfering RNAs (siRNAs) has become a powerful tool for the genetic analysis of many animals. However, the rapid degradation of siRNA and the limited duration of its action in vivo have called for an efficient delivery technology. Here, we describe that siRNA complexed with a synthetic collagen poly(Pro‐Hyp‐Gly) (SYCOL) is resistant to nucleases and is efficiently transferred into cells in vitro and in vivo, thereby allowing long‐term gene silencing in vivo. We found that the SYCOL‐mediated local application of siRNA targeting myostatin, coding a negative regulator of skeletal muscle growth, in mouse skeletal muscles, caused a marked increase in the muscle mass within a few weeks after application. Furthermore, in vivo administration of an anti‐luciferase siRNA/SYCOL complex partially reduced luciferase expression in xenografted tumors in vivo. These results indicate a SYCOL‐based non‐viral delivery method could be a reliable simple approach to knockdown gene expression by RNAi in vivo as well as in vitro.     

Chitosan hydrogels containing liposomes and cubosomes as particulate sustained release vaccine delivery systems

Sustained release depot systems have been widely investigated for their potential to improve the efficacy of subunit vaccines and reduce the requirement for boosting. The present study aimed to further enhance the immunogenicity of a sustained release vaccine by combining a depot formulation with a particulate antigen delivery system. Sustained release of the model subunit antigen, ovalbumin (OVA), was observed in vivo from chitosan thermogel-based formulations containing cationic, nanosized liposomes loaded with OVA and the immunopotentiator, Quil A (QA). Such formulations demonstrated the ability to induce cluster of differentiation (CD)8⁺ and CD4⁺ T-cell proliferation and interferon (IFN)-γ production, as well as the production of OVA-specific antibody. However, gel-incorporated liposomes showed evidence of instability and similar in vivo immune responses to liposomes in gel formulations were induced by gel-based systems loaded with soluble OVA and QA. The immunogenicity of chitosan thermogels containing cubosomes, a more stable lipidic particulate system, was therefore examined. Similarly, all gel-based formulations produced comparable effector immune responses in experimental mice, irrespective of whether the antigen and immunopotentiator were present in gels within cubosomes or in a soluble form. This work demonstrates the potential for sustained release thermogelling systems and highlights the importance of matching the physicochemical and immunological properties of the particulate system to that of the depot.     

Plasmid delivery in the rat brain

Neurodegenerative diseases as a class do not have effective pharmacotherapies. This is due in part to a poor understanding of the pathologies of the disease processes, and the lack of effective medications. Gene delivery is an attractive possibility for treating these diseases. For the paradigm to be effective, efficient, safe and versatile vectors are required. In this study we evaluated three plasmid delivery systems for transgene expression in the rat hippocampus. Two of these systems were designed to have enhanced intracellular biodegradability. It was hypothesized that this system would be less toxic and could increase the free (non-vector) associated plasmids within the cell, leading to increased transgene activity. Polyethylenimine (PEI) and r-AAV-2 (recombinant adeno associated virus-2) were used as positive, non-viral and viral controls respectively, in the in vivo experiments. The results from the studies indicate there is a distinct difference between the various vectors in terms of total cells transfected, type of cell transfected, and toxicity. Non-viral systems were effective at transfecting both neurons and glia cells within the hippocampus, while the r-AAV-2 transfected mainly neurons. In summary, plasmid-mediated systems are effective for transgene expression within the brain and deserve further study.     

Novel liposome-based formulations for prolonged delivery of proteins and vaccines

Abstract

Two strategies for increasing liposome stability in vivo are described in this review. The first strategy involves the encapsulation of liposomes within polymeric microcapsules of alginate-poly(L-lysine) that retained the liposomes inside but allowed the outward diffusion of proteins of 100 kDa or less, once they were released from the encapsulated liposomes. In vivo studies revealed that the microencapsulated liposome systems (MELs) extended the delivery of a model antigen, bovine serum albumin (BSA), for more that 80 days, resulting in the prolonged production of high levels of antigen-specific antibodies. The antibody levels were higher that those obtained with rats injected with BSA in complete Freund's adjuvant, or in liposomes. The unique construction of MELs enabled also the enzymatically-triggered pulsatile delivery of proteins from encapsulated liposomes, which was not possible before with liposomes.     

Antibody-mediated delivery of chimeric protein degraders which target estrogen receptor alpha (ERα)

Chimeric molecules which effect intracellular degradation of target proteins via E3 ligase-mediated ubiquitination (e.g., PROTACs) are currently of high interest in medicinal chemistry. However, these entities are relatively large compounds that often possess molecular characteristics which may compromise oral bioavailability, solubility, and/or in vivo pharmacokinetic properties. Accordingly, we explored whether conjugation of chimeric degraders to monoclonal antibodies using technologies originally developed for cytotoxic payloads might provide alternate delivery options for these novel agents. In this report we describe the construction of several degrader-antibody conjugates comprised of two distinct ERα-targeting degrader entities and three independent ADC linker modalities. We subsequently demonstrate the antigen-dependent delivery to MCF7-neo/HER2 cells of the degrader payloads that are incorporated into these conjugates. We also provide evidence for efficient intracellular degrader release from one of the employed linkers. In addition, preliminary data are described which suggest that reasonably favorable in vivo stability properties are associated with the linkers utilized to construct the degrader conjugates.     

Gene transfer to skeletal muscle using hydrodynamic limb vein injection: current applications,hurdles and possible optimizations

Hydrodynamic limb vein injection is an in vivo locoregional gene delivery method. It consists of administrating a large volume of solution containing nucleic acid constructs in a limb with both blood inflow and outflow temporarily blocked using a tourniquet. The fast, high pressure delivery allows the musculature of the whole limb to be reached. The skeletal muscle is a tissue of choice for a variety of gene transfer applications, including gene therapy for Duchenne muscular dystrophy or other myopathies, as well as for the production of antibodies or other proteins with broad therapeutic effects. Hydrodynamic limb vein delivery has been evaluated with success in a large range of animal models. It has also proven to be safe and well‐tolerated in muscular dystrophy patients, thus supporting its translation to the clinic. However, some possible limitations may occur at different steps of the delivery process. Here, we have highlighted the interests, bottlenecks and potential improvements that could further optimize non‐viral gene transfer following hydrodynamic limb vein injection.     

Ocular amphotericin B delivery by chitosan-modified nanostructured lipid carriers for fungal keratitis-targeted therapy

Abstract

Context: Fungal keratitis, a corneal fungal infection of the eye caused mainly by Candida species, has become the leading cause of blindness resulting from corneal disease in China. Present limitations in the management of ophthalmic fungal infections include the inability to provide long-term extraocular drug delivery without compromising intraocular structures and/or systemic drug exposure.

Objective: The aim of this study was to construct amphotericin B (AmB) loaded, chitosan-modified, nanostructured lipid carriers (AmB-CH-NLC) for prolonged ocular application and for the improvement of the targeted delivery of AmB to the ocular mucosa.

Materials and methods: The AmB-CH-NLC was produced by the method of emulsion evaporation-solidification at low temperature. The particle size, zeta potential, and encapsulation efficiency, drug-release behavior, and corneal penetration ability were performed in vitro and in vivo.

Results and discussion: The prepared AmB-CH-NLC nanoparticles exhibited a measured size of 185.4?nm, a zeta potential of 27.1?mV, and an entrapment efficiency of 90.9%. Sustained drug release behavior was observed in vitro. The in vivo ocular pharmacokinetic study indicated improved bioavailability of AmB-CH-NLC. The corneal penetration study showed that the AmB-CH-NLC could successfully penetrate into the cornea with no obvious irritation to the rabbits’ eyes.

Conclusion: The results support that this novel nanomedicine could be a promising system for effective ocular delivery of amphotericin B for fungal keratitis-targeted therapy.     

Imaging gene expression in the brain with peptide nucleic acid (PNA) antisense radiopharmaceuticals and drug targeting technology

Antisense oligomers are potential pharmaceutical and radiopharmaceutical agents that can be used to modulate and image gene expression. Progress with in vivogene targeting using antisense-based therapeutics has been slower than expected during the last decade, owing to poor trans-cellular delivery of antisense agents. This chapter suggests that if antisense pharmacology is merged with drug targeting technology, then membrane barriers can be circumvented and antisense agents can be delivered to tissues in vivo. Without the application of drug targeting, the likelihood of success for an antisense drug development program is low, particularly for the brain which is protected by the blood-brain barrier (BBB). Among the different classes of antisense agents, peptide nucleic acids (PNA) present advantages for in vivoapplications over conventional and modified oligodeoxynucleotides (ODN), including phosphorothioates (PS)-ODN. Some advantages of PNAs include their electrically neutral backbone, low toxicity to neural cells, resistance to nucleases and peptidases, and lack of binding to plasma proteins. PNAs are poorly transported through cellular membranes, however, including the BBB and the brain cell membrane (BCM). Because the mRNA target for the antisense agent lies within the cytosol of the target cell, the BBB and the BCM must be circumvented in vivo, which ispossible with the use of chimeric peptide drug targeting technology. Chimeric peptides are formed by conjugation of a non-transportable drug, such as a PNA, to a drug delivery vector. The vector undergoes receptor-mediated transcytosis (RMT) through the BBB and receptor-mediated endocytosis through the BCM in vivo. When labeled with a radioisotope (e.g., ¹²⁵I or ¹¹¹In), the antisense chimeric peptide provides imaging of gene expressionin the brain in vivoin a sequence-specific manner. Further development of antisense radiopharmaceutical agents may allow for in vivoimaging of genes in pathological states, and may provide tools for the analysis of novel genes with functional genomics.     

Non-viral gene therapy for bone tissue engineering

The possibilities of using gene therapy for bone regeneration have been extensively investigated. Improvements in the design of new transfection agents, combining vectors and delivery/release systems to diminish cytotoxicity and increase transfection efficiencies have led to several successful in vitro, ex vivo and in vivo strategies. These include growth factor or short interfering ribonucleic acid (siRNA) delivery, or even enzyme replacement therapies, and have led to increased osteogenic differentiation and bone formation in vivo. These results provide optimism to consider use in humans with some of these gene-delivery strategies in the near future.     

小鼠对量子点的透皮吸收和代谢

皮肤是身体的最大器官,能够直接与含纳米材料的防晒霜、化妆品等接触,但是人们对纳米材料的皮肤渗透性却了解不多．本文研究了水溶性硫硒化镉(CdSeS)量子点纳米颗粒的皮肤渗透性和在体内的代谢情况．将雄性ICR鼠背部脱毛,在脱毛部位涂抹直径约为5 nm、发光波长为620 nm的量子点0.32 nmol,然后检测皮肤和心、肝、脾、肺、肾中量子点沉积量随时间的变化情况．荧光显微像显示,量子点能够堆积在皮肤的表皮层中和真皮层的毛囊和腺体中,电感耦合等离子体质谱(inductively coupled plasma-mass spectrometry,ICP-MS)结果表明,透皮吸收的量子点能够沉积在器官中,并且肝和肾中沉积的量子点代谢缓慢,涂抹量子点5天之后,肾脏中残存的镉离子浓度仍超过14 ng/g．这些结果表明,量子点能够被小鼠透皮吸收,而且对肝和肾产生严重影响．     

Room temperature vulcanizing silicone sheaths on a reusable support for progesterone delivery in estrous synchronization treatments in cattle

High temperature vulcanizing silicone elastomers have been widely used in controlled delivery systems of steroid hormones with the aim of controlling estrous cycle in livestock. This paper reports experiments conducted to evaluate the possibility of using room temperature vulcanizing (RTV) silicone elastomers for the intravaginal administration of progesterone to cattle. In vitro studies showed that RTV silicones and high-temperature vulcanizing silicone release progesterone at a similar rate.Y-shaped inserts made of different polymers were designed as supports of RTV silicone sheaths to test the in vivo release of progesterone. Field evaluation showed that RTV silicone sheaths containing 0.75 g of progesterone were at least as effective at estrous synchronization as commercially available intravaginal inserts.     

Targeted delivery of insulin-modified immunoliposomes in vivo

The purpose of this study was to investigate the effect of liposomes conjugated with insulin to the surface on circulation time, biodistribution, and antitumor activity after intravenous injection in tumor-bearing mice. Immunoliposomes were constructed with insulin, which was covalently linked to liposomes containing anticancer drugs. In order to investigate the targeting performance of insulin-modified immunoliposomes (SILs) in vivo, plasma pharmacokinetics, biodistribution, and antitumor activity were tested. In comparison with nontargeted liposomes (SLs), SILs were cleared faster from circulation as a result of greater liver and tumor uptake. In addition, SILs retarded the growth of the tumor effectively, compared with the ZTO injection or SL. This is the first time for selective in vivo targeting of tumor vessels using insulin-modified immunoliposomes. SILs are candidate drug-delivery systems for therapeutic anticancer approaches.     

Imaging gene expression in the brain with peptide nucleic acid (PNA) antisense radiopharmaceuticals and drug targeting technology

Summary Antisense oligomers are potential pharmaceutical and radiopharmaceutical agents that can be used to modulate and image gene expression. Progress within vivo gene targeting using antisense-based therapeutics has been slower than expected during the last decade, owing to poor trans-cellular delivery of antisense agents. This chapter suggests that if antisense pharmacology is merged with drug targeting technology, then membrane barriers can be circumvented and antisense agents can be delivered to tissuesin vivo. Without the application of drug targeting, the likelihood of success for an antisense drug development program is low, particularly for the brain which is protected by the blood-brain barrier (BBB). Among the different classes of antisense agents, peptide nucleic acids (PNA) present advantages forin vivo applications over conventional and modified oligodeoxynucleotides (ODN), including phosphorothioates (PS)-ODN. Some advantages of PNAs include their electrically neutral backbone, low toxicity to neural cells, resistance to nucleases and peptidases, and lack of binding to plasma proteins. PNAs are poorly transported through cellular membranes, however, including the BBB and the brain cell membrane (BCM). Because the mRNA target for the antisense agent lies within the cytosol of the target cell, the BBB and the BCM must be circumventedin vivo, which is possible with the use of chimeric peptide drug targeting technology. Chimeric peptides are formed by conjugation of a non-transportable drug, such as a PNA, to a drug delivery vector. The vector undergoes receptor-mediated transcytosis (RMT) through the BBB and receptor-mediated endocytosis through the BCMin vivo. When labeled with a radioisotope (e.g.,¹²⁵I or¹¹¹In), the antisense chimeric peptide provides imaging of gene expression in the brainin vivo in a sequence-specific manner. Further development of antisense radiopharmaceutical agents may allow forin vivo imaging of genes in pathological states, and may provide tools for the analysis of novel genes with functional genomics.     

Carbon nanotubes in cancer diagnosis and therapy

During the past years, great progress has been made in the field of nanomaterials given their great potential in biomedical applications. Carbon nanotubes (CNTs), due to their unique physicochemical properties, have become a popular tool in cancer diagnosis and therapy. They are considered one of the most promising nanomaterials with the capability of both detecting the cancerous cells and delivering drugs or small therapeutic molecules to these cells. Over the last several years, CNTs have been explored in almost every single cancer treatment modality, including drug delivery, lymphatic targeted chemotherapy, thermal therapy, photodynamic therapy, and gene therapy. In this review, we will show how they have been introduced into the diagnosis and treatment of cancer. Novel SWNT-based tumor-targeted drug delivery systems (DDS) will be highlighted. Furthermore, the in vitro and in vivo toxicity of CNTs reported in recent years will be summarized.