Development of Delivery Systems for Introducing Endophytic Bacteria into Cotton

Experiments were designed to evaluate the effectiveness of several methods for delivering 15 endophytic bacteria into cotton stem and root tissues. The delivery methods included stab-inoculation of bacteria into stems, soaking seeds in bacterial suspensions, methyl cellulose seed coating, foliar spray, bacteria-impregnated granules applied in-furrow, vacuum infiltration and pruned-root dip. The success of delivery was gaged by recovery of the bacteria from internal plant tissues 2 weeks after the plants had been grown in a glasshouse potting mix. Following stab-inoculation into stems or radicles, 10 of the bacterial endophytes which previously exhibited biological control against fusarium wilt of cotton were successfully re-isolated from 50% of the plants inoculated; however, this method was labor-intensive, involved wounding the plant and sometimes reduced plant growth. Four of the other methods established from six to eight of the 15 strains, and, with some strains, all methods effectively established endophytic bacteria, based on re-isolation of strains from internal tissues 2 weeks after inoculation. A method was developed which allowed more convenient isolation of endophytes from a large number of plants. The results suggest that introduction of beneficial endophytic strains into cotton plants could be accomplished by practical methods chosen specifically for each strain.     

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos^1,2 that we have successfully adapted^3,4. It is largely based on the success of in ovo electroporation technologies that are commonly used in chick⁵. Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described⁶. Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells.To date, in utero electroporation has been most widely used for the analysis of neocortical development^1,2,3,4. More recent studies have targeted the embryonic retina^7,8,9 and thalamus^10,11,12. Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3^rd ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.Download video file.^{(82M, mov)}     

Nanotechnology-Based Drug Delivery Systems

In recent years, there has been a considerable interest in the development of novel drug delivery systems using nanotechnology. Nanoparticles represent a promising drug delivery system of controlled and targeted release. In this context, nanosuspensions will be effective in increasing the solubility and bioavailability of poorly soluble drugs. This review focuses on advantages, method of preparation, physical characteristics, and evaluation of drug nanosuspensions.     

Design and Evaluation of a Novel Matrix Type Multiple Units as Biphasic Gastroretentive Drug Delivery Systems

A biphasic gastroretentive floating drug delivery system with multiple-unit mini-tablets based on gas formation technique was developed to maintain constant plasma level of a drug concentration within the therapeutic window. The system consists of loading dose as uncoated core units, and prolonged-release core units are prepared by direct compression process; the latter were coated with three successive layers, one of which is seal coat, an effervescent (sodium bicarbonate) layer, and an outer polymeric layer of polymethacrylates. The formulations were evaluated for quality control tests, and all the parameters evaluated were within the acceptable limits. The system using Eudragit RL30D and combination of them as polymeric layer could float within acceptable time. The drug release was linear with the square root of time. The rapid floating and the controlled release properties were achieved in this present study. When compared with the theoretical release profile, the similarity factor of formulation with coating of RS:RL (1:3)–7.5%, was observed to be 74, which is well fitted into zero-order kinetics confirming that the release from formulation is close to desired release profile. The stability samples showed no significant change in dissolution profiles (p > 0.05). In vivo gastric residence time was examined by radiograms, and it was observed that the units remained in the stomach for about 5 h.     

Novel Delivery Systems for Interferons

ABSTRACT

Interferons, IFNs, are among the most widely studied and clinically used biopharmaceuticals. Despite their invaluable therapeutic roles, the widespread use of IFNs suffers from some inherent limitations, mainly their relatively short circulation lifespan and their unwanted effects on some non-target tissues. Therefore, both these constraints have become the central focus points for the research efforts on the development of a variety of novel delivery systems for these therapeutic agents with the ultimate goal of improving their therapeutic end-points. Generally, the delivery systems currently under investigation for IFNs can be classified as particulate delivery systems, including micro- and nano-particles, liposomes, minipellets, cellular carriers, and non-particulate delivery systems, including PEGylated IFNs, other chemically conjugated IFNs, immunoconjugated IFNs, and genetically conjugated IFNs. All these strategies and techniques have their own possibilities and limitations, which should be taken into account when considering their clinical application. In this article, currently studied delivery systems/techniques for IFN delivery have been reviewed extensively, with the main focus on the pharmacokinetic consequences of each procedure.     

Moisture Stability of Maltodextrin-Based Delivery Systems

The water sorption isotherms are exploited here for maltodextrin-based perfume-delivery systems with the aim of predicting water vapor stability of the samples at a given temperature. A combination of Couchman (glass-transition temperature) and Guggenheim-Anderson-deBoer (GAB) (fraction of water condensed) models is shown demonstrating important information hidden in a simple sorption experiment (rate of loss of perfume by diffusion and capacity to retain volatiles under humid air conditions). Pure maltodextrin and maltodextrin loaded with apolar and polar perfume components are treated using the same developed methodology. It is shown that apolar molecule release from classical carbohydrate spray dry particles follows a zero-order kinetic. This poster was presented at the 2nd International Symposium on Delivery of Functionality in Complex Food Systems, University of Massachusetts, Amherst, USA. October 8th to 10th, 2007.     

Multiple Delivery of siRNA against Endoglin into Murine Mammary Adenocarcinoma Prevents Angiogenesis and Delays Tumor Growth

Endoglin is a transforming growth factor-β (TGF- β) co-receptor that participates in the activation of a signaling pathway that mediates endothelial cell proliferation and migration in angiogenic tumor vasculature. Therefore, silencing of endoglin expression is an attractive approach for antiangiogenic therapy of tumors. The aim of our study was to evaluate the therapeutic potential of small interfering RNA (siRNA) molecules against endoglin in vitro and in vivo. Therapeutic potential in vitro was assessed in human and murine endothelial cells (HMEC-1, 2H11) by determining endoglin expression level, cell proliferation and tube formation. In vivo, the therapeutic potential of siRNA molecules was evaluated in TS/A mammary adenocarcinoma growing in BALB/c mice. Results of our study showed that siRNA molecules against endoglin have a good antiangiogenic therapeutic potential in vitro, as expression of endoglin mRNA and protein levels in mouse and human microvascular endothelial cells after lipofection were efficiently reduced, which resulted in the inhibition of endothelial cell proliferation and tube formation. In vivo, silencing of endoglin with triple electrotransfer of siRNA molecules into TS/A mammary adenocarcinoma also significantly reduced the mRNA levels, number of tumor blood vessels and the growth of tumors. The obtained results demonstrate that silencing of endoglin is a promising antiangiogenic therapy of tumors that could not be used as single treatment, but as an adjunct to the established cytotoxic treatment approaches.     

High-Titer Human Immunodeficiency Virus Type 1-Based Vector Systems for Gene Delivery into Nondividing Cells

Previously we designed novel pseudotyped high-titer replication defective human immunodeficiency virus type 1 (HIV-1) vectors to deliver genes into nondividing cells (J. Reiser, G. Harmison, S. Kluepfel-Stahl, R. O. Brady, S. Karlsson, and M. Schubert, Proc. Natl. Acad. Sci. USA 93:15266–15271, 1996). Since then we have made several improvements with respect to the safety, flexibility, and efficiency of the vector system. A three-plasmid expression system is used to generate pseudotyped HIV-1 particles by transient transfection of human embryonic kidney 293T cells with a defective packaging construct, a plasmid coding for a heterologous envelope (Env) protein, and a vector construct harboring a reporter gene such as neo, ShlacZ (encoding a phleomycin resistance/β-galactosidase fusion protein), HSA (encoding mouse heat-stable antigen), or EGFP (encoding enhanced green fluorescent protein). The packaging constructs lack functional Vif, Vpr, and Vpu proteins and/or a large portion of the Env coding region as well as the 5′ and 3′ long terminal repeats, the Nef function, and the presumed packaging signal. Using G418 selection, we routinely obtained vector particles pseudotyped with the vesicular stomatitis virus G glycoprotein (VSV-G) with titers of up to 8 × 10⁷ CFU/μg of p24, provided that a functional Tat coding region was present in the vector. Vector constructs lacking a functional Tat protein yielded titers of around 4 × 10⁶ to 8 × 10⁶ CFU/μg of p24. Packaging constructs with a mutation within the integrase (IN) core domain profoundly affected colony formation and expression of the reporter genes, indicating that a functional IN protein is required for efficient transduction. We explored the abilities of other Env proteins to allow formation of pseudotyped HIV-1 particles. The rabies virus and Mokola virus G proteins yielded high-titer infectious pseudotypes, while the human foamy virus Env protein did not. Using the improved vector system, we successfully transduced contact-inhibited primary human skin fibroblasts and postmitotic rat cerebellar neurons and cardiac myocytes, a process not affected by the lack of the accessory proteins.     

生物源性基因疫苗的运送系统

基因疫苗具有很多独特的优点,已经成为疫苗研究领域的热点。但由于其免疫原性相对较弱,限制了基因疫苗的广泛应用。人们一直在寻求一种理想的基因疫苗运送系统,它不仅能将基因疫苗导入体内,还能提高基因疫苗的免疫原性,诱导机体产生持续高水平的免疫应答反应。     

综述: siRNA脂质纳米输送载体的研究进展

siRNA能高效且特异地阻断内源性同源基因的表达即RNA干涉(RNAi)．RNAi在临床中的应用需要开发安全有效的输送系统,脂质纳米输送载体是一种具有发展潜力的siRNA输送系统．siRNA-脂质复合物的形成主要通过静电相互作用,静电作用必须足够强以至于载体在运输过程中不释放siRNA,而载体到达治疗部位时,解聚释放出siRNA．载体的粒径应小于100 nm,以利于细胞的摄取和透过特定部位的血管开窗．为了减少网状内皮系统(RES)的摄取和延长载体的循环时间,载体的表面由聚乙二醇修饰．本文主要综述了构建siRNA输送载体的基本要求．     

Establishment of Protein Delivery Systems Targeting Podocytes

Background

Podocytes are uniquely structured cells that are critical to the kidney filtration barrier. Their anatomic location on the outer side of the glomerular capillaries expose podocytes to large quantities of both plasma and urinary components and thus are reachable for drug delivery. Recent years have made clear that interference with podocyte-specific disease pathways can modulate glomerular function and influence severity and progression of glomerular disease.

Methodology/Principal Findings

Here, we describe studies that show efficient transport of proteins into the mammalian cells mouse 3T3 fibroblasts and podocytes, utilizing an approach termed profection. We are using synthetic lipid structures that allow the safe packing of proteins or antibodies resulting in the subsequent delivery of protein into the cell. The uptake of lipid coated protein is facilitated by the intrinsic characteristic of cells such as podocytes to engulf particles that are physiologically retained in the extracellular matrix. Profection of the restriction enzyme MunI in 3T3 mouse fibroblasts caused an increase in DNA degradation. Moreover, purified proteins such as β-galactosidase and the large GTPase dynamin could be profected into podocytes using two different profection reagents with the success rate of 95–100%. The delivered β-galactosidase enzyme was properly folded and able to cleave its substrate X-gal in podocytes. Diseased podocytes are also potential recipients of protein cargo as we also delivered fluorophore labeled IgG into puromycin treated podocytes. We are currently optimizing our protocol for in vivo profection.

Conclusions

Protein transfer is developing as an exciting tool to study and target highly differentiated cells such as podocytes.     

反义寡核苷酸递送方法研究进展

如何将反义寡核苷酸 (AS ODNs)有效递送进入细胞是反义核酸领域面临的一大难题。近年来 ,出现了多种寡核苷酸 (ODNs)的递送方法。在培养细胞中 ,使用的递送方法包括阳离子载体包裹、特异受体的配体导向、ODNs偶联修饰、细胞膜辅助穿透以及利用逆转录病毒载体转染等 ,其应用有效增强了AS ODNs的作用效果 ,大幅度降低了AS ODNs的使用浓度 ;在体内 ,由于临床使用裸露AS ODNs连续给药能达到一定的反义效果 ,而使递送方法的研究和应用尚处于初步尝试和探索之中 ,迄今报道的递送方法有脂类和非脂类两类。     

生物可降解聚合物纳米粒给药载体

生物可降解聚合物纳米粒用于给药载体具有广阔的前景。本文综述了生物可降解聚合物纳米粒给药载体领域的最新进展 :包括纳米粒表面修饰特性、药物释放、载多肽和蛋白质等生物大分子药物传输中的潜在应用。     

l-Aspartate Transport into Pea Chloroplasts : Kinetic and Inhibitor Evidence for Multiple Transport Systems

The kinetics of l-aspartate transport into pea chloroplasts was studied in the presence and absence of transport inhibitors to determine whether multiple aspartate carriers exist. Transport was measured by the silicone oil centrifugation technique. Reciprocal plots of concentration-dependent transport rates were biphasic, indicating the presence of two transport components, distinguishable on the basis of their affinity for aspartate. These transport components, called high affinity and low affinity transport could also be distinguished on the basis of their apparent substrate saturability and their sensitivity to media pH. The apparent K_m for high affinity transport was 30 micromolar. The K_m for low affinity transport was not determined. To test whether these transport components could also be distinguished on the basis of inhibitor sensitivity and to assess the value of inhibitors for distinguishing multiple aspartate translocators, a survey of several classes of potential inhibitors was conducted. High affinity aspartate transport was inhibited by p-chloromercuribenzenesulfonate and mersalyl, both sulfhydryl-reactive reagents; diethyl pyrocarbonate, a histidine-reactive reagent; and nigericin and carbonyl cyanide m-chlorophenylhydrazone, both ionophores. Low affinity aspartate transport was not inhibited by p-chloromercuribenzenesulfonate or nigericin, but preliminary results suggest it was sensitive to diethyl pyrocarbonate. Because the high and low affinity transport components could be distinguished not only by their sensitivity to media pH and substrate saturability, but also by their sensitivity to various inhibitors, we concluded that they may represent different transport systems or carriers.     

腺病毒载体与慢病毒载体感染骨髓间充质干细胞的比较

随着基因治疗的发展,建立高效稳定表达目的基因的载体和靶细胞成为目前研究的热点.骨髓间充质干细胞具有自我复制,高度的增殖能力,多向或定向分化的潜能等优势,是细胞和基因工程中的理想靶细胞.而腺病毒载体和慢病毒载体作为基因载体均能将目的基因导入宿主细胞,因此对腺病毒载体和慢病毒载体的结构、分类、优缺点以及感染骨髓间充质干细胞的转染效率等方面做简要的分析.