膜材性质及制备方法调控下的脂质体负载干扰素的研究

依据干扰素(IFN)分子、磷脂分子本身的理化性质和结构特点, 分别用三种制备方法, 以四种脂质体为膜材, 制备IFN脂质体, 考察了不同膜材、不同制备方法对脂质体粒径及包封率的影响. 结果表明, 以二肉豆蔻酰胆碱和二棕榈酰磷脂酰胆碱复合材料为主要膜材, 采用薄膜蒸发法制备的IFN脂质体有良好的稳定性, 60 d内其粒径可以保持在200~350 nm, 包封率可保持30%~40%.     

Intracellular distribution of fluorescent probes delivered by vesicles of different lipidic composition

In order to study mechanisms involved in liposome–cell interaction, this work attempted to assess the influence of vesicle composition on the delivery of liposomal content to Hela cells. In particular, to evaluate pH-sensitive properties and cell interaction of the prepared liposomes, the lipid formulations contained cholesterol (Chol) and they were varied by using phosphatidylcholines with different purity degree: soy lecithin (SL; 80% phosphatidylcholine), a commercial mixture of soy phosphatidylcholine (P90; 90% phosphatidylcholine) or dipalmitoylphosphatidylcholine (DPPC; 99% of purity). A second series of liposomes also contained stearylamine (SA). Dehydration-rehydration vesicles (DRV) were prepared and then sonicated to decrease vesicle size. Vesicle–cell interactions and liposomal uptake were examined by fluorescence microscopy using carboxyfluorescein (CF) and phosphatidylethanolamine-dioleoyl-sulforhodamine B (Rho-PE) as fluorescent markers. Fluorescence dequenching assay was used to study the influence of pH on CF release from the liposomal formulations. Liposome adhesion on the cell surface and internalization were strongly dependent on vesicle bilayer composition. SA vesicles were not endocytosed. DPPC/Chol liposomes were endocytosed but did not release their fluorescent content into the cytosol. SL/Chol and P90/Chol formulations displayed a diffuse cytoplasmic fluorescence of liposomal marker.     

Membrane properties of cationic liposomes composed of dipalmitoylphosphatidylcholine and dipalmityldimethylammonium bromide

Cationic liposomes composed of dipalmitoylphosphatidylcholine (DPPC) and dipalmityldimethylammmonium bromide (DPAB) were prepared by the Bangham method and the effect of DPAB on the membrane properties was examined in terms of liposomal shape, particle size, trapping efficiency, surface potential and dispersibility. The dispersibility of the mixed DPPC/DPAB liposomes (the mole fraction of DPAB (X_DPAB)  0.05) was excellent and the dispersibility was maintained for 6 months, since the zeta-potential of the mixed liposomes was approximately +40 mV. The trapping efficiency of the mixed DPPC/DPAB liposomes (X_DPAB = 0.05) was 10 times greater than that of the DPPC liposomes, and the value was largest among the mixed liposomes (X_DPAB = 0–1.0). Freeze-fracture electron micrographs indicated that the shape of the mixed DPPC/DPAB liposomes (X_DPAB = 0.05) was that of large unilamellar vesicles (LUVs) with a diameter of approximately 2 μm, while the shape of the DPPC liposomes was that of multilamellar vesicles (MLVs). The mixed liposomes had, therefore, a high trapping efficiency. Furthermore, the shape of the mixed DPPC/DPAB liposomes (X_DPAB = 0.75) was also that of LUVs with a diameter of approximately 2 μm and these had a high trapping efficiency. Whereas, the particle size (500 nm) of the mixed DPPC/DPAB liposomes (X_DPAB = 0.25) was smaller than that of the former and had the minimum trapping efficiency. The phase transition temperature of the liposomal bilayer membranes indicated a maximum value at 0.25–0.30 mole fractions of DPAB. These facts were considered to be due to the fact that DPPC and DPAB, whose molar ratio was 7.5:2.5, were tightly packed in the liposomal bilayer membranes and that the curvature of the liposomal particle was resultantly large. Nevertheless, LUVs having a high trapping efficiency were easily obtained by mixing a small amount of DPAB with the DPPC.     

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.     

Characterization of heat-induced interaction of neutral liposome with lipid membrane of Streptomyces griseus cell

The interaction between the neutral 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) liposomes and cell membrane of Streptomyces griseus induced by the heat treatment at specific temperature was investigated, focusing on the internalization of the neutral POPC liposomes with S. griseus cells. In an attempt to clarify the modes of liposome internalization, various kinds of inhibitors of endocytotic pathways were used to treat S. griseus cells. The efficiency of the heat treatment on liposome–cell membrane interactions was finally characterized based on the hydrophobic, electrostatic interactions and hydration effect. In fact, the internalization of the neutral liposomes into these cells was found to show higher rate and greater amount at higher temperatures. The kinetic study showed that the maximum amount of the internalized liposomes was, respectively, 469 × 10⁵ and 643 × 10⁵ liposomes/cell at 37 and 41 °C. The internalization of the neutral liposomes induced by the heat treatment was characterized, implying that the endocytosis occurred. The interactions involving the internalization, adsorption, and fusion of these liposomes with S. griseus cells were mainly contributed by the hydrophobic interaction and the unstable hydrogen bonds caused by the loss of water of surface hydration of cell membrane rather than the electrostatic interaction under the specific heat condition.     

Membrane interactions of ternary phospholipid/cholesterol bilayers and encapsulation efficiencies of a RIP II protein

Membrane interactions of liposomes of ternary phospholipid/cholesterol bilayers are investigated. These interactions lead to discoidal deformations and regular aggregations and are strongly enhanced by the presence of mistletoe lectin (ML), a RIP II type protein. The encapsulation of ML into liposomal nanocapsules is studied with a systematic variation of the lipid composition to monitor its effect on the physical properties: entrapment, mean size, morphology, and stability. Extrusion of multilamellar vesicles through filters 80 nm pore size was used for the generation of liposomes. The mean sizes of liposomes ranged between 120 and 200 nm in diameter with narrow size distributions. The increase in flow rate with pressure for three dioleoylphosphatidylcholine (DOPC)/cholesterol (Chol)/dipalmitoylphosphatidylcholine (DPPC) lipid mixtures was linear and allowed to extrapolate to the minimum burst pressure of the liposomal bilayers. From the minimum pressures P(min), the bilayer lysis tensions gamma(l) were determined. The increase in P(min) and gamma(l) with an increasing content of a saturated phosopholipid (DPPC) indicates that DPPC increases the mechanical strength of lipid bilayers. Apparently, DPPC, like cholesterol, leads to a less compressible surface and a more cohesive membrane. After preparation, vesicle solutions were purified by gel permeation chromatography to separate encapsulated ML from free ML in the extravesicular solution. Purified liposomes were then characterized. The content of entrapped and adsorbed ML was measured using ELISA. Repetitive freezing/thawing cycles prior to extrusion significantly increased ML uptake. On the contrary, adsorption was not affected neither by lipid composition, nor concentration and preparation. Differences in experimental encapsulation efficiency only reflect the differences in the mean vesicle sizes of the different samples as is revealed by a comparison to a theoretical estimate. Cryo-transmission electron microscopy (Cryo-TEM) images show that beside spherical, single-walled liposomes, there is a considerable fraction of discoidally deformed vesicles. Based on our results and those found in the literature, we speculate that the flattening of the vesicles is a consequence of lipid phase separation and the formation of condensed complexes and areas of different bending elasticities. This phenomenon eventually leads to agglomeration of deformed liposomal structures, becoming more pronounced with the increase in the relative amount of saturated fatty acids, presumably caused by hydrophobic interaction. For the same lipid mixture aggregation correlated linearly with the ML content. Finally, tested liposomal samples were kept at 4 degrees C to examine their stability. Only slight fluctuations in diameter and the increase in polydispersity after 3 weeks of storage occurred, with no statistically significant evidence of drug leakage during a time period of 12 days, illustrating physical stability of liposomes.     

Photo-responsive NIR-II biomimetic nanomedicine for efficient cancer-targeted theranostics

In pancreatic cancer, the special barrier system formed by a large number of stromal cells severely hinders drug penetration in deep tumor tissues, resulting in low treatment efficiency. Cell membrane protein-camouflaged liposomal nanomedicines have cancer cell targeting abilities, whereas near-infrared two-zone (NIR-II) fluorescence imaging can achieve deep tissue penetration due to its long light wavelength (1,000–1,700 nm). To combine the cell membrane-based biomimetic technology with NIR-II fluorescence imaging, we constructed a biomimetic nanomedicine (BLIPO-I/D) by camouflaging indocyanine green-doxorubicin (ICG-DOX) liposomes with SW1990 pancreatic cancer cell membrane. The nanomedicine exhibited light-controlled DOX release and high pancreatic cancer treatment efficiency in vitro and in vivo. BLIPO-I/D showed the ability of targeted delivery of a large number of liposomes to pancreatic tumor tissues through homologous targeting of SW1990 cell membranes, which increased the NIR-II fluorescence imaging intensity. Irradiation of the liposomes taken up by pancreatic tumor tissues with near-infrared light (808 nm) triggered the rapid release of DOX from the liposomes, induced the photothermal and photodynamic effects of ICG, which exerted anti-tumor effects. Therefore, the fabricated biomimetic liposomal nanomedicine BLIPO-I/D is expected to achieve precise theranostics of pancreatic cancer.     

Disulfide‐Unit Conjugation Enables Ultrafast Cytosolic Internalization of Antisense DNA and siRNA

Development of intracellular delivery methods for antisense DNA and siRNA is important. Previously reported methods using liposomes or receptor‐ligands take several hours or more to deliver oligonucleotides to the cytoplasm due to their retention in endosomes. Oligonucleotides modified with low molecular weight disulfide units at a terminus reach the cytoplasm 10 minutes after administration to cultured cells. This rapid cytoplasmic internalization of disulfide‐modified oligonucleotides suggests the existence of an uptake pathway other than endocytosis. Mechanistic analysis revealed that the modified oligonucleotides are efficiently internalized into the cytoplasm through disulfide exchange reactions with the thiol groups on the cellular surface. This approach solves several critical problems with the currently available methods for enhancing cellular uptake of oligonucleotides and may be an effective approach in the medicinal application of antisense DNA and siRNA.     

Metallomics in drug development: characterization of a liposomal cisplatin drug formulation in human plasma by CE–ICP–MS

A capillary electrophoresis inductively coupled plasma mass spectrometry method for separation of free cisplatin from liposome-encapsulated cisplatin and protein-bound cisplatin was developed. A liposomal formulation of cisplatin based on PEGylated liposomes was used as model drug formulation. The effect of human plasma matrix on the analysis of liposome-encapsulated cisplatin and intact cisplatin was studied. The presence of 1 % of dextran and 4 mM of sodium dodecyl sulfate in HEPES buffer was demonstrated to be effective in improving the separation of liposomes and cisplatin bound to proteins in plasma. A detection limit of 41 ng/mL of platinum and a precision of 2.1 % (for 10 μg/mL of cisplatin standard) were obtained. Simultaneous measurements of phosphorous and platinum allows the simultaneous monitoring of the liposomes, liposome-encapsulated cisplatin, free cisplatin and cisplatin bound to plasma constituents in plasma samples. It was demonstrated that this approach is suitable for studies of the stability of liposome formulations as leakage of active drug from the liposomes and subsequent binding to biomolecules in plasma can be monitored. This methodology has not been reported before and will improve characterization of liposomal drugs during drug development and in studies on kinetics.

Effect of TRX-liposomes size on their prolonged circulation in rats

Newly formulated cationic liposomes (TRX-liposomes) with four different mean diameters were injected into twelve male rats via the lateral tail vein in order to evaluate the effect of liposomal size on pharmacokinetic parameters. TRX-liposomes disappeared from the blood according to the one-compartment model and demonstrated maximum and minimum half-lives of ca. 14 h (mean diameter of 114.3 nm) and ca. 5 h (mean diameter of 285.9 nm), respectively. This prolonged half-life tended to decrease at the boundary of 114.3 nm mean diameter. The optimal size (114.3 nm) for prolonged circulation of TRX-liposomes was consistent with that of pegylated liposomes such as Doxil((R)), however, the half-life was different among these liposomes. The electric charge of the TRX-liposomal surface is assumed to be responsible for this difference. The results of the present study will be very useful in the design of long-circulating cationic liposomes.     

Synthesis, evaluation and incorporation into liposomes of 4-functionalised-2,5-diphenyloxazole derivatives for application in scintillation proximity assays

The preparation of a series of 4-functionalised-2,5-diphenyloxazoles is described. The scintillating efficiency of each of these ‘scintilipid’ molecules has been evaluated in the presence of ionising radiation. Each ‘scintilipid’ has been assessed for the ability to assemble, with other lipids, into liposomes, under a variety of preparative conditions. Each liposomal preparation has been monitored for the ability to scintillate in the presence of ionising radiation. The optimal ‘scintilipid’, both in terms of effective liposomal formation and scintillation efficiency, has been determined.     

Multifunctional magnetic calcium phosphate nanoparticles for targeted platin delivery

Magnetic mesoporous amorphous calcium phosphate nanoparticles with a size of 62 nm and abundant -COOH groups on the surface have been prepared by a simple method. The particles show excellent aqueous dispersion stability in physiological pH without any deterioration in hydrodynamic size and zetapotential. By virtue of the carboxylate groups on the surface, the platinum pharmacophore cis-diaquadiamine platinum(ii), folic acid and rhodamine isothiocyanate were conjugated on these magnetic calcium phosphate nanoparticles. The cytotoxicity and internalization efficiency of these nanocarriers have been evaluated on folate receptor overexpressed HeLa cells. These drug loaded nanoagents exhibit elevated cytotoxicity and induce apoptosis in HeLa cells.     

DNA-passivated CdS nanocrystals: luminescence, bioimaging, and toxicity profiles

A class of luminescent semiconductor quantum dots is described that exhibit low cellular toxicity without the use of bulky surface coatings. Nucleic acids, either in the form of mononucleotides or DNA oligonucleotides, are used as a ligand system in the aqueous synthesis of CdS nanocrystals. The materials produced exhibit diameters on the order of 4 nm and luminescence in the range of 500-700 nm. Importantly, DNA-CdS is stable in buffers of high ionic strength for many hours. When tested for toxicity in HeLa cells, minimal decreases in cell viability were observed, indicating that the DNA-CdS nanocrystals are highly stable in biological media. Uptake of the nanocrystals into unfixed mammalian cells was tested, and internalization was observed. The results reported indicate that the use of DNA as a ligand system for water-soluble semiconductor nanocrystals represents a worthwhile strategy for the production of new biological imaging agents.     

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.     

Topographic, electrochemical, and optical images captured using standing approach mode scanning electrochemical/optical microscopy

We developed a high-resolution scanning electrochemical microscope (SECM) for the characterization of various biological materials. Electrode probes were fabricated by Ti/Pt sputtering followed by parylene C-vapor deposition polymerization on the pulled optical fiber or glass capillary. The effective electrode radius estimated from the cyclic voltammogram of ferrocyanide was found to be 35 nm. The optical aperture size was less than 170 nm, which was confirmed from the cross section of the near-field scanning optical microscope (NSOM) image of the quantum dot (QD) particles with diameters in the range of 10-15 nm. The feedback mechanism controlling the probe-sample distance was improved by vertically moving the probe by 0.1-3 microm to reduce the damage to the samples. This feedback mode, defined as "standing approach (STA) mode" (Yamada, H.; Fukumoto, H.; Yokoyama, T.; Koike, T. Anal. Chem. 2005, 77, 1785-1790), has allowed the simultaneous electrochemical and topographic imaging of the axons and cell body of a single PC12 cell under physiological conditions for the first time. STA-mode feedback imaging functions better than tip-sample regulation by the conventionally available AFM. For example, polystyrene beads (diameter approximately 6 microm) was imaged using the STA-mode SECM, whereas imaging was not possible using a conventional AFM instrument.     

Uptake of amitriptyline and nortriptyline with liposomes, proteins, and serum: implications for drug detoxification

Liposomes composed of DOPG and DMPC were studied for their ability to sequester amitriptyline and nortriptyline under physiological conditions. The liposomes reduced the free drug concentration in protein mixtures and in human serum, but the drug uptake efficiency of liposomes was reduced in the presence of plasma proteins, perhaps due to adsorption of proteins on the liposomes. The reduction was significantly more for the pure DOPG liposomes. The 50:50 DMPC:DOPG liposomes (0.72 mg lipid/mL) reduced the free amitriptyline concentration by 50-60% in the presence of 7% proteins (4% albumin (w/w), 2% fibrinogen (w/w), 1% globulins (w/w)). In human serum, the free drug reduction was 35-70% with the same 50:50 liposomes (0.72 mg lipid/mL). The liposomal systems were equally efficient at sequestering nortriptyline, which is a major metabolite of amitriptyline. The drug binding to liposomes in the presence of serum proteins is also quick and reversible and the likely mechanism of drug sequestration is adsorption of drug on the surface of liposomes. Accordingly, the drug uptake increases with increased charge and lipid loading. Even though the serum proteins reduced the effectiveness of the liposomes at sequestering the drug, the 50:50 DMPC:DOPG liposomes may be effective at treating amitriptyline overdose patients.     

Characterization of defects and surface structures in microporous materials by HRTEM, HRSEM, and AFM.

High-resolution transmission (HRTEM) and high-resolution scanning electron microscopy as well as atomic force microscopy (AFM), X-ray diffraction, and electron diffraction were used for studying the zeolites MFI, MEL, and the MFIMEL intergrowth system. All three zeolites consisted of individual particles having a size in the range of approximately 0.5 m to 5 m. The particle habits varied from rather cubelike to almost spherelike with many intermediate habits. Typically, the particles of these three zeolites were assembled by many individual blocks that differed in the dimension from about 25 nm to 140 nm as well as in the shape from very frequently almost rectangular (for MFI, MEL, and MFIMEL) to sometimes roundish or irregular habits (mainly for MFIMEL). An estimate shows that some 104 up to more than 106 densely packed blocks typically may assemble each individual zeolite particle or, related to the corresponding unit cell dimension, about 108 up to 1010 unit cells. The fine surface structure of zeolite particles was terracelike with steps between adjacent terraces typically in the range of 20 nm to 60 nm; the minimum step measured was approximately 4 nm. A detailed study of the surface topography was performed by AFM, detecting organic molecules at the block intersections. The presence of topological defects was observed by HRTEM and electron diffraction.     

PR_b-targeted PEGylated liposomes for prostate cancer therapy

In recent years, there has been considerable effort in designing improved delivery systems by including site-directed surface ligands to further enhance their selective targeting. The goal of this study is to engineer alpha5beta1-targeted stealth liposomes (nanoparticles covered with poly(ethylene glycol) (PEG)) that will bind to alpha5beta1-expressing LNCaP human prostate cancer cells and efficiently release the encapsulated load intracellularly. For this purpose, liposomes (with and without PEG2000) were functionalized with a fibronectin-mimetic peptide (PR_b) and delivered to LNCaPs. The amount of PEG2000 and other liposomal components were characterized by 1H NMR, and the amount of peptide by the bicinchoninic acid protein assay. Fibronectin is the natural ligand for alpha5beta1, and a promising design for a fibronectinmimetic peptide includes both the primary binding site (RGD) and the synergy site (PHSRN) connected by a linker and extended off a surface by a spacer. We have previously designed a peptide-amphiphile, PRb, that employed a hydrophobic tail, connected to the N-terminus of a peptide headgroup composed of a spacer, the synergy site sequence, a linker mimicking both the distance and hydrophobicity/hydrophilicity present in the native protein fibronectin (thus presenting an overall "neutral" linker), and finally the primary binding sequence. We have examined different liposomal formulations, functionalized only with PR_b or with PR_b and PEG2000. For PR_b-targeted PEGylated liposomes, efficient cell binding was observed for peptide concentrations of 2 mol % and higher. When compared to GRGDSP-targeted stealth liposomes, PR_b functionalization was superior to that of GRGDSP as shown by increased LNCaP binding, internalization efficiency, as well as cytotoxicity after incubation of LNCaPs with tumor necrosis factor-alpha (TNFalpha)-encapsulated liposomes. More importantly, PR_b is alpha5beta1-specific, whereas many integrins bind to small RGD peptides. Thus, the proposed PR_b-targeted delivery system has the potential to deliver a therapeutic payload to prostate cancer cells in an efficient and specific manner.     

Liposomal structure: A comparative study on light scattering and chromatography techniques

Liposomes play an important role in medical and pharmaceutical science as, nanoscale drug carriers. One of the most important features is their size and size distribution, influencing both their bio-distribution and their targeting efficiency to tumors and also therapeutic activity of liposomal antitumor drugs. In this study, the effect of preparation method and molecular interaction on size and shape of liposome was studied. The size and shape characterization of liposomes was performed by asymmetrical flow field-flow fractionation coupled online with multi-angle light scattering (AF4-MALS). The size distributions obtained by AF4-MALS were compared to mean particle sizes and size distribution measured with other standard method such as Photon Correlation Spectroscopy (PCS). Furthermore, the effect of molecular interaction (hydrophilic and hydrophobic model drugs) on liposomal structure was assessed.