Influence of Particle Size on the Distributions of Liposomes to Atherosclerotic Lesions in Mice

ABSTRACT

In order to confirm the efficacy of liposomes as a drug carrier for atherosclerotic therapy, the influence of particle size on the distribution of liposomes to atherosclerotic lesions in mice was investigated. In brief, liposomes of three different particle sizes (500, 200, and 70 nm) were prepared, and the uptake of liposomes by the macrophages and foam cells in vitro and the biodistributions of liposomes administered intravenously to atherogenic mice in vivo were examined. The uptake by the macrophages and foam cells increased with the increase in particle size. Although the elimination rate from the blood circulation and the hepatic and splenic distribution increased with the increase in particle size in atherogenic mice, the aortic distribution was independent of the particle size. The aortic distribution of 200 nm liposomes was the highest in comparison with the other sizes. Surprisingly, the aortic distribution of liposomes in vivo did not correspond with the uptake by macrophages and foam cells in vitro. These results suggest that there is an optimal size for the distribution of liposomes to atherosclerotic lesions.     

Effect of surface-mannose modification on aerosolized liposomal delivery to alveolar macrophages

Purpose: The effect of surface-mannose modification on aerosolized liposomal delivery to alveolar macrophages (AMs) was evaluated in vitro and in vivo. Method: 4-Aminophenyl-α-D-mannopyranoside (Man) was used for surface-mannose modification, and mannosylated liposomes with various mannosylation rates (particle size: 1000 nm) were prepared. Results: In the in vitro uptake experiments, the uptake of mannosylated liposomes by AMs was increased with the increase in the mannosylation rate over the range 2.4–9.1 mol% Man and became constant at over 9.1%. Thus, the most efficient mannosylation rate was 9.1 mol% Man. Furthermore, free mannose inhibited the uptake of mannosylated liposomes by AMs. This indicates that the uptake mechanism of mannosylated liposomes by AMs is mannose receptor-mediated endocytosis. In the in vivo animal experiments, the mannosylated liposomes (mannosylation rate, 9.1 mol% Man) were more efficiently delivered to AMs after pulmonary aerosolization to rats than nonmodified liposomes and did not harm lung tissues. Conclusion: These results indicate that surface-mannose modification is useful for efficient aerosolized liposomal delivery to AMs.     

Liposome size analysis by dynamic/static light scattering upon size exclusion-/field flow-fractionation

The aim of the current study was to analyse the particle size distribution of a liposome dispersion, which contained small egg phosphatidylcholine vesicles and had been prepared by high-pressure homogenisation, by various size analysis techniques. Such liposomes were chosen since they can be looked at as a prototype of drug nano-carriers. Three sub-micron particle size analysis techniques were employed: (1) fixed-angle quasi-elastic laser light scattering or photon correlation spectroscopy (PCS), (2) size exclusion chromatographic (SEC) fractionation with subsequent (off-line) PCS size-analysis and quantification of the amount of particles present in the sub-fractions, and (3) field-flow-fractionation coupled on-line with a static light scattering and a refractive index (RI)-detector. When designing liposome-based drug carrier systems, a reliable and reproducible analysis of their size and size distribution is of paramount importance: Not only does liposome size influence the nanocarrier's in-vitro characteristics such as drug loading capacity, aggregation and sedimentation but also it is generally acknowledged that the pharmacokinetic behaviour and biodistribution of the carrier is strongly size-dependent. All three approaches of liposome size analysis used here were found to yield useful results, although they were not fully congruent. PCS indicated either a broad, mono-modal, log-normal size distribution in the range of below 20 to over 200 nm in diameter, or alternatively, a bimodal distribution with two discrete peaks at 30 to 70 nm and 100 to over 200 nm. Which of the two distribution models represented the best fit depended primarily on the data collection times used. In contrast, both fractionating techniques revealed a size distribution with a large, narrow peak well below 50 nm and a minor, broad, overlapping peak or tail extending to over 100 nm in diameter. The observed differences in liposome size distribution may be explained by the inherent limitations of the different size analysis techniques, such as the detection limit and the fact that PCS is overemphasizing bigger particle sizes.     

Fluorescent liposomes as contrast agents for in vivo optical imaging of edemas in mice

This study assesses if specially designed fluorescent liposomes can be used as contrast agent for near-infrared fluorescence (NIRF) optical imaging of cultured macrophages in vitro and for NIRF imaging of inflammatory processes, like edema, in an in vivo mouse model. Fluorescent liposomes are prepared by the film hydration and extrusion method using cholesterol, L-phosphatidylcholine, and the NIR fluorescent dye DY-676-C(18) ester. Photon correlation spectroscopy and flow cytometry reveal that fluorescent liposomes are structurally stable for up to 133 days. Distinct uptake/labeling of cultured murine J774 macrophages is demonstrated by confocal laser scanning microscopy (CLSM), flow cytometry, and macroscopic NIRF imaging system at wavelengths >670 nm. Moreover, CLSM analysis reveals fluorescence signals within intracellular compartments. Ear edema is induced in mice (n = 16) by subcutaneous injection of zymosan A. Whole-body NIRF imaging is performed after intravenous injection (0-24 h) of fluorescent liposomes (55 nmol dye per kg body weight). Distinctly higher fluorescence intensities (1613.6 +/- 61.7 a.u.) are detected at inflamed areas of diseased mice as compared to controls (892.8 +/- 19.4 a.u.). Furthermore, cell isolated from ear lavage reveals the presence of labeled F4/80 positive tissue macrophages. Taken together, the results indicate both that mouse macrophages labeled with fluorescent liposomes can be detected in vitro with fluoro-optical methods and that in vivo optical imaging of inflammatory processes with fluorescent liposomes as contrast agent is feasible. Possibly, early stages of other inflammatory diseases could also be detected by the proposed diagnostic tool in the long term.     

基于二聚酸多元醇的高固含量聚氨酯分散体的合成及乳液性能

以二聚酸二元醇、二羟甲基丙酸(DMPA)、三羟甲基丙烷(TMP)、异佛二酮二异氰酸酯(IPDI)、三乙胺(TEA)、乙二胺(EDA)为原料,采用预聚直接分散法合成了固含量50%以上的聚氨酯分散体(PUD)。研究结果表明,PUD平均粒径多为100 nm~110nm,粒径多分布于50 nm~200nm范围内,羧基和TMP含量的增加使胶粒粒径变小。透射电镜(TEM)表征显示分散体胶粒呈多分散性。PUD的黏度随剪切速率的增加而先增大,随后保持稳定,最后减小。     

Quantitative assessment of the comparative nanoparticle-uptake efficiency of a range of cell lines

The mechanism(s) of nanoparticle-cell interactions are still not understood. At present there is little knowledge of the relevant length- and timescales for nanoparticle intracellular entry and localization within cells, or the cell-specificity of nanoparticle uptake and localisation. Here, the effect of particle size on the in-vitro intracellular uptake of model fluorescent carboxyl-modified polystyrene nanoparticles is investigated in various cell lines. A range of micro- and nanoparticles of defined sizes (40 nm to 2 μm) are incubated with a series of cell types, including HeLa and A549 epithelial cells, 1321N1 astrocytes, HCMEC D3 endothelial cells, and murine RAW 264.7 macrophages. Techniques such as confocal microscopy and flow cytometry are used to study particle uptake and subcellular localisation, making significant efforts to ensure reproducibility in a semiquantitative approach. The results indicate that internalization of (nano)particles is highly size-dependent for all cell lines studied, and the kinetics of uptake for the same type of nanoparticle varies in the different cell types. Interestingly, even cells not specialized for phagocytosis are able to internalize the larger nanoparticles. Intracellular uptake of all sizes of particles is observed to be highest in RAW 264.7 cells (a specialized phagocytic cell line) and the lowest in the HeLa cells. These results suggest that (nano)particle uptake might not follow commonly defined size limits for uptake processes, and highlight the variability of uptake kinetics for the same material in different cell types. These conclusions have important implications for the assessment of the safety of nanomaterials and for the potential biomedical applications of nanoparticles.     

Increased oxidative stress in foam cells obtained from hemodialysis patients

Premature atherosclerosis represents the main cause of mortality among end‐stage renal disease patients (ESRD). Increased inflammation and oxidative stress are involved in initiation and progression of the atherosclerotic plaque. As foam cells are capable of producing significant amounts of inflammatory mediators and free radicals, we hypothesized that foam cells from uremic patients could produce more inflammation and oxidative stress than foam cells from normal people and be, somehow, involved in the accelerated atherosclerosis of uremia. To test this hypothesis, the levels of a few markers of inflammation and oxidative stress: Tumor necrosis factor‐α, inducible nitric oxide synthase, malondialdehyde, nitric oxide by‐products were measured in the supernatants of macrophage‐derived foam cells cultures from 18 hemodialysis patients and 18 apparently healthy individuals controls. Malondialdehyde levels in the supernatant of cell cultures (macrophages stimulated or not with native and oxidized lipoprotein) were significantly increased in uremic patients; no statistically significant difference was found between the supernatant concentrations of nitric oxide by‐products, inducible nitric oxide synthase activity, and tumor necrosis factor‐α between patients and controls. Our results, obtained with human macrophages and macrophage‐derived foam cells, are compatible with the theory that increased cellular oxidative stress and inflammatory activity in ESRD patients could accelerate the atherosclerotic process. The present culture protocol showed it is possible to use human mononuclear cells to evaluate the oxidative metabolism of foam cells, which are considered to be the initial step of atherosclerotic lesions.     

A Smart Responsive Dual Aptamers‐Targeted Bubble‐Generating Nanosystem for Cancer Triplex Therapy and Ultrasound Imaging

The absence of targeted, single treatment methods produces low therapeutic value for treating cancers. To increase the accumulation of drugs in tumors and improve the treatment effectiveness, near‐infrared 808 nm photothermal responsive dual aptamers‐targeted docetaxel (DTX)‐containing nanoparticles is proposed. In this system, DTX and NH₄HCO₃ are loaded in thermosensitive liposomes. The surface of liposomes is coated with gold nanoshells and connected with sulfydryl (SH? ) modified AS1411 and S2.2 aptamers. The nanosystem has good biocompatibility and uniform size (diameter about 200 nm). The drug is rapidly released, reaching a maximum amount (84%) at 4 h under 808 nm laser irradiation. The experiments conducted in vitro and in vivo demonstrate the nanosystem can synergistically inhibit tumor growth by combination of chemotherapy, photothermal therapy, and biological therapy. Dual ligand functionalization significantly increases cellular uptake on breast cancer cell line (MCF‐7) cells and achieves ultrasound imaging (USI) at tumor site. The results indicate that this drug delivery system is a promising theranostic agent involving light‐thermal response at tumor sites, dual ligand targeted triplex therapy, and USI.     

Synthesis and hydrothermal treatment of nanostructured hydroxyapatite of controllable sizes

Nanoparticulate systems have been studied for targeted and controlled release of therapeutic agents; and size is one of the major determinants of their in vivo clearance kinetics by the MPS macrophages. As such, it is important to control the size of hydroxyapatite nanoparticles during synthesis. The results show that the size of hydroxyapatite nanoparticles, synthesized through chemical precipitation, increases with increasing synthesis time. Particle sizes were also observed to increase in a linear correlation with temperature. Crystallinity and carbonate-substitution of the nanoparticles also increased with temperature. Hydrothermal, performed as a post-synthesis treatment, improves particle morphology, giving particles with regular surface contours, well-defined sizes and lower particle agglomeration. By controlling synthesis temperature and time, hydroxyapatite nanoparticles with well-defined sizes and morphology can be obtained.     

The effects of reaction factors on the fabrication of nano-sized indium tin oxide powder by spray pyrolysis process

In this study, waste ITO target is dissolved into hydrochloric acid to generate a complex acid solution. Nano-sized ITO powder with the average particle size below 50 nm is generated from this complex solution by spray pyrolysis process. This study also examines the influences of reaction parameters such as nozzle tip size and air pressure on the properties of the generated ITO powder. When the nozzle tip size is at 1 mm, the particle size distribution becomes more uniform in contrast to the case of other tip sizes, and the average particle size is around 50 nm. When the nozzle tip size increases up to 5 mm, the average particle size increases slightly, yet the particle size distribution becomes extremely irregular. Along with the change of nozzle tip size, the changing tendencies of XRD peak intensity and specific surface area are almost consistent with that of average particle size. Along with the increase of air pressure, the average particle size of the ITO powder gradually decreases, and the particle size distribution becomes more uniform. When the air pressure is at 0.1 kg/cm2, the particle size distribution appears extremely irregular, yet the average particle size is around 70 nm. When the air pressure is at 3 kg/cm2, the average particle size decreases down to 40 nm. Along with the increase of air pressure, the XRD peak intensity gradually decreases and the specific surface area increases.     

Adriamycin-incorporated nanoparticles of deoxycholic acid-conjugated dextran: antitumor activity against CT26 colon carcinoma

In this study, we prepared adriamycin (ADR)-encapsulated nanoparticles using deoxycholic acid-conjugated dextran (DexDA). Its antitumor activity was evaluated using CT 26 tumor cells in vitro and in vivo. ADR-incorporated DexDA nanoparticles have spherical shapes and their particle sizes were ranged about 50-200. Their particle sizes were changed according to the preparation conditions, i.e., the higher substitution degree (DS) of deoxycholic acid (DA) and higher drug feeding ratio induced increased particle size and zeta potential. Furthermore, the higher DS of DA and higher drug feeding ratio induced increased drug contents and loading efficiency of drug. The higher DS of DA and higher drug feeding ratio induced decreased drug release rate. Futhermore, acidic pH of release media accelerated the drug release rate compared to alkaline pH. At in vitro cytotoxicity test using CT26 tumor cells, the nanoparticles showed higher antitumor activity than free ADR. In fluorescence microscopic observation, nanoparticles were properly entered into tumors cells and maintained in the cells compared to ADR itself. At in vivo animal tumor model using CT-26 cells, nanoparticles resulted in survivability increase of mice even though free ADR showed higher effectiveness to inhibit tumor growth. These results suggested that ADR-incorporated DexDA nanoparticles are promising vehicles for anti-tumor drug delivery.     

In‐Vitro Permeability of Neutral Polystyrene Particles via Buccal Mucosa

Drugs can be absorbed well in the oral cavity, which eliminates problems related to intestinal and hepatic first‐pass metabolism. Although it is well‐established that nanoparticles are small enough to penetrate/permeate epithelial barriers, there is no clear understanding of how they interact with the buccal mucosa. This work provides useful information regarding particle properties with regard to mucosal uptake and can be used for the rational design of nanocarriers. In the buccal mucosa, the uptake of neutral polystyrene nanoparticles (PP) is size‐dependent. Compared to 25 and 50 nm particles, 200 nm PP particles penetrate into deeper regions of the mucosa. This is attributed to the structure of the buccal mucosa, i.e., mucus layer and microplicae. The particles permeate the mucus layer and deposit in ridge‐like folds of superficial buccal cells. Thus, the effects of thermodynamic driving forces and/or interparticle electrostatic repulsion are enhanced and cellular uptake might be reduced for smaller particle sizes.     

In vivo biodistribution and urinary excretion of mesoporous silica nanoparticles: effects of particle size and PEGylation

The in vivo biodistribution and urinary excretion of spherical mesoporous silica nanoparticles (MSNs) are evaluated by tail-vein injection in ICR mice, and the effects of the particle size and PEGylation are investigated. The results indicate that both MSNs and PEGylated MSNs of different particle sizes (80-360 nm) distribute mainly in the liver and spleen, a minority of them in the lungs, and a few in the kidney and heart. The PEGylated MSNs of smaller particle size escape more easily from capture by liver, spleen, and lung tissues, possess longer blood-circulation lifetime, and are more slowly biodegraded and correspondingly have a lower excreted amount of degradation products in the urine. Neither MSNs nor PEGylated MSNs cause tissue toxicity after 1 month in vivo.     

Mie理论在静态光散射粒度测量的应用下限研究

测量下限是光散射颗粒测试技术的关键问题。本文通过理论分析、比较归一化散射光强的分布图和构造方差函数F(d)对颗粒散射光的光强分布进行了定性和定量的讨论,对Mie散射向Rayleigh散射趋近的情况进行了分析,讨论了散射光光强大小的分布,分析了测量不同粒径的颗粒的可行性,最终得到在入射光源是波长为0.6328μm的He-Ne激光器的情况下,当粒径d取200nm以上时,不同粒径颗粒的M ie散射光强分布有较大差别,适合用静态光散射的方法来判断颗粒粒径。     

Size characterization of bentonite colloids by different methods

The size and shape of colloids released from a natural bentonite into a low-mineralized groundwater are investigated using various colloid characterization methods. For the applied methods such as atomic force microscopy (AFM), laser-induced breakdown detection (UBD), photon correlation spectroscopy (PCS), and flow field-flow fractionation coupled to ICP-mass spectrometric detection (FFFF-ICPMS), the respective raw size data have to be corrected in order to consider chemical composition and shape of the colloids as well as instrumental artifacts. Noncontact mode AFM of the bentonite colloids shows disklike shapes of stacked smectite platelets with a mean height-to-diameter proportion (aspect ratio) of approximately 1/10. A broad particle number size distribution is determined by image processing with a mean particle diameter of 73 nm. In agreement with AFM, a broad size distribution is also found by PCS and FFFF-ICPMS. Likewise, mean particle sizes found by LIBD (67 +/- 13 nm) and FFFF-ICPMS (maximum in the number size distribution, approximately 70 nm) are in fair agreement with the AFM data. Somewhathigher values are obtained by PCS, where mean particle diameters of the intensity-weighted size distributions of larger than 200 nm are found (depending on the algorithm used for data processing). The influence of the disklike particle shape on the results of the individual methods is discussed. As a conclusion, the application of different colloid characterization methods is a prerequisite to get complementary information about colloid size and shape, which is essential for the understanding of natural colloidal systems.     

Role of alumina nanoporosity in acute cell response

This work studied the effect of nanoporous alumina in acute cellular response in an in vivo model. Nanoporous alumina membranes, with pore size diameters of 20 and 200 nm, were fabricated by anodic oxidation of aluminium. The membranes were thereafter characterized in terms of pore size distribution and chemical composition. To evaluate acute inflammatory response, the membranes were implanted in the peritoneal cavity of mice. Cell recruitment to the implant site was determined by fluorescence activated cell sorting (FACS) analysis. Cell adhesion to material surfaces was studied in terms of cell number, type, and morphology using scanning electron microscopy (SEM) and immunocytochemical staining followed by fluorescence microscopy. The fabricated nanoporous alumina membranes were found to have narrow pore size distribution. The in vivo study showed that 200 nm alumina membranes induced stronger inflammatory response than 20 nm membranes. This was reflected by the number of implant-associated phagocytes and the number of cells recruited to the implantation site. Since both pore-size membranes possess similar chemical composition, we believe that the observed difference in cell recruitment and adhesion is an effect of the material nanotopography. Our results suggest that nanotopography can be used to subtly control the recruitment and adherence of phagocytic cells during the acute inflammatory response to alumina membranes.     

不同粒径超顺磁性氧化铁纳米粒子的合成及其在交变磁场中的磁热效应

采用多醇热解法制备3种不同粒径的超顺磁性氧化铁纳米粒子(SPIONs),合成的SPIONs含Fe_3O_4晶相,分散性好,平均粒径分别为8.7,12.6nm和15.3nm,且在300K下,3种SPIONs均呈超顺磁性。将不同粒径、不同浓度的SPIONs水分散液置于频率为425kHz、磁场强度为5.3kA·m^-1的交变磁场(ACMF)中进行升温实验。探讨比能量吸收率值与SPIONs粒径之间的关系,计算布朗弛豫时间及尼尔弛豫时间。结果表明:SPIONs水分散液的升温速率随SPIONs的粒径增大而增大,初始温度为20℃时,粒径为8.7,12.6nm和15.3nm的SPIONs水分散液(2mg·mL^-1)在480s内温度分别升高了25,27,35℃。尼尔弛豫时间比布朗弛豫时间小,说明磁热效应主要来自于尼尔弛豫损耗。SPIONs粒径越大,比能量吸收率SAR值越高,最高可达810W·g^-1,且SAR值与SPIONs水分散液的浓度呈负相关关系。     

Fusogenic liposomal formulation of sirolimus: improvement of drug anti-proliferative effect on human T-cells

Context: Fusogenic liposomes are unique delivery vehicles capable of introducing their contents directly and efficiently into the cytoplasm.

Objective: The objective of this study was to evaluate the potential of fusogenic liposomes containing Sirolimus to improve its anti-proliferative effect on T-lymphocyte cells.

Materials and methods: Conventional liposomes containing Sirolimus were prepared from Dipalmitoylphosphatidylcholine (DPPC) and cholesterol using the modified ethanol injection method. To prepare fusogenic liposomes, dioleoylphosphatidylethanolamine (DOPE) was added to the conventional liposome formulation. The liposomes were characterized by their size, zeta potential, encapsulation efficiency percent (EE%) and chemical stability during 6 months. The in vitro release of liposomes, anti-proliferative effect and liposome uptake of both types of liposomes with optimized formulations were studied on human T-lymphocyte cells employing the MTT assay and fluorescein isothiocyanate-loaded liposomes.

Results and discussion: The particle size of the liposomes was evaluated between 138 and 650?nm and mean zeta potential was in the range of ?32.95 to ?45.60?mV. The average EE% of the prepared conventional and fusogenic liposomes were 76.9% and 80.5%, respectively. Liposomal formulations released only 10–20% of encapsulated drug without any burst effect. In vitro immunosuppressive evaluation on T-cells showed that fusogenic liposomes have the best anti-proliferative effects and uptake on T-lymphocyte cell compared to the conventional liposomes.

Conclusion: Our results indicated that fusogenic liposomes can be useful carriers for improving the inhibition of T-cell proliferation.     

Physical characterization and cellular uptake of propylene glycol liposomes in vitro

In order to facilitate the intracellular delivery of therapeutic agents, a new type of liposomes–propylene glycol liposomes (PGL) were prepared, and their cell translocation capability in vitro was examined. PGL was composed of hydrogenated egg yolk lecithin, cholesterol, Tween 80 and propylene glycol. With curcumin as a model drug, characterization of loaded PGL were measured including surface morphology, particle size, elasticity, encapsulation efficiency of curcumin and physical stability. Using curcumin-loaded conventional liposomes as the control, the cell uptake capacity of loaded PGL was evaluated by detection the concentration of curcumin in cytoplasm. Compared with conventional liposomes, PGL exhibited such advantages as high encapsulation efficiency (92.74% ± 3.44%), small particle size (182.4?±?89.2?nm), high deformability (Elasticity index?=?48.6) and high stability both at normal temperature (about 25°C) and low temperature at 4°C. From cell experiment in vitro, PGL exhibited the highest uptake of curcumin compared with that of conventional liposomes and free curcumin solution. Little toxic effect on cellular viability was observed by methyl tetrazolium assay. In conclusion, PGL might be developed as a promising intracellular delivery carrier for therapeutic agents.     

Physical characterization and cellular uptake of propylene glycol liposomes in vitro

In order to facilitate the intracellular delivery of therapeutic agents, a new type of liposomes-propylene glycol liposomes (PGL) were prepared, and their cell translocation capability in vitro was examined. PGL was composed of hydrogenated egg yolk lecithin, cholesterol, Tween 80 and propylene glycol. With curcumin as a model drug, characterization of loaded PGL were measured including surface morphology, particle size, elasticity, encapsulation efficiency of curcumin and physical stability. Using curcumin-loaded conventional liposomes as the control, the cell uptake capacity of loaded PGL was evaluated by detection the concentration of curcumin in cytoplasm. Compared with conventional liposomes, PGL exhibited such advantages as high encapsulation efficiency (92.74% ± 3.44%), small particle size (182.4?±?89.2?nm), high deformability (Elasticity index?=?48.6) and high stability both at normal temperature (about 25°C) and low temperature at 4°C. From cell experiment in vitro, PGL exhibited the highest uptake of curcumin compared with that of conventional liposomes and free curcumin solution. Little toxic effect on cellular viability was observed by methyl tetrazolium assay. In conclusion, PGL might be developed as a promising intracellular delivery carrier for therapeutic agents.