三种方法提取血清外泌体的比较研究

目的　比较超速离心法、沉淀法和尺寸排阻色谱法提取血清外泌体的得率和纯度。方法　选取8例健康体检受试者血清,用三种方法提取外泌体,采用纳米流式检测仪、透射电子显微镜和Pierce BCA 蛋白检测试剂盒分别检测外泌体颗粒数、粒径分布、形态特征和蛋白浓度;分析外泌体颗粒数。外泌体颗粒数和蛋白总量比值、Triton X-100处理前后颗粒数变化来评估外泌体提取的得率和纯度。结果　超速离心法、沉淀法和尺寸排阻色谱法提取外泌体粒径均分布在40~150 nm;透射电子显微镜下均能观察到呈明显茶托状的双层膜结构,但沉淀法提取的外泌体混有聚乙二醇聚合物及蛋白大颗粒;蛋白质印迹结果显示三种方法提取的外泌体均表达外泌体标记蛋白;沉淀法提取的血清外泌体颗粒数高于超速离心法和尺寸排阻色谱法[(1.11±0.07)×1011 particley/ml vs (2.54±0.04)×1010particley/ml, (7.51±0.89)×1010particley/ml],差异均有统计学意义（P<0.05）,尺寸排阻色谱法提取外泌体颗粒数高于超速离心法（P<0.001） ;尺寸排阻色谱法提取外泌体纯度高于超速离心法和沉淀法[(5.66±0.45)×107particles/μg vs (7.65±0.42)×106 particles/μg, (3.18±0.45)×106 particles/μg],超速离心法提取外泌体纯度高于沉淀法,差异均有统计学意义（P<0.05）。结论　超速离心法、沉淀法和尺寸排阻色谱法均能提取出血清外泌体,超速离心法步骤繁琐,耗时较长,提取外泌体得率与纯度较低;沉淀法操作简单,耗时短,提取外泌体得率最高,但外泌体纯度最低;尺寸排阻色谱法操作简单,耗时短,提取外泌体的纯度最高;相比而言,尺寸排阻色谱法显然优于超速离心法和沉淀法。     

超速离心法与QIAGEN膜亲和柱法提取前列腺癌细胞培养上清外泌体的方法学比较

目的 对超速离心法与QIAGEN膜亲和柱法提取前列腺癌细胞上清外泌体优缺点进行比较,为外泌体相关基础实验研究及临床检测提供方法学参考。方法 收集前列腺癌细胞上清分别用两种方法提取外泌体,进行电镜鉴定、BCA定量测定蛋白浓度、Western blot检测标志蛋白以及Zetaview颗粒粒径、浓度及电势分析,比较两种方法提取外泌体的优缺点。结果 电镜下两种方法均可观察到具有膜结构的典型的类似于茶托状结构的外泌体,但膜亲和柱法提取外泌体背景中有类似蛋白聚合物等杂质的存在,超离背景纯净,每个视野下的外泌体数量要明显多于QIAGEN膜亲和柱法; Western blot测定标志蛋白,根据BCA定量同等上样量超速离心法可观察到明显的β-actin,ALIX,CD63以及EGFR条带,而QIAGEN膜亲和柱法仅可观察到β-actin,ALIX以及EGFR条带,但均较超速离心法条带弱; Zetaview粒径分析,超速离心法粒径分布峰值为116 nm,膜亲和柱法提取外泌体分布峰值为122 nm,均符合外泌体的粒径分布范围; 电势分析两者均为负值,符合外泌体的电势分布; 而颗粒数与蛋白浓度比值超速离心法提取外泌体要高于QIAGEN膜亲和柱法(t=13.47,P<0.01)。结论 超速离心法与膜亲和柱法均可以从细胞上清中提取外泌体,超速离心法步骤繁琐,时间较长,但外泌体纯度高,杂质少,BCA蛋白定量与实际所含外泌体蛋白量基本一致; QIAGEN膜亲和柱法方法简单,可快速从细胞上清中提取到外泌体,但纯度不及超速离心法,BCA蛋白定量比实际所含外泌体蛋白量要高,超速离心法适用于外泌体的各方面研究,而QIAGEN膜亲和柱法由于有杂蛋白的污染,因此更适用于外泌体核酸分析相关研究。     

试剂盒法与差速超速离心法所提血清外泌体的形态学比较

目的:比较常用的外泌体提取方法,获得可靠有效的血清外泌体提取方法。方法:电镜下观察鉴定差速超速离心法、试剂盒法及组合优化方法所提外泌体的形态;蛋白印迹法检测外泌体标志蛋白CD63的表达。结果:差速超速离心法提取的外泌体呈茶托样双层膜结构,直径100 nm左右;试剂盒法提取的外泌体颗粒多,大小50~200 nm之间,无明显的立体结构,经差速超速离心法纯化后形态与差速超速离心法类似,经试剂盒法纯化后与仅使用一次试剂盒提取的外泌体无明显形态差异;四种方法所提外泌体表达标志蛋白CD63。结论:差速超速离心法是更为可靠且有效的外泌体提取方法。     

背根神经节持续受压大鼠血浆外泌体中差异表达的miRNAs及其功能分析

目的：探究背根神经节持续受压致神经病理性疼痛大鼠血浆外泌体中差异表达的miRNAs及其功能。方法：采用超速离心法提取并纯化健康对照大鼠及背根神经节持续受压大鼠血浆外泌体,通过透射电镜获取外泌体形态,采用Western Blot法进行外泌体标记蛋白鉴定;提取外泌体总RNA,Illumina HiSeq高通量测序检测miRNAs表达谱;生物信息学分析背根神经节持续受压后外泌体miRNAs的差异表达,预测其靶基因并分析其靶基因的潜在功能。结果：透射电镜、纳米粒径分析及Western Blot的结果均显示外泌体提取成功,电镜下形态为典型茶托样,平均粒径为136.2±1.56nm,标记蛋白TSG101、CD9均为阳性;高通量测序分析显示,实验组有152个血浆外泌体来源miRNAs表达显著改变,其中104个表达显著上调,48个表达显著下调。GO分析显示靶基因功能富集细胞器、细胞质上,与离子结合、ATP结合、催化活性等功能相关,参与系统的发展、细胞器物质代谢过程等。差异表达miRNA靶基因的KEGG通路分类统计显示在细胞间紧密连接、RAP1信号通路、PI3K-AKt信号通路及轴突导向、突触囊泡周期等...     

胆汁中外泌体及外泌体RNA提取方法比较

目的比较超速离心法和聚合沉淀法分离胆汁外泌体及外泌体RNA的差异。方法收集2017年6月至2018年12月在南京医科大学第二附属医院消化内科行内窥镜逆行胆囊-胰腺造影术(ERCP)患者术中胆汁,分别采用超速离心法和聚合沉淀法对胆汁进行分离。采用透射电镜法观察提取外泌体的形态;采用粒径分析所得粒子的直径;采用流式细胞术检测表面标记物CD63和CD81的表达差异;Trizol法提取RNA,分析RNA水平及A260/A280;采用实时荧光定量(RT-PCR)检测4种miRNA表达,分析两种方法所得平均CT值的差异。结果超速离心法和聚合沉淀法所得外泌体形态和粒径分布无明显差异;流式细胞术分析显示,超速离心法所得外泌体CD63和CD81阳性率较高。RNA分析显示,聚合沉淀法提取RNA水平较高,检测的miR-671-5p、miR-126-3p、miR-210-3p、miR-483-5p平均CT值较小。结论超速离心法和聚合沉淀法均可提取出一定数量的外泌体,其中超速离心法得到的RNA较纯,而聚合沉淀法得到的RNA水平较高,可用于稀缺标本的检测。因此,进一步寻找高敏感、高特异、经济便捷的方法仍然十分必要。     

3种分离尿外体方法比较

目的对超速离心法、超滤法和沉淀法3种分离尿液中外体(exosome)方法进行比较,为临床研究选择合适的分离方法提供依据。方法用超速离心法、超滤法和沉淀法3种方法分别从4例健康人和2例慢性肾脏病(CKD)患者尿液中分离exosome。用透射电镜观察尿exosome形态结构,BCA法检测exosome总蛋白质浓度,分别在尿体积及exosome总蛋白质浓度相同上样条件下,用western blot(WB)检测exosome标志蛋白TSG101和Alix。结果 3种分离方法均能获得直径为40~100 nm的圆形膜性囊泡;WB能检测到超速离心法及超滤法分离得到的exosome标志蛋白,而沉淀法未检测到;超滤法尿exosome总蛋白质浓度远高于超速离心法及沉淀法;超速离心法尿体积及exosome总蛋白质浓度相同情况下上样时所有样本均能检测到exosome标志蛋白,且表达量在3种方法中均最多;超滤法尿体积相同情况下上样,4例健康人均能检测到exosome标志蛋白,而以exosome总蛋白质浓度相同上样时,仅有2例健康人能检测到标志蛋白,但2例CKD患者在此两种上样方式未检测到标志蛋白;沉淀法两种上样条件下所有样本均未检测到exosome标志蛋白。结论超速离心法分离尿exosome效果较好,开展尿exosome临床研究时建议选择超速离心法。     

人角膜基质间充质干细胞外泌体的分离制备及鉴定

目的　制备人角膜基质间充质干细胞（human corneal stromal mesenchymal stem cells,HC-MSCs)外泌体并进行生物学鉴定。方法　采用组织块培养技术获得HC-MSCs,利用超速离心法从HC-MSCs培养上清中分离制备外泌体,通过蛋白免疫印迹、透射电镜、纳米颗粒跟踪分析法进行鉴定。结果　分离的纳米囊泡表达外泌体特异性标志蛋白CD63,CD81和TSG101,在透射电子显微镜下可见其呈圆形、卵圆形和杯盘状囊泡,直经113.3 nm。结论　从HC-MSCs培养上清中可分离出具有典型结构及特征的外泌体,为HC-MSCs外泌体的作用机制及其在眼科的转化应用研究奠定坚实的基础。     

脐带间质干细胞来源外泌体的安全性评估

目的评估脐带间质干细胞(huc-MSC)来源的外泌体作为一种临床应用生物制品的自身安全性。方法超速离心法分离外泌体,利用蛋白质印迹法(WB)检测外泌体表面标记CD9、CD63,利用纳米颗粒跟踪分析技术(NTA)检测外泌体的大小及浓度。溶血试验、全身过敏性试验和血常规检测验证外泌体的自身安全性。结果外泌体表达外泌体特异性分子CD9、CD63,外泌体不会引起溶血、全身过敏反应及血常规异常。结论脐带间质干细胞来源的外泌体具有外泌体的一般特征,且具有一定的安全性,为外泌体今后在临床上的应用提供了实验依据。     

尿液外泌体提取优化及冷冻对外泌体RNA含量的影响

目的对现有沉淀法提取尿液外泌体进行优化,并探讨不同的储存条件对尿液外泌体RNA含量的影响。方法分别用先浓缩后沉淀法和直接沉淀法提取人尿液标本中的外泌体,比较两种方法的分离效率和成本;采用ExoQuick-TC~(TM)沉淀试剂盒提取外泌体;纳米粒子跟踪分析技术(NTA)检测外泌体浓度及粒径分布;动态光散射技术(DLS)检测外泌体电位;透射电镜(TEM)观察外泌体形态;western blot检测外泌体标记分子CD63及Alix蛋白的表达;另取10例临床尿液标本验证先浓缩后沉淀的提取方法,以证实优化方法的可靠性。实时荧光定量聚合酶链反应(qRT-PCR)检测20例前列腺癌患者尿液外泌体经过反复冻融(新鲜、1、3、5次)及9例前列腺癌患者尿液外泌体在-80℃冷冻不同时间(新鲜、1周、2周、1个月)时外泌体RNA标志物let-7c和PSA mRNA的表达量,并采用Wilcoxon秩和检验进行统计学分析。结果 NTA检测两种方法提取的外泌体粒径分布为30～150 nm,均为单峰;DLS结果显示,两种方法提取的外泌体电位均呈负值;透射电镜观察两种方法提取的外泌体大小较为一致,直径分布为30～150 nm;western blot检测证实优化方法存在外泌体标记分子CD63及Alix蛋白;10例尿液标本提取的外泌体浓度约为10~9～10~(11) particles/mL。外泌体经过5次冻融后let-7c和PSA mRNA含量明显下降,Z值分别为-1.69、-1.73(P均0.05)。而外泌体在-80℃冷冻1个月后其RNA含量保持稳定。结论优化后的外泌体提取方法在保证外泌体浓度和质量的前提下极大地降低成本,分离得到的外泌体在-80℃短时间冷冻后其RNA含量保持稳定,但不能反复冻融超过5次。     

外泌体通过促进神经轴突再生治疗神经损伤性疾病

目的探索骨髓间充质干细胞外泌体对神经轴突再生的影响规律。方法提取SD大鼠骨髓间充质干细胞并通过细胞形态及流式细胞学进行鉴定,利用超速离心法提取细胞上清液中的外泌体并通过透射电镜、粒径及蛋白免疫印迹法进行鉴定。培养肾上腺嗜铬细胞瘤(PC12)细胞及提取SD大鼠原代神经干细胞。将PC12细胞分为空白对照组、30μg外泌体组及60μg外泌体组。通过荧光标记大鼠外泌体观察靶细胞吞噬情况。采用免疫荧光染色检测PC12细胞及神经干细胞轴突生长情况,通过蛋白免疫印迹法检测细胞神经丝蛋白(NF-200)和生长相关蛋白(GAP-43)的表达水平。结果细胞形态及流式细胞学证实成功提取出大鼠骨髓间充质干细胞,透射电镜、粒径及蛋白免疫印迹法检查证实成功提取出骨髓间充质干细胞外泌体,外泌体可以被神经细胞摄取促进PC12细胞及神经干细胞轴突生长及轴突蛋白表达,并且这一促进作用随着外泌体量的增加而增强,3组PC12细胞及神经干细胞轴突长度及轴突蛋白表达比较差异均有统计学意义(P均<0.001)。结论骨髓间充质干细胞外泌体可通过促进神经轴突再生进而发挥神经损伤修复作用。     

Analysis of the regenerative capacity of human serum exosomes after a simple multistep separation from lipoproteins

Due to the abundance of lipoproteins in blood, it is challenging to characterize the biological functions and components of blood‐derived extracellular vesicles. The aim of this study was to develop a multiple‐step purification protocol to separate serum exosomes from serum proteins and lipoproteins and assess their regenerative potential. Exosomes were isolated by concentrating them in human serum using ultracentrifugation (UC), followed sequentially by density gradient (DG) UC and size exclusion chromatography (SEC). Purity and characterization were assessed by western blots, Lipoprint®, enzyme‐linked immunosorbent assay, electron microscopy, mass spectrometry, and nanoparticle tracking analysis. Functionality was assessed by cell proliferation analysis and with an in vivo subcutaneous angiogenesis model. SEC alone isolated nano‐sized vesicles possessing vesicle markers TSG101 and CD9, but there was a substantial presence of apolipoprotein B, predominantly derived from very‐low‐ and intermediate‐density lipoprotein particles. This was reduced to an undetectable level using the combined UC DG SEC approach. Mass spectrometry identified 224 proteins in UC DG SEC isolates relative to the 135 from SEC, with considerable increases in exosome‐related proteins and reductions in lipoproteins. A consistent but limited increase in human dermal fibroblast proliferation and evidence of neovascularization enhancement were observed after exposure to UC DG SEC exosomes. An UC DG SEC purification protocol considerably improved the removal of lipoproteins during isolation of serum exosomes. The purified exosomes stimulated cell proliferation and potentially increased an in vivo angiogenic response. This multistep purification allows for more accurate identification of serum exosome functional activity and composition.     

Inter-Laboratory Comparison of Extracellular Vesicle Isolation Based on Ultracentrifugation

Background/AimsExtracellular vesicles (EVs), including microvesicles and exosomes, deliver bioactive cargo mediating intercellular communication in physiological and pathological conditions. EVs are increasingly investigated as therapeutic agents and targets, but also as disease biomarkers. However, a definite consensus regarding EV isolation methods is lacking, which makes it intricate to standardize research practices and eventually reach a desirable level of data comparability. In our study, we performed an inter-laboratory comparison of EV isolation based on a differential ultracentrifugation protocol carried out in 4 laboratories in 2 independent rounds of isolation.MethodsConditioned medium of colorectal cancer cells was prepared and pooled by 1 person and distributed to each of the participating laboratories for isolation according to a pre-defined protocol. After EV isolation in each laboratory, quantification and characterization of isolated EVs was collectively done by 1 person having the highest expertise in the respective test method: Western blot, flow cytometry (fluorescence-activated cell sorting [FACS], nanoparticle tracking analysis (NTA), and transmission electron microscopy (TEM).ResultsEVs were visualized with TEM, presenting similar cup-shaped and spherical morphology and sizes ranging from 30 to 150 nm. NTA results showed similar size ranges of particles in both isolation rounds. EV preparations showed high purity by the expression of EV marker proteins CD9, CD63, CD81, Alix, and TSG101, and the lack of calnexin. FACS analysis of EVs revealed intense staining for CD63 and CD81 but lower levels for CD9 and TSG101. Preparations from 1 laboratory presented significantly lower particle numbers (p < 0.0001), most probably related to increased processing time. However, even when standardizing processing time, particle yields still differed significantly between groups, indicating inter-laboratory differences in the efficiency of EV isolation. Importantly, no relation was observed between centrifugation speed/k-factor and EV yield.ConclusionsOur findings demonstrate that quantitative differences in EV yield might be due to equipment- and operator-dependent technical variability in ultracentrifugation-based EV isolation. Furthermore, our study emphasizes the need to standardize technical parameters such as the exact run speed and k-factor in order to transfer protocols between different laboratories. This hints at substantial inter-laboratory biases that should be assessed in multi-centric studies.     

人骨髓来源间充质干细胞分泌外泌体特性研究

本研究旨在探讨正常人骨髓间充质干细胞（bonemarrowmesenchymalstemcells,BMMSC）分泌的外泌体（exosome）免疫调节功能及支持血管形成能力。收集第4—6代BMMSC上清液提取外泌体,应用透射电镜观察其形态,蛋白印迹法检测其表面特异标志CD9,bicinchoninicacid（BCA）方法定量检测外泌体分泌量;与正常人外周血单个核细胞（peripheralbloodmononuclearcells,PBMNC）作用72h后用酶联免疫吸附试验（ELISA）法检测PBMNC分泌的γ-干扰素（IFN-γ）;实时定量PCR检测外泌体内免疫相关microRNA表达;共聚焦显微镜观察外泌体与人脐带静脉内皮细胞（humanumbilicalveinendothelialcells,HUVEC）的作用方式,与HUVEC共培养后观察其网状结构形成;应用裸鼠体内试验检测外泌体对血管形成能力的作用。结果表明,正常人骨髓间充质干细胞分泌的外泌体近似圆形,直径在40—160nm之间,表达表面标志CD9,其分泌量与接种细胞数呈线性关系,可抑制PBMNC分泌IFN-γ（P〈0．01）,内含有免疫相关microRNA如miR301、miR22和miR-let-7a等,能促进HUVEC网状结构形成和血管形成（P〈0．01）。结论：BMMSC分泌外泌体具有免疫调节功能与支持血管形成作用。     

Reproducibility and efficiency of serum-derived exosome extraction methods

ObjectivesExosomes are emerging as a source of biomarkers with putative prognostic and diagnostic value. However, little is known about the efficiency, reproducibility and reliability of the protocols routinely used to quantify exosomes in the human serum.Design and methodsWe used increasing amounts of the same serum sample to isolate exosomes using two different methods: ultracentrifugation onto a sucrose cushion and ExoQuick™. Quantitative analysis of serum-derived exosomes was performed by determining protein concentration (BCA assay) and the number of nanoparticles (Nanosight™ technology). Exosome quality was assessed by Coomassie staining and Western blotting for CD9, LAMP2 exosomal markers and a negative marker Grp94.ResultsCorrelation between serum volume and the number of isolated exosomes is significant for both methods when exosomes are quantified using protein concentration. However, when the number of nanoparticles is used to quantify exosomes, ExoQuick™ is the only reproducible and efficient method. CD9, LAMP2 and Grp94 exosomal markers are equivalently expressed in both methods. However, exosomes isolated using ultracentrifuge method are strongly contaminated with albumin and IgG.ConclusionExoQuick™ is an efficient and reproducible method to isolate exosomes for quantitative studies, whereas ultracentrifugation is not. Moreover, high albumin contamination of ultracentrifuged-derived exosomes impairs the use of protein concentration as a mean to quantify serum-derived exosomes.     

静电吸附法与生物素-亲和素桥联法制备PSMA抗体靶向纳米微泡的对比实验研究

目的比较静电吸附法和生物素-亲和素桥联法制备前列腺特异性膜抗原（PSMA）抗体靶向纳米微泡的物理特性和靶向黏附率。方法分别应用静电吸附法和生物素一亲和素桥联法制备PSMA抗体靶向的纳米微泡,检测其物理性状;光镜下观察两种微泡与前列腺癌细胞LNCaP和c4—2的靶向能力,并与胃癌细胞MKN45对照;对比分析两种靶向纳米微泡的粒径、浓度及与前列腺癌细胞结合能力。结果两种靶向纳米微泡均呈圆形,表面光滑,大小均匀,分散度好,无聚集,粒径均〈1000nm。静电吸附法制备的靶向纳米微泡平均粒径为（702．96±66．65）nm,平均浓度为（3．46±0．30）×10^8／ml,与前列腺癌细胞LNCaP黏附率（77．2±3．19）％,与C4-2细胞黏附率（61．00±4．47）％;生物素-亲和素桥联法制备的靶向纳米微泡平均粒径为（609．90±37．40）nm,平均浓度约（4．52±0．19）×10^8／ml,与前列腺癌细胞LNCaP黏附率（96．60±1．14）％,与C4-2细胞黏附率（94．00±1．58）％。两种靶向纳米微泡的粒径、浓度及与前列腺癌细胞黏附率比较,差异均有统计学意义（均P〈0．05）;与胃癌细胞MKN45均无黏附。结论生物素-亲和素桥联法是一种高效、灵敏、稳定、特异性强的PSMA抗体靶向微泡制备方法,较静电吸附法具有更大的优势。     

树突细胞分泌的exosomes生物学特性及功能的初步研究

目的建立树突细胞（DC）分泌的exosomes的制备方法，分析其生物学特性及其在抗肿瘤免疫中的功能。方法从正常人外周血单个核细胞中诱导未成熟DC（imDC），并使其负载K562细胞的抗原，然后用脂多糖（LPS）诱导DC成熟（mDC），通过超速离心结合膜超滤的方法分别提取imDC和mDC分泌的exosomes。检测exosomes的粒径并通过电子显微镜分析exosomes的形态，Western blot法检测exosomes的表面分子。比较mDC和imDC分泌的exosome8引起的针对K562抗原特异性T细胞的增殖、CD69的上调、细胞因子IFN．1的分泌及对肿瘤细胞的杀伤能力。结果制备的exosomes为碟状小囊泡，平均直径为72．3nm，表达CD80、CD86、HLA．DR、FasL、CD54和乳脂肪球-表皮生长因子-因子Ⅷ（MFG-E8）。与imDC相比，mDC分泌的exosomes CD80表达较高而MFG-E8的表达较低，并且在体外mDC分泌的exosomes能够更显著地引起特异性T细胞的增殖和免疫应答：在exosomes最适浓度下，T细胞增殖的吸光度值为O．50±0．01，激活T细胞的CD69上调，并且有（13．4±5．8）％T细胞扩增，（22．8±2．4）％的T细胞产生细胞因子IFN-1，对肿瘤细胞的杀伤率为（21．3±8．6）％。结论建立了简便、快速的exosomes分离纯化方法，所分离的exosomes具有引起抗肿瘤免疫应答的功能     

Influence of experimental parameters on the characteristics of poly(lactic acid) nanoparticles prepared by a double emulsion method

Nanoparticles were prepared by the double emulsion method (w/o/w), using methylene chloride as an organic solvent and polyvinyl alcohol (PVA) or human serum albumin (HSA) as a surfactant. Experimental parameters such as the preparation temperature, the solvent evaporation method, the internal aqueous phase volume, the surfactant concentration and the polymer molecular weight were investigated for particle size, the zeta potential, the residual surfactant percentage and the polydispersity index. Preparation parameters leading to particles with well-defined characteristics such as an average size around 200 nm and a polydispersity index lower than 0.1 were identified. The conditions were optimized to ensure protein encapsulation: a cool temperature, a short processing time, a sufficient internal aqueous phase and careful washing. It appeared that the higher the surfactant concentration in the external aqueous phase was, the smaller the particles, the lower the polydispersity index and the higher the residual amount of surfactant were. For PVA or HSA, the agreement between the convenient surfactant concentration and its critical aggregation concentration could be emphasized. Otherwise, an increased polymer molecular weight led both to a slightly decreased particle size and to a lower polydispersity index. Moreover, multilayer adsorption of PVA which does not depend on Poly(lactic-acid) molecular weight was exhibited. Finally, the zeta potential resulted from the polymer molecular weight and the residual PVA.