Rational design of amphiphile-based drug carriers and sterically stabilized carriers

Abstract

This paper describes the parameters recommended for rational design of amphiphile-based drug carriers. The main advantage of a carrier is its ability to modify the pharmacokinetics and biodistribution of the drug, so that the drug level at the target is sufficient for therapeutic benefits. Three parameters are described. Two of them, the drug-to-carrier partition coefficient (Ky_iC) and the rate of drug release from the carrier (k_ff), are related to drug-carrier interactions; the third one is the rate of carrier clearance (k_c). We demonstrate that carrier performance for drugs associated with the carrier amphiphile(s) is determined to a large extent by K_c, while for drugs encapsulated in the aqueous phase of the carrier it is important that k_off will be similar to k_c These conclusions are based on two examples: (i) Amphotericin B as a drug associated with five dosage forms which represent different types of amphiphile-based carriers: micelles (Fungizone), stable micelle-like disks (Amphocil), a complex with phospholipids (ABPLC), liposomes (AmBisome), and a submicronized emulsion, (ii) Liposomal doxorubicin which consisted of either doxorubicin associated with the membrane of negatively-charged, fluid oligolamellar liposomes (L-DOX) or doxorubicin loaded by an ammonium sulfate gradient into small, unilamellar, rigid liposomes having steric stabilizing lipid grafted in their lipid bilayer, (S-DOX). To better understand what contributes to k, we also describe the effect of bilayer acyl chain composition and the role of precipitation of the drug inside the liposomes.     

Development of a tactical screening method to investigate the characteristics of functional peptides

Using spot-synthesized peptide arrays, a functional peptide can be screened as a high-binding peptide for a target molecule. We have developed a rational screening method for functional peptides by analyzing the physicochemical rules of high-binding peptide sequences. To screen the peptides simply and strategically, we prepared an exhaustive 4-mer peptide library consisting of 256 peptides (4⁴ = 256) characterized by four physicochemical groups of 20 amino acids: Group 1, non-charged hydrophobic amino acids; Group 2, non-charged hydrophilic amino acids; Group 3, positive-charged hydrophilic amino acids; Group 4, negative-charged hydrophilic amino acids. First, our previous screening data from cell adhesion, bile acid-binding, and nanoparticle-binding peptides were applied to the four-category analysis, and target-specific physicochemical characteristics were obtained. We then prepared an exhaustive 4-mer peptide library using these four physicochemical groups, and screened for high-binding peptides that bind model proteins interleukin-2 and IgG. We obtained individual physicochemical rules for high-binding peptides: group 1 or 4 amino acids in position (P) 1, group 1 in P2 and P4 for IL-2, and group 2 and 3 amino acids at all position for IgG. Therefore, this system, which employs the use of a simple and strategic peptide library, will be useful in the development of functional peptides.     

Francisella strains express hemolysins of distinct characteristics

Historically, Francisella strains have been described as nonhemolytic. In this study, we show by use of solid and liquid hemolysis assays that some Francisella strains have hemolytic properties. The Francisella novicida type strain U112 is hemolytic to horse erythrocytes and Francisella philomiragia type strain FSC144 is hemolytic towards both human and horse erythrocytes. The F. novicida strain U112 released a protein (novilysin A) into the culture supernatant which cross-reacted with antiserum against Escherichia coli HlyA whereas there was no similar protein detectable with this cross-reactive property from the supernatant of the F. philomiragia strain.     

Evolutionary design principles and functional characteristics based on kingdom-specific network motifs

BACKGROUND: Network motifs within biological networks show non-random abundances in systems at different scales. Large directed protein networks at the cellular level are now well defined in several diverse species. We aimed to compare the nature of significantly observed two- and three-node network motifs across three different kingdoms (Arabidopsis thaliana for multicellular plants, Saccharomyces cerevisiae for unicellular fungi and Homo sapiens for animals). RESULTS: 'Two-node feedback' is the most significant motif in all three species. By considering the sign of each two-node feedback interaction, we examined the enrichment of the three types of two-node feedbacks [positive-positive (PP), negative-negative (NN) and positive-negative (PN)]. We found that PN is enriched in the network of A.thaliana, NN in the network of S.cerevisiae and PP and NN in the network of H.sapiens. Each feedback type has characteristic features of robustness, multistability and homeostasis. Conclusions: We suggest that amplification of particular network motifs emerges from contrasting dynamical and topological properties of the motifs, reflects the evolutionary design principles selected by the characteristic behavior of each species and provides a signature pointing to their behavior and function.     

GAIP participates in budding of membrane carriers at the trans-Golgi network

Galpha interacting protein (GAIP) is a regulator of G protein signaling protein that associates dynamically with vesicles and has been implicated in membrane trafficking, although its specific role is not yet known. Using an in vitro budding assay, we show that GAIP is recruited to a specific population of trans -Golgi network-derived vesicles and that these are distinct from coatomer or clathrin-coated vesicles. A truncation mutant (NT-GAIP) encoding only the N-terminal half of GAIP is recruited to trans -Golgi network membranes during the formation of vesicle carriers. Overexpression of NT-GAIP induces the formation of long, coated tubules, which are stabilized by microtubules. Results from the budding assay and from imaging in live cells show that these tubules remain attached to the Golgi stack rather than being released as carrier vesicles. NT-GAIP expression blocks membrane budding and results in the accumulation of tubular carrier intermediates. NT-GAIP-decorated tubules are competent to load vesicular stomatitis virus protein G-green fluorescent protein as post-Golgi, exocytic cargo and in cells expressing NT-GAIP there is reduced surface delivery of vesicular stomatitis virus protein G-green fluorescent protein. We conclude that GAIP functions as an essential part of the membrane budding machinery for a subset of post-Golgi exocytic carriers derived from the trans -Golgi network.     

Biological activity and characteristics of triamcinolone-acetonide formulated with the self-regulating drug carriers,Transfersomes

Novel formulations of the halogenated corticosteroid, triamcinolone-acetonide, based on ultradeformable mixed lipid vesicles, Transfersomes, are described. Their performance was tested in vivo using radioactive label measurements, to study the drug biodistribution, and murine ear edema, to determine the drug bioactivity. Sparse use of drug-loaded Transfersomes on the skin ensures an almost exclusive delivery of triamcinolone-acetonide into the organ, thus arguably increasing the treatment safety. Delivery of triamcinolone-acetonide in the skin with ultradeformable vesicles prolongs the anti-inflammatory drug action several times compared to drug usage in a conventional crème or an ointment, the robustness of biological response for the former being at least identical to the latter. The required dose of Transfersome-based triamcinolone-acetonide is also greatly reduced. The drug dose of 0.2 microg cm(-2) suppresses 75% of arachidonic acid-induced murine ear edema for at least 48 h. In contrast, a conventional formulation of triamcinolone-acetonide requires a 10-fold higher drug dosage to achieve a similar effect. In either case, increasing the applied corticosteroid amount delays the onset of anti-edema action.     

Enriching the analysis of genomewide association studies with hierarchical modeling

Genomewide association studies (GWAs) initially investigate hundreds of thousands of single-nucleotide polymorphisms (SNPs), and the most promising SNPs are further evaluated with additional subjects, for replication or a joint analysis. Deciding which SNPs merit follow-up is one of the most crucial aspects of these studies. We present here an approach for selecting the most-promising SNPs that incorporates into a hierarchical model both conventional results and other existing information about the SNPs. The model is developed for general use, its potential value is shown by application, and tools are provided for undertaking hierarchical modeling. By quantitatively harnessing all available information in GWAs, hierarchical modeling may more clearly distinguish true causal variants from noise.     

Biological activity and characteristics of triamcinolone-acetonide formulated with the self-regulating drug carriers, Transfersomes®

Novel formulations of the halogenated corticosteroid, triamcinolone-acetonide, based on ultradeformable mixed lipid vesicles, Transfersomes®, are described. Their performance was tested in vivo using radioactive label measurements, to study the drug biodistribution, and murine ear edema, to determine the drug bioactivity. Sparse use of drug-loaded Transfersomes® on the skin ensures an almost exclusive delivery of triamcinolone-acetonide into the organ, thus arguably increasing the treatment safety. Delivery of triamcinolone-acetonide in the skin with ultradeformable vesicles prolongs the anti-inflammatory drug action several times compared to drug usage in a conventional crème or an ointment, the robustness of biological response for the former being at least identical to the latter. The required dose of Transfersome®-based triamcinolone-acetonide is also greatly reduced. The drug dose of 0.2 μg cm⁻² suppresses 75% of arachidonic acid-induced murine ear edema for at least 48 h. In contrast, a conventional formulation of triamcinolone-acetonide requires a 10-fold higher drug dosage to achieve a similar effect. In either case, increasing the applied corticosteroid amount delays the onset of anti-edema action.     

Ultrastructure of long-range transport carriers moving from the trans Golgi network to peripheral endosomes

The delivery of mannose 6-phosphate receptors carrying lysosomal hydrolases from the trans-Golgi network (TGN) to the endosomal system is mediated by selective incorporation of the receptor-hydrolase complexes into vesicular transport carriers (TCs) that are coated with clathrin and the adaptor proteins, GGA and AP-1. Previous electron microscopy (EM) and biochemical studies have shown that these TCs consist of spherical coated vesicles with a diameter of 60-100 nm. The use of fluorescent live cell imaging, however, has revealed that at least some of this transport relies on a subset of apparently larger and highly pleiomorphic carriers that detach from the TGN and translocate toward the peripheral cytoplasm until they meet with distally located endosomes. The ultrastructure of such long-range TCs has remained obscure because of the inability to examine by conventional EM the morphological details of rapidly moving organelles. The recent development of correlative light-EM has now allowed us to obtain ultrastructural 'snapshots' of these TCs immediately after their formation from the TGN in live cells. This approach has revealed that such carriers range from typical 60- to 100-nm clathrin-coated vesicles to larger, convoluted tubular-vesicular structures displaying several coated buds. We propose that this subset of TCs serve as vehicles for long-range distribution of biosynthetic or recycling cargo from the TGN to the peripheral endosomes.     

电诱导棕色固氮菌表达抗氨阻遏特性的初步研究

采用循环伏安技术电诱导棕色固氮菌成为抗氨阻遏的菌株,其菌株最高耐氨率可高达８ｍＭＮＨ＾４＋左右。农田实验结果表明,直接施用该固菌于农田中,能使玉米及花生增产５％～８％,菌体的抗氨阻遏能力与封土质及成分有着密切关系。     

Protein kinase D regulates the fission of cell surface destined transport carriers from the trans-Golgi network

When a kinase inactive form of Protein Kinase D (PKD-K618N) was expressed in HeLa cells, it localized to the trans-Golgi network (TGN) and caused extensive tubulation. Cargo that was destined for the plasma membrane was found in PKD-K618N-containing tubes but the tubes did not detach from the TGN. As a result, the transfer of cargo from TGN to the plasma membrane was inhibited. We have also demonstrated the formation and subsequent detachment of cargo-containing tubes from the TGN in cells stably expressing low levels of PKD-K618N. Our results suggest that PKD regulates the fission from the TGN of transport carriers that are en route to the cell surface.     

A network aware approach for the scheduling of virtual machine migration during peak loads

Live virtual machine migration can have a major impact on how a cloud system performs, as it consumes significant amounts of network resources such as bandwidth. Migration contributes to an increase in consumption of network resources which leads to longer migration times and ultimately has a detrimental effect on the performance of a cloud computing system. Most industrial approaches use ad-hoc manual policies to migrate virtual machines. In this paper, we propose an autonomous network aware live migration strategy that observes the current demand level of a network and performs appropriate actions based on what it is experiencing. The Artificial Intelligence technique known as Reinforcement Learning acts as a decision support system, enabling an agent to learn optimal scheduling times for live migration while analysing current network traffic demand. We demonstrate that an autonomous agent can learn to utilise available resources when peak loads saturate the cloud network.     

Morphology and dynamics of clathrin/GGA1-coated carriers budding from the trans-Golgi network

Sorting of transmembrane proteins and their ligands at various compartments of the endocytic and secretory pathways is mediated by selective incorporation into clathrin-coated intermediates. Previous morphological and biochemical studies have shown that these clathrin-coated intermediates consist of spherical vesicles with a diameter of 60-100 nm. Herein, we report the use of fluorescent imaging of live cells to demonstrate the existence of a different type of transport intermediate containing associated clathrin coats. Clathrin and the adaptors GGA1 and adaptor protein-1, labeled with different spectral variants of the green fluorescent protein, are shown to colocalize to the trans-Golgi network and to a population of vesicles and tubules budding from it. These intermediates are highly pleiomorphic and move toward the peripheral cytoplasm for distances of up to 10 microm with average speeds of approximately 1 microm/s. The labeled clathrin and GGA1 cycle on and off membranes with half-times of 10-20 s, independently of vesicle budding. Our observations indicate the existence of a novel type of trans-Golgi network-derived carriers containing associated clathrin, GGA1 and adaptor protein-1 that are larger than conventional clathrin-coated vesicles, and that undergo long-range translocation in the cytoplasm before losing their coats.     

Oxime linkage: a robust tool for the design of pH-sensitive polymeric drug carriers

Oxime bonds dispersed in the backbones of the synthetic polymers, while young in the current spectrum of the biomedical application, are rapidly extending into their own niche. In the present work, oxime linkages were confirmed to be a robust tool for the design of pH-sensitive polymeric drug delivery systems. The triblock copolymer (PEG-OPCL-PEG) consisting of hydrophilic poly(ethylene glycol) (PEG) and hydrophobic oxime-tethered polycaprolactone (OPCL) was successfully prepared by aminooxy terminals of OPCL ligating with aldehyde-terminated PEG (PEG-CHO). Owing to its amphiphilic architecture, PEG-OPCL-PEG self-assembled into the micelles in aqueous media, validated by the measurement of critical micelle concentration (CMC). The MTT assay showed that PEG-OPCL-PEG exhibited low cytotoxicity against NIH/3T3 normal cells. Doxorubicin (DOX) as a model drug was encapsulated into the PEG-OPCL-PEG micelles. Drug release study revealed that the DOX release from micelles was significantly accelerated at mildly acid pH of 5.0 compared to physiological pH of 7.4, suggesting the pH-responsive feature of the drug delivery systems with oxime linkages. Flow cytometry and confocal laser scanning microscopy (CLSM) measurements indicated that these DOX-loaded micelles were easily internalized by living cells. MTT assay against HeLa cancer cells showed DOX-loaded PEG-OPCL-PEG micelles had a high anticancer efficacy. All of these results demonstrate that these polymeric micelles self-assembled from oxime-tethered block copolymers are promising carriers for the pH-triggered intracellular delivery of hydrophobic anticancer drugs.     

Viscoelastic characteristics of protein solutions and the micromechanics of their motion in porous carriers

The interactions of model proteins with porous matrices in biosensors are considered. The viscoelastic properties of casein and albumin were assessed by a dynamic method of a piezoquartz resonator by applying thin layers of the studied solutions to a piezocrystal. The experimental data on the viscoelastic characteristics of protein solutions of various concentrations were compared with the characteristics of their tangential motion in the porous carriers of cellulose nitrate. It was demonstrated that the parameters of dynamic viscosity correlated with the motion time of the protein solutions in the porous polymeric carrier.     

壁蜂生物学特性与传粉服务功能

壁蜂是对蜜蜂总科(Apoidae)切叶蜂科(Megachilidae)壁蜂属(Osmia)的统称,主要分布北半球的温带区域,在自然生态系统和农田生态系统中均发挥着重要的传粉生态服务功能。壁蜂群居筑巢、接受人类提供的巢穴、一年一代的习性,使它们很容易被大量饲养,并且人们可以通过调节其在春节破茧羽化的时间与作物花期同步,为多种目标作物授粉。本文综述壁蜂的生物学和生态学特性、传粉功能及其影响因子,旨在于为发挥壁蜂的传粉功能提供参考。