拉链型脂肽的温控开关效应

亮氨酸拉链型脂肽是由两条肽链以螺旋结构依靠疏水作用并列结合形成的二聚体,当温度升至其相变温度时,其螺旋结构解旋继而变为无序链状结构。利用该类脂肽的温敏性能,本文设计、合成得到一组具有温敏性的拉链型脂肽,将其与磷脂混合制备温敏性脂质体。用圆二色谱测定磷脂双分子层上脂肽的二级结构,动态光散射测定脂肽-脂质体的粒径及电位;荧光偏振法测定脂质体膜的流动性;采用紫外分光光度计考察阿霉素(DOX)在37.0、45.0°C下的释放行为。结果表明,含有脂肽的脂质体具备较好的温敏性,胆固醇含量、脂质体膜的流动性,对脂肽的温控开关效应有一定的影响。脂肽-脂质体作为一种新型的温敏性药物载体展现了其较好的应用前景。     

Use of Edman degradation sequence analysis and matrix-assisted laser desorption/ionization mass spectrometry in designing substrates for matrix metalloproteinases

The matrix metalloproteinase (MMP) family has been implicated in the process of a variety of diseases such as arthritis, atherosclerosis, and tumor cell metastasis. We have been designing single-stranded peptides (SSPs) and triple-helical peptides (THPs) as potential discriminatory MMP substrates. Edman degradation sequence and matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analyses of proteolytic activity have been utilized to aid in further substrate design. THP models of the alpha1(I)772-786 sequence from type I collagen were synthesized to examine the triple-helical substrate specificity of MMP family members. Sequence and MALDI-MS analyses were used in conjunction with a fluorometric assay to determine the exact point of cleavage by each MMP. MMP-1 (interstitial collagenase) cleaved the substrates at a single Gly-Ile bond, analogous to the cleavage site in type I collagen. MMP-2 (Mr 72 000 type IV collagenase; gelatinase A) was found to cleave the substrates at two sites, a Gly-Ile bond and a Gly-Gln bond. MMP-3 (stromelysin 1) was found to cleave only one of the substrates after reaction for 48 h. Ultimately, sequence and MALDI-MS analyses allowed us to detect an additional cleavage site for MMP-2 in comparison to MMP-1, while MMP-3 was found to cleave a substrate after an extended time period. The second cleavage site would cause the kinetic parameters for MMP-2 to be overestimated by the fluorometric assay. Further design variations for these substrates need to consider the presence of more stable triple-helical conformation (to eliminate MMP-3 binding) and the removal of Gly-Gln bonds that may be susceptible to MMP-2.     

Recognition and catabolism of synthetic heterotrimeric collagen peptides by matrix metalloproteinases

BACKGROUND: The general consensus is that interstitial collagens are digested by collagenases and denatured collagen by gelatinases, although processing of fibrillar and acetic-acid-soluble collagen by gelatinase A has also been reported. One of the main difficulties in studying the mechanism of action of these matrix metalloproteinases (MMPs) derives from the physicochemical properties of the natural triple-helical collagen, which makes it difficult to handle. RESULTS: Synthetic heterotrimeric collagenous peptides that contain the collagenase cleavage site of human collagen type I and differ in the thermal stability of the triple-helical fold were used to mimic natural collagen and gelatin, respectively. Results from digestion of these substrates by fibroblast and neutrophil collagenases (MMP-1 and MMP-8), as well as by gelatinase A (MMP-2), confirmed that the two classes of enzymes operate within the context of strong conformational dependency of the substrates. It was also found that gelatinases and collagenases exhibit two distinct proteolytic mechanisms: gelatinase digests the gelatin-like heterotrimer rapidly in individual steps with intermediate releases of partially processed substrate into the medium, whereas collagenases degrade the triple-helical heterotrimer by trapping it until scission through all three alpha chains is achieved. CONCLUSIONS: The results confirm the usefulness of synthetic heterotrimeric collagenous peptides in the folded and unfolded state as mimics of the natural substrates collagen and gelatin, respectively, to gain a better a insight into the proteolytic mechanisms of matrix metalloproteinases.     

Interaction of amphipathic model lipopeptides with phospholipid bilayers.

In order to investigate the conformation and localization of lipopeptides in lipid bilayers, a basic model peptide with a long alkyl chain, Ac-Ser-Val-Lys-Amy-Ser-Trp-Lys-Val-NHCH3 Amy-1; Amy = alpha-aminomyristic acid) was synthesized. Its interaction with neutral and acidic phospholipid bilayers was studied by circular dichroism (CD) spectroscopy, dye leakage and fluorescence measurements. Another peptide, Ac-Leu-Ala-Arg-Leu-Trp-Amy-Arg-Leu-Leu-Ala-Arg-Leu-NHCH3 (Amy-2), which was prepared previously, was used for comparison. The CD data indicated that Amy-1 took a beta-turn and/or a beta-structure in the absence and presence of liposomes. Amy-2 formed a beta-structure in aqueous solution and an alpha-helical structure in liposomes. The dye leakage ability of Amy-1 was much weaker than that of Amy-2. Fluorescence spectroscopic data suggest that the peptides are immersed in lipid bilayers. Based on these results, discussion is made in terms of localization of the peptides in lipid bilayers.     

Formulation of photocleavable liposomes and the mechanism of their content release

In pursuit of designing photocleavable liposomes as drug delivery vehicles, we synthesized several amphiphilic lipids by connecting stearyl amine (as the non-polar tail) and charged amino acids (as polar heads) via the o-nitrobenzyl derivatives. The lipids containing Glu, Asp, and Lys amino acids were subjected to photocleavage reaction by UV light, and the overall spectral changes of the chromophoric o-nitrobenzyl conjugates were determined as a function of time. The experimental data revealed that the feasibility of the cleavage reaction, nature and magnitude of the spectral changes during the course of the cleavage reaction, and their overall kinetic profiles were dictated by the type of amino acid constituting the polar head groups. The cleavage reactions of the Asp and Glu containing lipids were found to be more facile than that of the lysine-containing lipid. Using these lipids, we formulated photocleavable liposomes, and investigated the photo-triggered release of an encapsulated (within the liposomal lumen) dye as a function of time. The kinetic data revealed that the release of the liposomal content conformed to a two-step mechanism, of which the first (fast) step involved the photocleavage of lipids followed by the slow release of the liposomal content during the second step. The overall mechanistic features intrinsic to the photocleavage of Asp, Glu and Lys containing o-nitrobenzyl conjugated lipids, and their potential applications in formulating liposomes (whose contents can be "unloaded" by the UV light) as drug delivery vehicles are discussed.     

亮氨酸拉链型脂肽对脂质体温敏性调节的分子模拟

含亮氨酸拉链型脂肽的温敏性脂质体被认为是抗癌药物的优良载体。亮氨酸拉链型脂肽的主要氨基酸残基序列为[VAQLEVK-VAQLESK-VSKLESK-VSSLESK],嵌入脂质体后可以有效改善脂质体的温敏性。本文首先采用隐式溶剂副本交换分子动力学方法,对N端修饰的亮氨酸拉链单链的折叠状态进行了模拟,得到了亮氨酸拉链单链的转变温度。并对包含该种新型亮氨酸拉链型脂肽的DPPC脂质体进行常规分子动力学模拟,研究了2种不同头基的亮氨酸拉链型脂肽(ALA,C3CO)二聚体嵌入后DPPC脂质体的相转变温度变化,证明了亮氨酸拉链型脂肽对于该脂质体温敏性的控制作用。利用这一规律,可以对亮氨酸拉链型脂肽进行优化改良,得到效果更佳的温敏脂质体,对于抗癌药物载体的开发有着重要的意义。     

A General Approach to Enzyme-Responsive Liposomes

Liposomes are effective nanocarriers due to their ability to deliver encapsulated drugs to diseased cells. Nevertheless, liposome delivery would be improved by enhancing the ability to control the release of contents at the target site. While various stimuli have been explored for triggering liposome release, enzymes provide excellent targets due to their common overexpression in diseased cells. We present a general approach to enzyme-responsive liposomes exploiting targets that are commonly aberrant in disease, including esterases, phosphatases, and β-galactosidases. Responsive lipids correlating with each enzyme family were designed and synthesized bearing an enzyme substrate moiety attached via a self-immolating linker to a non-bilayer lipid scaffold, such that enzymatic hydrolysis triggers lipid decomposition to disrupt membrane integrity and release contents. Liposome dye leakage assays demonstrated that each enzyme-responsive liposome yielded significant content release upon enzymatic treatment compared to minimal release in controls. Results also showed that fine-tuning liposome composition was critical for controlling release. DLS analysis showed particle size increases in the cases of esterase- and β-galactosidase-responsive lipids, supporting alterations to membrane properties. These results showcase an effective modular strategy that can be tailored to target different enzymes, providing a promising new avenue for advancing liposomal drug delivery.     

Triple-helical transition state analogues: a new class of selective matrix metalloproteinase inhibitors

Alterations in activities of one family of proteases, the matrix metalloproteinases (MMPs), have been implicated in primary and metastatic tumor growth, angiogenesis, and pathological degradation of extracellular matrix (ECM) components, such as collagen and laminin. Since hydrolysis of the collagen triple-helix is one of the committed steps in ECM turnover, we envisioned modulation of collagenolytic activity as a strategy for creating selective MMP inhibitors. In the present study, a phosphinate transition state analogue has been incorporated within a triple-helical peptide template. The template sequence was based on the alpha1(V)436-450 collagen region, which is hydrolyzed at the Gly(439)-Val(440) bond selectively by MMP-2 and MMP-9. The phosphinate acts as a tetrahedral transition state analogue, which mimics the water-bound peptide bond of a protein substrate during hydrolysis. The phosphinate replaced the amide bond between Gly-Val in the P1-P1' subsites of the triple-helical peptide. Inhibition studies revealed Ki values in the low nanomolar range for MMP-2 and MMP-9 and low to middle micromolar range for MMP-8 and MMP-13. MMP-1, MMP-3, and MT1-MMP/MMP-14 were not inhibited effectively. Melting of the triple-helix resulted in a decrease in inhibitor affinity for MMP-2. The phosphinate triple-helical transition state analogue has high affinity and selectivity for the gelatinases (MMP-2 and MMP-9) and represents a new class of protease inhibitors that maximizes potential selectivity via interactions with both prime and nonprime active site subsites as well as with secondary binding sites (exosites).     

"Uncorking" of liposomes by matrix metalloproteinase-9

A triggered release methodology of liposomal contents via the enzyme MMP-9 is described.     

Detachable immobilization of liposomes on polymer gel particles

Immobilization of liposomes on hydrophobized Sephacryl gel and controlled detachment of the liposomes from the gel were examined. The gel was chemically modified and bore octyl, hexadecyl or cholesteryl moiety via disulfide linkage as anchors to liposomal bilayer membrane. Upon interaction with the gel, egg phosphatidylcholine liposomes were successfully immobilized onto the gel. The gel with cholesteryl moiety showed 1.7 times higher liposome immobilization per anchor moiety than the gels with the alkyl moieties. The immobilization of liposomes on the gel was stable, and no significant spontaneous detachment of phospholipid or leakage of fluorescein isothiocyanate-conjugated dextran encapsulated in the immobilized liposomes was observed in 24h. Reductive cleavage of the disulfide linkage by dithiothreitol resulted in detachment of the liposomes from the gel. The majority of the detached liposomes were found encapsulating the dextran derivative, and these liposomes should have kept their structural integrity throughout the immobilization and the detachment processes. The release of the liposomes was insignificant until the ratio of the dithiothreitol to the hydrophobic anchor reached a threshold. The presence of the threshold suggests that the immobilization of liposomes should require a certain minimum number of the hydrophobic moieties anchored in the liposomal membrane. By applying the present immobilization-detachment system, preparation of liposomes encapsulating the dextran derivative without using costly gel filtration or ultracentrifugation procedure was successfully demonstrated.     

Redox-triggered contents release from liposomes

An exciting new direction in responsive liposome research is endogenous triggering of liposomal payload release by overexpressed enzyme activity in affected tissues and offers the unique possibility of active and site-specific release. Bringing to fruition the fully expected capabilities of this new class of triggered liposomal delivery system requires a collection of liposome systems that respond to different upregulated enzymes; however, a relatively small number currently exist. Here we show that stable, approximately 100 nm diameter liposomes can be made from previously unreported quinone-dioleoyl phosphatidylethanolamine (Q-DOPE) lipids, and complete payload release (quenched fluorescent dye) from Q-DOPE liposomes occurs upon their redox activation when the quinone headgroup possesses specific substituents. The key component of the triggerable, contents-releasing Q-DOPE liposomes is a "trimethyl-locked" quinone redox switch attached to the N-terminus of DOPE lipids that undergoes a cleavage event upon two-electron reduction. Payload release by aggregation and leakage of "uncapped" Q-DOPE liposomes is supported by results from liposomes wherein deliberate alteration of the "trimethyl-locked" switch completely deactivates the redox-destructible phenomena (liposome opening). We expect that Q-DOPE liposomes and their variants will be important in treatment of diseases with associated tissues that overexpress quinone reductases, such as cancers and inflammatory diseases, because the quinone redox switch is a known substrate for this group of reductases.     

Targeted drug delivery utilizing protein-like molecular architecture

Nanotechnology-based drug delivery systems (nanoDDSs) have seen recent popularity due to their favorable physical, chemical, and biological properties, and great efforts have been made to target nanoDDSs to specific cellular receptors. CD44/chondroitin sulfate proteoglycan (CSPG) is among the receptors overexpressed in metastatic melanoma, and the sequence to which it binds within the type IV collagen triple-helix has been identified. A triple-helical "peptide-amphiphile" (alpha1(IV)1263-1277 PA), which binds CD44/CSPG, has been constructed and incorporated into liposomes of differing lipid compositions. Liposomes containing distearoyl phosphatidylcholine (DSPC) as the major bilayer component, in combination with distearoyl phosphatidylglycerol (DSPG) and cholesterol, were more stable than analogous liposomes containing dipalmitoyl phosphatidylcholine (DPPC) instead of DSPC. When dilauroyl phosphatidylcholine (DLPC):DSPG:cholesterol liposomes were prepared, monotectic behavior was observed. The presence of the alpha1(IV)1263-1277 PA conferred greater stability to the DPPC liposomal systems and did not affect the stability of the DSPC liposomes. A positive correlation was observed for cellular fluorophore delivery by the alpha1(IV)1263-1277 PA liposomes and CD44/CSPG receptor content in metastatic melanoma and fibroblast cell lines. Conversely, nontargeted liposomes delivered minimal fluorophore to these cells regardless of the CD44/CSPG receptor content. When metastatic melanoma cells and fibroblasts were treated with exogeneous alpha1(IV)1263-1277, prior to incubation with alpha1(IV)1263-1277 PA liposomes, to potentially disrupt receptor/liposome interactions, a dose-dependent decrease in the amount of fluorophore delivered was observed. Overall, our results suggest that PA-targeted liposomes can be constructed and rationally fine-tuned for drug delivery applications based on lipid composition. The selectivity of alpha1(IV)1263-1277 PA liposomes for CD44/CSPG-containing cells represents a targeted-nanoDDS with potential for further development and application.     

Sterol-modified phospholipids: cholesterol and phospholipid chimeras with improved biomembrane properties

We synthesized a family of sterol-modified glycerophospholipids (SML) in which the sn-1 or sn-2 position is covalently attached to cholesterol and the alternative position contains an aliphatic chain. The SML were used to explore how anchoring cholesterol to a phospholipid affects cholesterol behavior in a bilayer. Notably, cholesterol in the SML retains the membrane condensing properties of free cholesterol regardless of the chemistry or position of its attachment to the glycerol moiety of the phospholipid. SMLs by themselves formed liposomes upon hydration and in mixtures between an SML and diacylglycerophospholipids (C14 to C18 chain length) the thermotropic phase transition is eliminated at the SML equivalent of about 30 mol % free cholesterol. Osmotic-induced contents leakage from SML (C14-C18) liposomes depends upon the linkage and position of cholesterol but in general is similar to that observed in 3/2 diacylphosphatidylcholine/cholesterol (mole ratio) liposomes. SML liposomes are exceptionally resistant to contents release in the presence of serum at 37 degrees C. This is probably due to the fact that SML exchange between bilayers is more than 100 fold less than the exchange rate of free cholesterol in the same conditions. Importantly, SML liposomes containing doxorubicin are as effective in treating the murine C26 colon carcinoma as Doxil, a commercial liposome doxorubicin formulation. SMLs stabilize bilayers but do not exchange and hence provide a new tool for biophysical studies on membranes. They may improve liposomal drug delivery in organs predisposed to the extraction of free cholesterol from bilayers, such as the skin, lung, or blood.     

Triggered Liposomal Release through a Synthetic Phosphatidylcholine Analogue Bearing a Photocleavable Moiety Embedded within the sn‐2 Acyl Chain

Liposomes represent promising carriers for drug delivery applications. To maximize this potential, there has been significant interest in developing liposomal systems encapsulating molecular cargo that are highly stable until their contents are released remotely in a controlled manner. Herein, we describe the design, synthesis, and analysis of a photocleavable analogue of the ubiquitous lipid phosphoatidylcholine (PC) for the development of highly stable and controllable photodisruptable membranes. Our strategy was to develop a lipid that closely mimics the structure of PC to optimize favorable properties including biocompatibility and stability of subsequent liposomes when mixed with lipids possessing a broad range of physicochemical properties. Thus, NB‐PC was designed, which contains a photocleavable 2‐nitrobenzyl group embedded within the acyl chain at the sn‐2 position. Following the synthesis of NB‐PC , liposome disruption efficacy was evaluated through photolysis studies involving the detection of nile red release. Studies performed using a range of liposomes with different percentages of NB‐PC , PC, phosphatidylethanolamine (PE), cholesterol, and polyethylene glycol‐PE (PEG‐PE) demonstrated minimal background release in controls, release efficacies that correlate directly with the amount of NB‐PC incorporation, and that release is only minimally impacted by the inclusion of the lipids PE and cholesterol that possess disparate properties. These results demonstrate that the NB‐PC system is a highly stable, flexible, and tunable system for photoinitiated release from liposomal systems.     

Chip gel electrophoresis as a tool for study of matrix metalloproteinase 9 interaction with metallothionein

Matrix metalloproteinases (MMPs) play numerous roles in physiological and pathological processes including cancer. Interactions of MMPs with other zinc-binding proteins are of great interest mainly from the point of view of the participation of these proteins in carcinogenesis. The aim of this study was to utilize chip gel electrophoresis for investigation of matrix metalloproteinase 9 (gelatinase B, MMP-9) interactions with collagen. The interaction was observed and the effect of temperature was tested. We further focused on the study of activation of MMP-9 by the presence of zinc-binding proteins called metallothioneins (MT). We confirmed the basic presumption of the activation of MMP-9 by the presence of MT similar to the temperature effect. Moreover, we chose the method of differential pulse voltammetry Brdicka reaction to verify our results. It can be concluded that the activity of MMP-9 was higher in the presence of MT in comparison with the experimental collagen(type I)-MMP-9 mixture in vitro.     

Bispidinone-based molecular switches for construction of stimulus-sensitive liposomal containers

It is demonstrated that new lipid-like amphiphilic compounds, derivatives of 3,7-diazabicyclo[3.3.1]nonan-9-one (bispidinone) with long alkyl substituents, can be integrated into liposomal membranes. They can serve as molecular switches changing the conformation from the chair–boat to chair–chair on addition of an aqueous solution of a bivalent copper salt, and thus enhancing the permeability of the lipid bilayer of liposomes and the release of encapsulated compounds.     

Reactive Oxygen Species (ROS) Activated Liposomal Cell Delivery using a Boronate-Caged Guanidine Lipid

We report boronate-caged guanidine-lipid 1 that activates liposomes for cellular delivery only upon uncaging of this compound by reactive oxygen species (ROS) to produce cationic lipid products. These liposomes are designed to mimic the exceptional cell delivery properties of cell-penetrating peptides (CPPs), while the inclusion of the boronate cage is designed to enhance selectivity such that cell entry will only be activated in the presence of ROS. Boronate uncaging by hydrogen peroxide was verified by mass spectrometry and zeta potential (ZP) measurements. A microplate-based fluorescence assay was developed to study the ROS-mediated vesicle interactions between 1 -liposomes and anionic membranes, which were further elucidated via dynamic light scattering (DLS) analysis. Cellular delivery studies utilizing fluorescence microscopy demonstrated significant enhancements in cellular delivery only when 1 -liposomes were incubated with hydrogen peroxide. Our results showcase that lipid 1 exhibits strong potential as an ROS-responsive liposomal platform for targeted drug delivery applications.     

Controlling liposomal drug release with low frequency ultrasound: mechanism and feasibility

The ability of low-frequency ultrasound (LFUS) to release encapsulated drugs from sterically stabilized liposomes in a controlled manner was demonstrated. Three liposomal formulations having identical lipid bilayer compositions and a similar size ( approximately 100 nm) but differing in their encapsulated drugs and methods of drug loading have been tested. Two of the drugs, doxorubicin and methylpredinisolone hemisuccinate, were remote loaded by transmembrane gradients (ammonium sulfate and calcium acetate, respectively). The third drug, cisplatin, was loaded passively into the liposomes. For all three formulations, a short exposure to LFUS (<3 min) released nearly 80% of the drug. The magnitude of drug release was a function of LFUS amplitude and actual exposure time, irrespective of whether irradiation was pulsed or continuous. Furthermore, no change in liposome size distribution or in the chemical properties of the lipids or of the released drugs occurred due to exposure to LFUS. Based on our results, we propose that the mechanism of release is a transient introduction of porelike defects in the liposome membrane, which occurs only during exposure to LFUS, after which the membrane reseals. This explains the observed uptake of the membrane-impermeable fluorophore pyranine from the extraliposomal medium during exposure to LFUS. The implications of these findings for clinical applications of controlled drug release from liposomes are discussed.     

A new type of pH-sensitive phospholipid

We describe the synthesis and characterization of a type of pH-sensitive pentaerythritol phospholipids, using a trialkoxybenzylidene acetal as the acid-labile moiety. This lipid was prepared by an eight-step synthesis via a latentiation strategy. Liposomes were prepared via the thin film extrusion method. The changes of liposomal sizes were measured by dynamic light scattering. Content release rates of the liposomes as a function of pH were monitored by using a calcein fluorescence dequenching assay. These results indicated that this new liposomal system was capable of releasing its contents under mildly acidic conditions. At last, in vitro cytotoxicity was assayed against three cell lines, suggesting this type of phospholipids was low toxic.