纳米银凝胶在体内分布特性的初步研究

目的:研究纳米银妇女外用抗菌凝胶在兔体内的吸收和分布情况,以了解纳米银凝胶是否具有在体内迁移和蓄积的特性。方法:将成年新西兰雌兔经阴道注入纳米银凝胶,给药剂量为0.575 g.kg-1.d-1,连续给药1个疗程后,在预定时间点解剖并取各动物血液和主要组织器官,利用超高压微波消解系统消解这些组织器官,再用原子吸收光谱仪检测消解液中的银含量。结果:在给药1个疗程后的8 h,纳米银在试验组各组织中的银含量达高峰,与对照组各脏器中银含量进行比较,结果有显著性差异(P<0.05)。其中阴道是纳米银主要的分布器官,其银含量高于其他组织达3倍以上。1周以后各组织中的银含量均大幅度下降,到1个月和3个月后,各试验组脏器中的银含量与对照组无显著性差异(P>0.05)。结论:纳米银凝胶中的纳米银可以通过血液循环系统迁移,并在兔体内各主要脏器肝、肾、脾、子宫、阴道等中分布,其中阴道是其主要的分布器官。在采用5倍临床给药剂量1个疗程的情况下,纳米银凝胶1周后在这些脏器中无明显蓄积。在下一步的研究中还需进一步了解纳米银凝胶在体内的状态、进入血液系统的方式和对各部位细胞的影响。     

纳米银抗菌凝胶在大鼠体内主要脏器的分布

目的研究纳米银抗菌凝胶治疗大鼠的大面积烧伤（包括深二度、三度）及阴道黏膜给药后大鼠对银的吸收,分布和蓄积情况。方法采用火焰原子吸收法测定实验组及正常对照组的大鼠的心、肝、脾、肺、肾、子宫、皮肤及阴道粘膜中的痕量银。结果创面皮肤及阴道黏膜连续给药纳米银抗菌凝胶6d,停药8h后各主要脏器都检测出了痕量银,皮肤的银含量为（0.089±0.031）μg/g;阴道黏膜中银的含量为（0.135±0.036）μg/g。24h后只有肾,脾,子宫,皮肤及阴道粘膜有痕量银,72h后未检测出痕量银。结论纳米银抗菌凝胶中的纳米银颗粒可以进入血液循环,并进一步分布到重要脏器。随着时间的推移,各脏器中的银含量趋于下降,这说明吸收分布到各脏器中的纳米银基本可以很快被代谢到体外,而不会在体内长期蓄积。     

纳米银抗菌水凝胶大鼠阴道给药后体内银元素的代谢与分布

目的研究纳米银抗菌水凝胶阴道给药后银元素在大鼠组织器官中的代谢与分布情况。方法雌性Wistar大鼠随机分为试验组与对照组,试验组每天阴道给纳米银抗菌水凝胶1次,剂量为1750μg,连续给药5 d;对照组不给药。采用原子吸收光谱法测定血清与器官中的银元素。结果试验组给药后血清银元素浓度逐渐上升,至第5天达到高峰,后呈下降趋势;银元素在大鼠体内的分布情况是:脾>肾>肾上腺>肝>肺>心>子宫和卵巢>脑。结论纳米银抗菌水凝胶阴道给药后在短时间内会产生一定浓度的蓄积。     

ICP-MS法测定家兔阴道给予纳米银凝胶后血浆中银的浓度

目的建立测定家兔血浆中银(Ag)浓度的电感耦合等离子体质谱(ICP-MS)方法,用以研究家兔阴道给予纳米银凝胶后其在体内的吸收特征。方法样品经高压消解,以铑(Rh)为内标,采用ICP-MS法测定血浆中Ag的浓度。结果以水为替代基质建立标准曲线,Ag的线性为1.25⁴0.00μg·L-1(r=0.997 9),日内和日间精密度RSD≤4.7%。以水为替代基质时Ag的平均提取回收率在96.6%40.00μg·L-1(r=0.997 9),日内和日间精密度RSD≤4.7%。以水为替代基质时Ag的平均提取回收率在96.6%⁹8.6%内,以空白血浆为基质时Ag的提取回收率为94.8%。以水和空白血浆为基质的基质效应均符合有关规定。家兔血浆中Ag的本底水平较低。家兔多次阴道给予纳米银凝胶后,部分家兔血浆中银的浓度相比于本底水平有一定的增加。结论本方法可用于研究纳米银医用材料在家兔体内的吸收特征。     

乙酸阴道热敏凝胶剂在家兔阴道内的药动学

目的研究乙酸阴道热敏凝胶剂经阴道给药后药物在家兔阴道内的药代动力学特征,探索适合阴道用药的新剂型。方法 3 ml含乙酸36.23 mg的乙酸阴道热敏凝胶剂推入家兔阴道内,给药后0.25,0.5,1,1.5,2,4,8,12,16,20和24 h,对家兔阴道进行灌洗并测定灌洗液中乙酸的含量、用微电极插入阴道测定阴道pH值、取阴道组织匀浆后测定组织中乙酸含量。采用非线性最小二乘法进行计算机拟合获得药动学参数。结果乙酸阴道热敏凝胶剂置入家兔阴道后,阴道pH值由给药前中性(pH 6.67)转变为酸性(pH 3.56),阴道pH 3.5～4.0可保持约6 h;pH 3.5～4.5可保持约12 h;pH 3.5～5.0可保持19 h;给药后24 h,pH值为5.32±0.24,乙酸含量为(0.38±0.15)g.L-1,为给药量的11%。阴道组织中乙酸浓度峰值为600μg.g-1,达峰时间为2 h,平均保留时间为7.73 h,药物动力学参数符合二室模型。结论乙酸阴道热敏凝胶剂符合阴道用药的要求。     

羧甲基壳聚糖阴道温敏性原位凝胶在家兔细菌性及霉菌性阴道炎中的疗效考察

摘要：目的:考察羧甲基壳聚糖（CCS）阴道温敏原位凝胶治疗家兔细菌性及霉菌性阴道炎的疗效,并观察其对阴道的刺激性。方法:选取健康雌性家兔分别制造细菌性及霉菌性阴道炎模型,并各随机分为5组:模型组、阳性对照组（碘伏组或保妇康栓组）、CCS原位凝胶低、中、高剂量组,每组10只,另分别取10只健康雌性家兔作为空白对照组;模型组及空白对照组给予无菌生理盐水,碘伏组给予2 ml市售碘伏消毒液,保妇康栓组给予0.08 g·kg-1栓剂,CCS原位凝胶组分别给予0.06,0.12,0.24 g·kg-1CCS原位凝胶;各组家兔每天给药1次,连续7 d。每日观察家兔一般行为,并于末次给药12 h后观察外阴、阴道红肿及分泌物情况,临床微生物检验阴道及宫颈分泌物含染病病源菌。另取36只雌性家兔随机分为阴性对照组、碘伏组、保妇康栓组、CCS低、中、高剂量组,连续给药7 d后处死家兔,取出完整阴道纵向切开,肉眼观察,并选取阴道组织HE染色后,显微镜下观察,按阴道黏膜刺激反应评分标准评分,并按阴道黏膜刺激强度分级标准确定刺激性强度。结果:各药物治疗组在治疗7 d后均取得显著疗效,与模型组相比,阴道红肿率及病原菌阳性率均显著下降（P<0.05）。与阳性对照组相比,CCS低剂量组疗效较差,高剂量组疗效更好,差异有统计学意义（P<0.05）,中剂量组效果相当（P>0.05）;刺激性实验表明,阳性对照药品及CCS高浓度组对家兔阴道有极轻微刺激,CCS低、中浓度均对阴道无刺激。结论:CCS温敏性原位凝胶对家兔细菌性和霉菌阴道炎有较好的治疗作用,且无明显阴道刺激性。     

纳米银凝胶在家兔体内的生殖器官毒性及体外细胞毒性研究

目的:为了明确纳米银凝胶的体内生殖器官毒性和体外细胞毒性。方法:通过检测和观察生殖器官内银的蓄积、组织病理学及超微病理的变化,对纳米银凝胶在家兔体内生殖器官的吸收、蓄积和组织细胞的病理变化以及体外细胞毒性进行了系统研究。结果:研究结果显示兔子阴道内连续给药6 d后生殖器官内银含量均较对照组显著增加,在生殖器官组织内造成不同程度的纳米银蓄积,引起了阴道粘膜组织和子宫内膜组织,乃至卵巢组织不同程度的超微病理变化,诸如线粒体肿胀、内质网扩张及空泡形成等。体外细胞试验同样显示了纳米银凝胶暴露后,纳米银在细胞内的蓄积、细胞内大量的空泡形成及微核现象。结论:这些结果提示纳米银凝胶持续大量应用会产生一定的毒性反应。子宫内膜及卵巢组织内许多凋亡细胞的存在,提示纳米银凝胶可能存在遗传毒性及生殖毒性风险。结合既往研究的DNA微阵列分析,ROS相关的金属结合蛋白家族基因的显著增高,JAK-STAT信号通路的激活可能是纳米银凝胶引起生殖器官组织超微病理变化的主要作用机制。     

采用优化家兔阴道刺激模型评价乙酸阴道热敏凝胶剂的阴道免疫炎症毒性研究

目的 采用优化家兔阴道刺激模型评价乙酸阴道热敏凝胶剂(Acetic acid vaginal thermosensitive in situ gel,Acid-TISG)对家兔阴道黏膜的免疫炎症毒性。方法 将24只雌性家兔随机分为空白凝胶组、Acid-TISG组(12.11mg.ml_-1)、阳性壬苯醇醚(Nonoxynol-9,N-9)凝胶对照组(5.6 mg.ml_-1),阴道给予3 ml凝胶,每天一次,连续10天,给药前和给药后24h收集家兔阴道灌洗液(Cervicovaginal Lavage,CVL),检测CVL中白介素6(Interleukin-6,IL-6)和肿瘤坏死因子α(Tumor Necrosis Factor α,TNF-α)的含量。家兔处死后取阴道组织做病理学检查,HE染色观察家兔阴道黏膜组织损伤并进行评分。运用免疫组化技术检测阴道黏膜内白细胞共同抗原-CD45RB和核增殖抗原(Proliferating Cell Nuclear Antigen, PCNA)的表达。结果 与空白凝胶组相比,Acid-TISG组黏膜上皮完整,无明显炎症浸润、充血和水肿(P＞0.05),N-9凝胶组黏膜上皮出现脱落,固有层炎性细胞明显增多(P＜0.05);空白凝胶组和Acid-TISG组阴道黏膜无明显CD45RB阳性表达,N-9凝胶组阴道粘膜固有层出现较多的CD45RB阳性表达细胞。与空白凝胶组相比,Acid-TISG组的阴道黏膜PCNA表达增多,但无显著性差异(P＞0.05),N-9凝胶组的阴道黏膜PCNA阳性表达细胞明显增多(P＜0.05)。与给药前相比,给药后24h时CVL中IL-6和TNF-α含量在空白凝胶组和Acid-TISG组无明显改变(P＞0.05),N-9凝胶组明显升高(P＜0.05)。结论Acid-TISG对家兔阴道黏膜无明显免疫炎症毒性,优化的家兔阴道刺激模型可用来评价生殖道外用药物对阴道黏膜的免疫炎症毒性。     

dl-扁桃酸热敏凝胶对家兔阴道黏膜刺激性的组织病理学研究

目的运用组织病理学方法评价dl-扁桃酸热敏凝胶(MA Gel)对家兔阴道黏膜的刺激性。方法 40只雌性日本大耳兔随机分为基质对照组、MA Gel低剂量组(10 mg/ml)、中剂量组(20 mg/ml)、高剂量组(40 mg/ml)和市售壬苯醇醚凝胶(40 mg/ml, N-9 Gel)组。阴道给予凝胶2.5 ml,1次/d,连续给药10 d。末次给药72 h后处死家兔,取阴道组织,观察阴道黏膜组织病理学改变;运用原位缺口末端标记法(TdT-mediated dUTP Nick-End Labeling, TUNEL)检测阴道黏膜上皮细胞凋亡;运用免疫组织化学法检测阴道黏膜内增殖细胞核抗原(Proliferating cell nuclear antigen,PCNA)的阳性表达。结果家兔阴道黏膜组织苏木素-伊红(HE)染色显示,MA Gel的黏膜刺激性呈剂量依赖性,高剂量组可造成黏膜上皮轻度损伤、固有层毛细血管充血和炎症细胞浸润(病理学评分4.35±1.04),与N-9 Gel组7.88±1.67相比,差异有统计学意义(P0.05)。TUNEL检测结果显示,MA Gel 3个剂量组均出现不同数量的凋亡细胞阳性表达,分布于上皮层和黏膜固有层。与基质对照组相比,MA Gel各组的阴道黏膜细胞凋亡细胞数目差异无统计学意义(P0.05)。MA Gel 3个剂量组黏膜上皮固有层均出现PCNA阳性表达,且表达量与扁桃酸浓度呈正相关,提示MA Gel可诱发炎症细胞轻度的活化增殖,但较N-9 Gel组为轻。结论 MA Gel对家兔阴道黏膜有轻度的刺激性,但刺激性远低于市售N-9 Gel。MA Gel安全性较好,作为阴道外用避孕新药具有潜在开发前景。     

结合雌激素阴道软胶囊初步药效学及局部毒性研究

目的研究结合雌激素阴道软胶囊的局部毒性及初步药效学。方法家兔阴道给药后考察结合雌激素软胶囊内容物的急性毒性和对阴道黏膜的刺激性;小鼠阴道给药后考察结合雌激素软胶囊内容物对未成年雌性小鼠子宫指数的影响。结果家兔阴道给药7 d后,家兔阴道外观及家兔阴道组织病理切片未见异常;一次性给予家兔相当于人体临床用量75倍剂量时未见家兔出现死亡及饮食、自主活动、行为改变,皮肤、眼睛、黏膜、毛色均无异常,呼吸、心率等指标与对照组比较差异无统计学意义(P>0．05);按低、中、高不同剂量软胶囊内容物小鼠阴道给药后,受试物中剂量组与阳性对照组之间小鼠子宫指数差异无统计学意义(P>0．05),各组与阴性对照组之间差异有统计学意义(P<0．01)。结论结合雌激素阴道软胶囊无局部毒性,具有雌激素活性。     

Assessment of total silver and silver nanoparticle extraction from medical devices

There is concern over the release of silver nanoparticles (AgNPs) from medical devices due to their potential toxicological consequences inside the body. Towards developing the exposure component of a risk assessment model, the purpose of this study was to determine the amount and physical form of silver released from medical devices. Scanning electron microscopy was used to confirm that three of five marketed medical devices contained nanosilver coatings (mean feature sizes 115–341 nm). Aqueous device extracts (water, saline and human plasma) were analyzed with inductively coupled plasma mass spectrometry, ultraviolet–visible spectroscopy, dynamic light scattering, transmission electron microscopy, and nanoparticle tracking analysis. The amount of silver extracted from the devices ranged from 1 × 10⁻¹ to 1 × 10⁶ ng/cm² (conditions ranged from 37 to 50 °C, over one hour to seven days). The results further indicated that one of the five devices (labeled MD1) released significantly more AgNPs than the other devices. This data suggests that some but not all devices that are formulated with nanosilver may release detectable levels of AgNPs upon extraction. Further work is underway to quantitate the proportion of silver released as AgNPs and to incorporate this data into a risk assessment for AgNP exposure from medical devices.     

Structures of silver sulfonamides

The structures of silver sulfonamides were found to depend highly on the substituent at the amide nitrogen of the sulfonamide. Silver is coordinated to that nitrogen and the sulfonamide is in the amido form if no substituent is present or if the substituent is a phenyl, acetyl, or 2-pyrimidyl group. If the substituent is a 2-thiazolyl or 2-pyridinyl group, the sulfonamide is in the imido form and silver coordinates to the nitrogen of the substituent. Depending on the number of suitable donor atoms per sulfonamide, the silver compounds are charged or uncharged and the primary amino group may be involved in complexation.     

Uptake and elimination kinetics of silver nanoparticles and silver nitrate by Raphidocelis subcapitata: The influence of silver behaviour in solution

Abstract

Raphidocelis subcapitata is a freshwater algae species that constitutes the basis of many aquatic trophic chains. In this study, R. subcapitata was used as a model species to investigate the kinetics of uptake and elimination of silver nanoparticles (AgNP) in comparison to silver nitrate (AgNO₃) with particular focus on the Ag sized-fractions in solution. AgNP used in this study were provided in a suspension of 1?mg Ag/l, with an initial size of 3–8?nm and coated with an alkane material. Algae was exposed for 48?h to both AgNP and AgNO₃ and sampled at different time points to determine their internal Ag concentration over time. Samples were collected and separated into different sized fractions: total (Ag_tot), water column Ag (Ag_water), small particulate Ag (Ag_small.part.) and dissolved Ag (Ag_dis). At AgNO₃ exposures algae reached higher bioconcentration factor (BCF) and lower elimination rate constants than at AgNP exposures, meaning that Ag is more readily taken up by algae in its dissolved form than in its small particulate form, however slowly eliminated. When modelling the kinetics based on the Ag_dis fraction, a higher BCF was found. This supports our hypothesis that Ag would be internalised by algae only in its dissolved form. In addition, algae images obtained by Coherent Anti-stokes Raman Scattering (CARS) microscopy demonstrated large aggregates of nanoparticles external to the algae cells with no evidence of its internalisation, thus providing a strong suggestion that these AgNP were not able to penetrate the cells and Ag accumulation happens through the uptake of Ag ions.     

Influence of hardness on the bioavailability of silver to a freshwater snail after waterborne exposure to silver nitrate and silver nanoparticles

Abstract

The release of Ag nanoparticles (AgNPs) into the aquatic environment is likely, but the influence of water chemistry on their impacts and fate remains unclear. Here, we characterize the bioavailability of Ag from AgNO₃ and from AgNPs capped with polyvinylpyrrolidone (PVP AgNP) and thiolated polyethylene glycol (PEG AgNP) in the freshwater snail, Lymnaea stagnalis, after short waterborne exposures. Results showed that water hardness, AgNP capping agents, and metal speciation affected the uptake rate of Ag from AgNPs. Comparison of the results from organisms of similar weight showed that water hardness affected the uptake of Ag from AgNPs, but not that from AgNO₃. Transformation (dissolution and aggregation) of the AgNPs was also influenced by water hardness and the capping agent. Bioavailability of Ag from AgNPs was, in turn, correlated to these physical changes. Water hardness increased the aggregation of AgNPs, especially for PEG AgNPs, reducing the bioavailability of Ag from PEG AgNPs to a greater degree than from PVP AgNPs. Higher dissolved Ag concentrations were measured for the PVP AgNPs (15%) compared to PEG AgNPs (3%) in moderately hard water, enhancing Ag bioavailability of the former. Multiple drivers of bioavailability yielded differences in Ag influx between very hard and deionized water where the uptake rate constants (k_uw, l?g^?1?d^?1?±?SE) varied from 3.1?±?0.7 to 0.2?±?0.01 for PEG AgNPs and from 2.3?±?0.02 to 1.3?±?0.01 for PVP AgNPs. Modeling bioavailability of Ag from NPs revealed that Ag influx into L. stagnalis comprised uptake from the NPs themselves and from newly dissolved Ag.     

Antimicrobial effects of silver nanoparticles

The antimicrobial effects of silver (Ag) ion or salts are well known, but the effects of Ag nanoparticles on microorganisms and antimicrobial mechanism have not been revealed clearly. Stable Ag nanoparticles were prepared and their shape and size distribution characterized by particle characterizer and transmission electron microscopic study. The antimicrobial activity of Ag nanoparticles was investigated against yeast, Escherichia coli, and Staphylococcus aureus. In these tests, Muller Hinton agar plates were used and Ag nanoparticles of various concentrations were supplemented in liquid systems. As results, yeast and E. coli were inhibited at the low concentration of Ag nanoparticles, whereas the growth-inhibitory effects on S. aureus were mild. The free-radical generation effect of Ag nanoparticles on microbial growth inhibition was investigated by electron spin resonance spectroscopy. These results suggest that Ag nanoparticles can be used as effective growth inhibitors in various microorganisms, making them applicable to diverse medical devices and antimicrobial control systems.     

Importance of surface coatings and soluble silver in silver nanoparticles toxicity to Daphnia magna

Silver nanoparticles (AgNPs) are now widely used in antibacterial and personal care products. However, the underlying physicochemical mechanisms leading to the toxicity of AgNPs are still under debate. The present study revealed the different effects of three surface coatings (including lactate, polyvinylpyrrolidone, and sodium dodecylbenzene sulfonate, as AgNPs-L, AgNPs-P and AgNPs-S, respectively) on the acute toxicity of AgNPs to a model freshwater cladoceran Daphnia magna. Significant difference in mortality was observed among these three surface coatings of AgNPs, with the 48-h 50% lethal concentrations (48-h LC50s) of AgNPs-L, AgNPs-P and AgNPs-S being 28.7, 2.0 and 1.1 μg/L, respectively. In contrast, when the daphnids were exposed to soluble Ag released from AgNPs-L and AgNPs-P, the difference in the 48-h LC50s between the two surface coatings (1.1 μg/L and 0.57 μg/L, respectively) decreased significantly. These 48-h LC50s were comparable to that of AgNO? (0.88 μg/L), indicating that soluble Ag was the primarily cause of the observed toxicity of AgNPs. Indeed, the surface coatings influenced the dissolution of AgNPs into soluble Ag, resulting in the different toxicities of AgNP to the daphnids. Additionally, the 48-h bioaccumulation of AgNPs in daphnids was also dependent on the characteristics of the nanoparticles, such as particle size and surface coating. Our results point to the need to consider the effects of surface coating on the toxicity of AgNPs in environmental risk assessments.     

Comparison of in vitro toxicity of silver ions and silver nanoparticles on human hepatoma cells

Scientific information on the potential harmful effects of silver nanoparticles (AgNPs) on human health severely lags behind their exponentially growing applications in consumer products. In assessing the toxic risk of AgNP usage, liver, as a detoxifying organ, is particularly important. The aim of this study was to explore the toxicity mechanisms of nano and ionic forms of silver on human hepatoblastoma (HepG2) cells. The results showed that silver ions and citrate‐coated AgNPs reduced cell viability in a dose‐dependent manner. The IC50 values of silver ions and citrate‐coated AgNPs were 0.5 and 50 mg L^?1, respectively. The LDH leakage and inhibition of albumin synthesis, along with decreased ALT activity, indicated that treatment with either AgNP or Ag ions resulted in membrane damage and reduced the cell function of human liver cells. Evaluation of oxidative stress markers demonstrating depletion of GSH, increased ROS production, and increased SOD activity, indicated that oxidative stress might contribute to the toxicity effects of nano and ionic forms of silver. The observed toxic effect of AgNP on HepG2 cells was substantially weaker than that caused by ionic silver, while the uptake of nano and ionic forms of silver by HepG2 cells was nearly the same. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 679–692, 2016.