Reverse Biomembrane Vesicles for Effective Controlled Delivery of Doxorubicin HCl

The stability of liposome preparations under the action of the nonionic detergent Triton X-100 was measured using the fluorescent molecular probe octadecylrhodamine B (R18). The probe inserted in the lipid bilayer shows a self-quenched fluorescence and the degree of quenching depends both on the probe concentration and the phase state of the lipid membrane. The addition of detergent to the liposomes produces a steep decrease in self-quenching caused by dilution of the probe in the bilayer. The curves of steady-state fluorescence intensity show an abrupt change in slope that corresponds to the point at which liposomes break down into lipid-detergent mixed entities that are different from the earlier liposome-monodisperse population. The lytic process was followed in parallel by dynamic light scattering (DLS), and the analysis of the DLS results agree with the interpretation of the fluorescence measurements. The probe R18 therefore is a useful marker to test the stability of liposome preparations. The advantages of the present method are discussed by comparison with other techniques.     

A stability test of liposome preparations using steady-state fluorescent measurements

The stability of liposome preparations under the action of the nonionic detergent Triton X-100 was measured using the fluorescent molecular probe octadecylrhodamine B (R18). The probe inserted in the lipid bilayer shows a self-quenched fluorescence and the degree of quenching depends both on the probe concentration and the phase state of the lipid membrane. The addition of detergent to the liposomes produces a steep decrease in self-quenching caused by dilution of the probe in the bilayer. The curves of steady-state fluorescence intensity show an abrupt change in slope that corresponds to the point at which liposomes break down into lipid-detergent mixed entities that are different from the earlier liposome-monodisperse population. The lytic process was followed in parallel by dynamic light scattering (DLS), and the analysis of the DLS results agree with the interpretation of the fluorescence measurements. The probe R18 therefore is a useful marker to test the stability of liposome preparations. The advantages of the present method are discussed by comparison with other techniques.     

Characterization of a Nanoparticulate Drug Delivery System Using Scanning Ion Occlusion Sensing

Purpose

To explore the application of scanning ion occlusion sensing (SIOS) as a novel technology for characterization of nanoparticles.

Methods

Liposomes were employed as model nanoparticles. The size distribution of the liposomes was measured by both SIOS and dynamic light scattering (DLS). Particle number concentration was determined based on particle translocation rate. The ability of SIOS and DLS to resolve bimodal samples was evaluated by measuring a mixture of 217 and 355 nm standard nanoparticles. Opsonization of liposomes by plasma was also studied using SIOS.

Results

SIOS was shown to measure the size of different liposomes with higher sensitivity than DLS and it requires a smaller sample volume than DLS. With appropriate calibration, SIOS could be used to determine particle number concentrations. In comparison, SIOS analysis of the mixture showed accurate resolution of the population as a bimodal distribution over a wide range of number ratios of the particles. SIOS could detect plasma opsonization of liposomes by demonstrating a increase in particle size and also changes in the particle translocation rate.

Conclusion

SIOS is a useful technology for nanoparticle characterization. It shows some advantages over DLS and is clearly a useful tool for the study of nanoparticle drug delivery systems.     

Chitosan nanoparticles as a novel delivery system for ammonium glycyrrhizinate

The ammonium glycyrrhizinate-loaded chitosan nanoparticles were prepared by ionic gelation of chitosan with tripolyphosphate anions (TPP). The particle size and zeta potential of nanoparticles were determined, respectively, by dynamic light scattering (DLS) and a zeta potential analyzer. The effects, including chitosan molecular weight, chitosan concentration, ammonium glycyrrhizinate concentration and polyethylene glycol (PEG) on the physicochemical properties of the nanoparticles were studied. These nanoparticles have ammonium glycyrrhizinate loading efficiency. The encapsulation efficiency decreased with the increase of ammonium glycyrrhizinate concentration and chitosan concentration. The introduction of PEG can decrease significantly the positive charge of particle surface. These studies showed that chitosan can complex TPP to form stable cationic nanoparticles for subsequent ammonium glycyrrhizinate loading.     

Triton-X-100-modified polymer and microspheres for reversal of multidrug resistance.

Triton X-100 is a non-ionic detergent capable of reversing multidrug resistance (MDR) due to its interaction with cell membranes. However, it interacts with cells in a non-specific way, causing cytotoxicity. This work aimed to develop polymeric chemosensitizers that possess the ability to reverse MDR and lower toxic side effects. When being delivered to tumours, the polymeric chemosensitizers may also have longer retention times in tumours than the free detergent. Triton-X-100-immobilized dextran microspheres (T-MS) and inulin (T-IN) were prepared and characterized. Their cytotoxicity against multidrug-resistant Chinese hamster ovary cells (CH(R)C5) was compared with that of free Triton X-100 solutions. The in-vitro effect of the products on 3H-vinblastine accumulation by CH(R)C5 cells was determined. Both T-MS and T-IN showed a marked decrease in the cytotoxicity, as compared with free Triton solutions at equivalent concentrations. Drug accumulation by CH(R)C5 cells was increased over two fold in the presence of T-MS or T-IN. These results suggest that polymeric drug carriers with MDR-reversing capability and lower cytotoxicity may be prepared by immobilization of chemosensitizers.     

Formulation of tenofovir-loaded functionalized solid lipid nanoparticles intended for HIV prevention

The objective of this study is to engineer polylysine-heparin functionalized solid lipid nanoparticles (fSLNs) for the use of a vaginal microbicide delivery template for HIV prevention. The fSLNs are prepared using a modified phase-inversion technique followed by a layer-by-layer deposition method. The Box-Behnken experimental design is used to analyze the influence of three factors (X(1) = bovine serum albumin concentration, X(2) = pH of the aqueous phase, and X(3) = lipid amount) on the particle mean diameter (PMD) measured by dynamic light scattering (DLS). Tenofovir is used as a model anti-HIV microbicide. The SLNs are also characterized for morphology, zeta potential (ζ ), percent drug encapsulation efficiency (EE%), and cytotoxicity on a human vaginal epithelial cell line by electron microscopy, DLS, ultraviolet, and fluorescence spectroscopy, respectively. The statistical model predicts particle size (Y) with 90% confidence and the Y values are significantly affected by X(1) and X(2) . The produced fSLNs appear noncytotoxic and exhibit a platelet-like shape with respective PMD, EE%, and ζ value of 153 nm, 8.3%, and - 51 mV. These fSLNs intended to be administered topically have the potential to enhance cellular uptake of hydrophobic microbicides and outdistance the virus during the HIV/AIDS infection process, possibly leading to more effective prevention of the disease transmission.     

Comparison of scanning electron microscopy, dynamic light scattering and analytical ultracentrifugation for the sizing of poly(butyl cyanoacrylate) nanoparticles.

Nanoparticles represent promising carriers for controlled drug delivery. This work focuses on the size and molecular mass characterization of polyalkylcyanoacrylate nanoparticles formed by anionic emulsion polymerization of butylcyanoacrylate in the presence of poloxamer 188 as a stabilizer. Three different methods were used to determine the size and size distribution of the particle populations: scanning electron microscopy (SEM), dynamic light scattering (DLS), and analytical ultracentrifugation (ANUC). SEM on freeze-dried and Au-shadowed samples showed a relatively narrow distribution of virtually spherical particles with a mean diameter of 167 nm. DLS yielded a monomodal distribution with hydrodynamic diameters around 199 nm (in the absence of additional stabilizer) or 184 nm (in the presence of 1% poloxamer 188). The size distribution determined by ANUC using sedimentation velocity analysis was somewhat more complex, the size of the most abundant particles being around 184 nm. Molar particle mass distributions centered around 2.3x10(9) g/mol. The advantages and disadvantages of the three sizing techniques are discussed.     

Analyzing Subvisible Particles in Protein Drug Products: a Comparison of Dynamic Light Scattering (DLS) and Resonant Mass Measurement (RMM)

Aggregation is common in protein drug manufacture, and while the effects of protein particulates are under investigation, many techniques applicable for their characterization have been recently developed. Among the methods available to characterize and quantify protein aggregates, none is applicable over the full size range and different methods often give conflicting results. The studies presented here compare two such methods: dynamic light scattering (DLS) and resonant mass measurement (RMM). The performance of each method was first characterized using polystyrene particle size standards (20, 60, 100, 200, 400, and 1,000 nm) over a range of concentrations. Standard particles were measured both singly and in binary mixtures containing 20 nm particles at a fixed concentration (10¹⁴ particles/mL) and various concentrations of one of the other particle sizes (i.e., 60, 100, 200, 400, or 1,000 nm). DLS and RMM were then used to detect unknown aggregate content in stressed samples of IgG. Both instruments were shown to have a working range that depends on particle size and concentration. In binary mixtures and polydisperse solutions, DLS was able to resolve two species in a manner dependent on both concentration and particle size. RMM was able to resolve particles above 200 nm (150 nm for protein) at concentrations below 10⁹ particles/mL. In addition, dilution was evaluated as a technique to confirm and quantify the number of particles in solution.     

Stabilization of C60 nanoparticles by protein adsorption and its implications for toxicity studies

Dispersion stability of nanoparticles of C60 under a model condition simulating a physiological environment was studied by dynamic light scattering. Although the C60 nanoparticles at a concentration of 9.3 x 10(-6) M (6.7 microg/mL) coagulated and precipitated out rapidly in phosphate buffered saline, coagulation was suppressed completely when HSA was present at concentrations above 1 mg/mL. DLS results show that the HSA molecules adsorb onto the surfaces of the C60 nanoparticles, thereby forming a protective layer, and prevent salt-induced coagulation. DLS results also indicate that the HSA molecules take an expanded conformation on the surface. Our findings suggest that C60 nanoparticles can be stabilized in the physiological environment even if they are not deliberately stabilized by using stabilizers and are of significant implications for the on-going efforts to evaluate the cytotoxicity of C60 nanoparticles in which no such effect has been considered.     

中心组合设计法优化载基因壳聚糖纳米粒的最佳转染制备区域

目的：采用中心组合设计法优化载基因壳聚糖纳米粒的最佳转染制备区域。方法采用复凝聚法制备载质粒基因的壳聚糖纳米粒,选择壳聚糖浓度和质粒基因浓度作为实验考察因素,应用两因素五水平中心组合设计优化最佳转染制备区域,优化指标选择平均粒径和基因转染率。通过透射电镜观察纳米粒的形态;通过动态光散射和电泳光散射技术分别测量纳米粒的粒径和Zeta电位;通过凝胶电泳分析考察质粒在纳米粒制备过程中的稳定性;通过倒置荧光显微镜观察质粒基因在细胞内的表达;通过流式细胞技术测定纳米粒的转染效率。结果成功优化了载基因壳聚糖纳米粒的最佳转染制备区域。优选条件下制备的纳米粒大多呈球形,纳米粒平均粒径为217．6 nm,粒径多分散系数为0．241,表明粒径分布较窄。纳米粒zeta电位为＋22．4 mV,表明纳米粒表面带有正电荷,可以增加纳米粒混悬液的稳定性。凝胶电泳分析结果表明质粒基因在纳米粒制备过程中没有遭到破坏。纳米粒的细胞转染效率比较高,能够高效地将绿色荧光蛋白质粒基因递送到细胞内,并且基因表达产生绿色荧光蛋白。结论本研究建立的数学模型具有良好的预测性。在优化的制备区域内制备的载基因壳聚糖纳米粒的转染性能比较理想。     

Preparation of nanocapsules containing the two-phase core materials

In this study, copolymer nanocapsules containing the two-phase core materials were prepared by mini-emulsion polymerization using styrene (St) and methylmethacrylate (MMA) as comonomer, in which the ZnO nanoparticles organic suspension was prepared by solvothermal synthesis method. The obtained materials were characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), atomic force microscopy (AFM), UV-visible spectrophotometry and fluorescence spectrometer. The particle size of the prepared ZnO nanoparticles dispersed in the organic solvent was less than 20 nm. The resulting polymer nanocapsules have 100-200 nm in diameter and approximately 10-20 nm in the wall thickness. The surface of the polymer nanocapsules is smooth and clear.     

Multimodal Dispersion of Nanoparticles: A Comprehensive Evaluation of Size Distribution with 9 Size Measurement Methods

Purpose

Evaluation of particle size distribution (PSD) of multimodal dispersion of nanoparticles is a difficult task due to inherent limitations of size measurement methods. The present work reports the evaluation of PSD of a dispersion of poly(isobutylcyanoacrylate) nanoparticles decorated with dextran known as multimodal and developed as nanomedecine.

Methods

The nine methods used were classified as batch particle i.e. Static Light Scattering (SLS) and Dynamic Light Scattering (DLS), single particle i.e. Electron Microscopy (EM), Atomic Force Microscopy (AFM), Tunable Resistive Pulse Sensing (TRPS) and Nanoparticle Tracking Analysis (NTA) and separative particle i.e. Asymmetrical Flow Field-Flow Fractionation coupled with DLS (AsFlFFF) size measurement methods.

Results

The multimodal dispersion was identified using AFM, TRPS and NTA and results were consistent with those provided with the method based on a separation step prior to on-line size measurements. None of the light scattering batch methods could reveal the complexity of the PSD of the dispersion.

Conclusions

Difference between PSD obtained from all size measurement methods tested suggested that study of the PSD of multimodal dispersion required to analyze samples by at least one of the single size particle measurement method or a method that uses a separation step prior PSD measurement.

     

阿霉素修饰纳米银的制备及其体外抗肿瘤活性研究

摘要： 目的 设计合成一类新型的具有pH响应性的阿霉素-纳米银 （DOX-Ag NPs） 联合抗肿瘤药物, 对其理化性质进行表征, 并研究其体外响应性释药行为和抗肿瘤活性。方法 通过硫辛酰肼 （LA-NHNH2 ） 连接纳米银 （Ag NPs） 和阿霉素 （DOX）, 得到DOX-Ag NPs。利用核磁氢谱 （1 H NMR） 和高分辨质谱 （HRMS） 对硫辛酰肼-阿霉素 （LA- NHN=DOX） 进行结构确证; 通过动态光散射 （DLS） 和透射电镜 （TEM） 分析纳米粒的粒径和形貌; 通过紫外-可见吸收光谱和荧光光谱表征纳米粒的光学性质; 通过透析法结合荧光光谱检测DOX-Ag NPs在不同pH下的DOX释放行为; 采用噻唑蓝比色法研究DOX-Ag NPs对HepG2肿瘤细胞的增殖抑制效果。结果 LA-NHN=DOX的1 H NMR数据及HRMS检测到746.275 6处的分子离子峰均证明LA-NHN=DOX成功合成。DOX-Ag NPs为粒径 （40.4±3.8） nm的球形纳米粒; 在弱酸性条件下DOX-Ag NPs能够快速响应性释放DOX; DOX-Ag NPs对HepG2肿瘤细胞增殖抑制呈现浓度依赖性, 当DOX浓度为0.5~20 mg/L （Ag浓度为0.45~18 mg/L） 时, DOX-Ag NPs组细胞生存率均明显低于DOX 组和Ag NPs组 （均P＜0.05）。结论 DOX-Ag NPs是一种具有pH响应性的联合抗肿瘤纳米制剂, 能在肿瘤组织快速释放DOX, 并通过与Ag NPs的协同治疗, 发挥良好的体外抗肿瘤作用。     

还原响应型透明质酸/聚己内酯纳米粒子的制备及其体外抗肺癌效应研究

目的： 构建一种具有良好生物安全性、肿瘤主动靶向性及能实现药物快速释放的药物纳米载体。方法： 通过点击化学反应制备由二硫键连接的两亲性透明质酸/聚己内酯接枝聚合物;采用动态光散射、透射电镜对聚合物的自组装行为进行研究;通过体外药物释放实验探究药物载体还原响应控制释放特性;通过流式细胞术、激光共聚焦显微镜等技术对A549细胞内吞噬载药纳米粒子的机制进行研究;结果： 所制备的含二硫键的接枝聚合物可自组装形成粒径大约为75 nm的球形纳米粒子;该纳米粒子在质量浓度为200 μg·mL^-1时仍具有较好的生物安全性;当纳米粒子包载阿霉素后其粒径增大至128 nm,且药物在还原性条件下可实现快速释放;载药后的纳米粒子经透明质酸/CD44受体间的相互作用快速进入肿瘤细胞内,并能显著抑制肿瘤细胞生长。结论： 本研究合成的含二硫键的透明质酸/聚己内酯接枝聚合物具有良好生物相容性、肿瘤靶向性及药物控制释放特性,作为抗肿瘤药物载体具有一定优势。     

A novel method for measuring rigidity of submicron-size liposomes with atomic force microscopy

There are many useful colloidal drug delivery systems that use liposomes. The rigidity of the carrier particle is one of the most important properties affecting drug delivery effectiveness, assessed by particle stability, release profile of encapsulated drug, and blood circulation time. However, it is difficult to evaluate the rigidity of such fine particles; so far, no useful methods have been reported. We demonstrate a unique method to evaluate the rigidity of liposomes using atomic force microscopy (AFM) and dynamic light scattering (DLS) in this report. We showed that the combination of two types of particle-size measurements, tapping mode AFM in buffer solution with another conventional method such as DLS, is useful for evaluating the rigidity of submicron-size particles such as liposomes.     

Preparation of nanocapsules containing the two-phase core materials

In this study, copolymer nanocapsules containing the two-phase core materials were prepared by mini-emulsion polymerization using styrene (St) and methylmethacrylate (MMA) as comonomer, in which the ZnO nanoparticles organic suspension was prepared by solvothermal synthesis method. The obtained materials were characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), atomic force microscopy (AFM), UV-visible spectrophotometry and fluorescence spectrometer. The particle size of the prepared ZnO nanoparticles dispersed in the organic solvent was less than 20?nm. The resulting polymer nanocapsules have 100–200?nm in diameter and?～?10–20?nm in the wall thickness. The surface of the polymer nanocapsules is smooth and clear.     

Reliable size determination of nanoparticles using dynamic light scattering method for in vitro toxicology assessment

Dynamic light scattering (DLS) is widely used for the evaluation of the particle size in the toxicity assessment of nanoparticles. However, the many types of DLS instruments and analytical procedures sometimes give different apparent sizes of particles and make it complicated to understand the size dependence on particles for the toxicity assay. In this study, we established an evaluation method of secondary nanoparticle sizes using a DLS analysis. First, we established a practical method for determining size with an appropriate evaluation of uncertainties. This proposed method could be a universal protocol for toxicity assessment that would allow researchers to achieve some degree of concordance on the size of nanoparticles for an assessment. Second, we investigated the processes associated with particles in suspension by examining the changes in the size and the light scattering intensity of secondary nanoparticles during an in vitro toxicity assessment, since the transport mode of particles to cells is significant in understanding in vitro nano-toxicity. In this study, these two points were investigated on TiO₂ nanoparticles suspension as an example. The secondary particles of TiO₂ with a light scattering intensity-averaged diameter (d_l) of 150–250 nm were characterized with appropriate uncertainties. The sizes were found to be comparable with values determined using other analytical procedures and other instruments. It is suggested that d_l could be an effective size parameter for toxicity assessments. Furthermore, TiO₂ secondary nanoparticle suspensions are well dispersed with slow gravity settling, no agglomeration, with the diffusion process as the primary transport mode of particles to cells.     

Release kinetics from bio-polymeric nanoparticles encapsulating protein synthesis inhibitor- cycloheximide, for possible therapeutic applications

Cycloheximide, a protein synthesis inhibitor, was encapsulated in cross-linked gelatin nanoparticles (Type B, Bovine skin, 75 Bloom) of 168 nm diameter with 26% entrapment efficiency. In-vitro release kinetics of the drug from the nanoparticles was done in phosphate buffer saline (PBS) at pH 7.4 and pH 5.8. The release kinetics showed a bi-phasic curve. Interestingly, the release of drug is approx 90% in acidic pH as compared to 50% release in neutral pH. The particle size was determined by Dynamic Light Scattering (DLS) technique, and size distribution spectra at different pH were observed to vary inversely with increase in pH. These drug loaded nanoparticles were found to be stable in whole blood showing negligible haemolysis. Cytotoxicity in HBL-100 and MCF-7, breast cancer cell lines was done in a 24-72 hrs assay, showing increased anti-tumour activity over a period of time indicating slow release. Dose dependent cytotoxicity was observed after 24 hours upto 72 hours of incubation of nanoparticles while the drug per se (<4 microg) showed 93% toxicity within 24 hours. Phase contrast microscopy of nanoparticle-cell interaction, clearly indicated aggregation along the lipid cell-membrane. Electron Microscopy (TEM, SEM) studies revealed its size and spherical shape. The stability of the particle, the slow and controlled release of drug from the gelatin nanoparticles indicate that it is a good candidate to deliver bio-pharmaceuticals. These behave as "intelligent" carriers for drug delivery, and can be exploited to empty their drug load in acidic medium. The paper focuses on the release kinetics of the gelatin nanoparticles that can be successfully exploited to treat solid tumors.     

Time‐dependent biodistribution and excretion of silver nanoparticles in male Wistar rats

Silver nanoparticles (AgNPs) are the most commonly used nanoparticles owing to their antimicrobial properties. The motivation of the present study was (1) to analyze the effect of silver particle size on rat tissue distribution at different time points, (2) to determine the accumulation of AgNPs in potential rat target organs, (3) to analyze the intracellular distribution of AgNPs and (4) to examine the excretion of AgNPs by urine and feces. AgNPs were characterized by dynamic light scattering (DLS), zeta potential measurements, BET surface area measurements, transmission and scanning electron microscopy. AgNPs (20 and 200 nm) were administered intravenously (i.v.) to male Wistar rats at a dose of 5 mg kg^–1 of body weight. Biological material was sampled 24 h, 7 and 28 days after injection. Using inductively coupled plasma‐mass spectrometry (ICP‐MS) and transmission electron microscopy (TEM) it was observed that AgNPs translocated from the blood to the main organs and the concentration of silver in tissues was significantly higher in rats treated with 20 nm AgNPs as compared with 200 nm AgNPs. The highest concentration of silver was found in the liver after 24 h. After 7 days, a high level of silver was observed in the lungs and spleen. The silver concentration in the kidneys and brain increased during the experiment and reached the highest concentration after 28 days. Moreover, the highest concentration of AgNPs was observed in the urine 1 day after the injection, maintained high for 14 days and then decreased. The fecal level of silver in rats was the highest within 2 days after AgNPs administration and then decreased. Copyright © 2012 John Wiley & Sons, Ltd.     

Physicochemical characterization of protamine-phosphorothioate nanoparticles

Protamine-oligonucleotide nanoparticles represent effective colloidal drug carriers for antisense phosphorothioate oligonucleotides (PTO). This study describes improvements in particle preparation and the physicochemical properties of the complexes prepared. The influence of component concentrations, length of the PTO chain and the PTO/protamine weight ratio on particle formation and size, shape and surface charge of the particles were studied in detail. Nanoparticles with diameters of 90-200nm were obtained, using protamine free base (PFB) and phosphorothioate in water. The chemical composition of the nanoparticles was analysed. More than 90% of the PTO could be assembled in the particle matrix using a > or = 1:2 ratio (w/w) of PTO and PFB. About 53-68% of the PFB was incorporated in the particle matrix. The complexes had a zetapotential of -19 up to +32 mV, depending on the PTO/PFB ratio. The kinetics of the assembly of this binary system were observed by dynamic light scattering (DLS) measurements and by sedimentation velocity analysis in the analytical ultracentrifuge (AUC). In addition, scanning electron microscopy (SEM) and atomic force microscopy (AFM) were applied to verify the results of DLS and the ultracentrifuge measurements. According to sedimentation velocity analysis, the particles were only moderately stable in water and unstable in salt solutions. However, the colloidal solution in water could be stabilized by polyethylenglycol 20000 (PEG), which also led to an increase of stability in cell medium.