他克莫司载药胶束的制备与制剂学性质

目的以聚乙二醇单甲醚-聚乳酸[methoxy poly(ethylene glycol)-poly(lactide),m PEG-PLA]嵌段共聚物为载体材料,制备他克莫司载药胶束,考察其制剂学性质。方法采用薄膜分散法制备他克莫司载药胶束。超速离心法测定他克莫司载药胶束的载药量和包封率。以载药量、包封率、粒径以及粒径分布为评价指标,采用单因素试验和正交试验优化他克莫司载药胶束制备处方。应用透射电镜、粒度测定仪和zeta电位分析仪对他克莫司载药胶束的形态、粒径和电位进行表征。结果在优化条件下制备的他克莫司载药胶束载药量为7.77%,包封率为84.31%,胶束粒子外观圆整且分散良好,平均粒径为23.20 nm,PDI为0.058,zeta电位为-0.831 m V。结论单因素试验联合正交试验可有效地优化他克莫司m PEG-PLA胶束处方和制备工艺。优化条件下制备的他克莫司载药胶束具有较高的载药量和包封率,粒径较小且分布均匀,24 h内稳定性良好。     

白藜芦醇pH敏感共聚物胶束的制备与大鼠体内药动学

目的以pH敏感聚乙二醇单甲醚-聚乳酸-聚组氨酸(poly(ethylene glycol)-poly(D,L-lactide)-poly(L-histidine),m PEG-PLA-PHis)两亲性嵌段共聚物为载体制备白藜芦醇载药胶束,并进行大鼠体内药动学研究。方法采用薄膜分散法制备白藜芦醇载药胶束,分别采用动态光散射法、透射电子显微镜法和差示扫描量热法对载药胶束进行表征,以白藜芦醇溶液为对照,测定p H敏感载药胶束在大鼠体内药动学。结果白藜芦醇p H敏感胶束的载药量为10.25%,包封率为90.69%,平均粒径为54.1 nm,zeta电位为-12.7 m V。透射电子显微镜照片显示载药胶束呈类球形,分布均匀。白藜芦醇在胶束内核以分子或无定型形式存在。大鼠药动学结果显示,与白藜芦醇溶液相比,白藜芦醇载药胶束组的血药质量浓度-时间曲线下面积显著提高(P<0.05),清除率显著下降(P<0.05),平均滞留时间显著增加(P<0.05)。结论白藜芦醇胶束具有较小的粒径且分布较窄,包封率较高,具有较好的长循环效果。     

胆甾醇基-普鲁兰多糖疏水改性材料的制备及其自组装性质的研究

目的：经琥珀酸间隔臂将胆甾醇连接到普鲁兰分子链上,对普鲁兰多糖进行疏水改性,获得不同取代度的胆甾醇基-普鲁兰 (cholesterol-modified pullulan,CHSP)改性材料,并研究CHSP材料在水中的自组装性质。方法：利用1-乙基-（3-二甲基氨基丙基）碳酰二亚胺盐酸盐（EDC）和4-二甲氨基吡啶（DMAP）催化琥珀酰胆甾醇（cholesterol succinate,CHS）与普鲁兰多糖反应,将琥珀酰胆甾醇接枝在普鲁兰分子链的羟基上,得到疏水改性的普鲁兰多糖衍生物。应用傅立叶红外光谱仪(Fourier transform infrared spectroscopy,FT-IR),核磁共振仪(proton nuclear magnetic resonance,1H-NMR)对产物进行表征。利用透析法制备自组装纳米球。通过透射电镜(transmission electron microscopy,TEM),动态激光粒度分析仪(dynamic laser light scattering,DLS)表征了纳米粒的形态和粒径。以芘为荧光探针,通过荧光检测分析,测定CHSP的临界胶束浓度(critical micellar concentration,CMC)。结果：通过FT-IR,1H-NMR确证,合成了胆甾醇接枝普鲁兰多糖材料。制备材料在水溶液中能够自组装成纳米粒。结论：利用EDC和DMAP催化能够有效合成胆甾醇基-普鲁兰多糖改性材料,此制备方法简单且易于操作。改性材料在水中有效自组装成纳米粒,制备球形CHSP纳米粒有望成为疏水性药物载体。     

pH敏感多西紫杉醇纳米胶束的制备及其增强小鼠的抑瘤活性研究

抗肿瘤药物的非特异性释药是化疗药物对正常组织产生毒副作用的主要原因。利用纳米技术实现抗肿瘤药物的特异性与响应性递药是提高药物治疗效果、减少毒副反应的重要途径之一。本研究合成了pH敏感聚乙二醇单甲醚-聚乳酸-聚-β-氨基酯[poly(methoxy-ethylene glycol)-poly(lactic acid)-poly-(β-amino ester), PBAE]三嵌段共聚物,并制备胶束负载多西紫杉醇(DTX),以提高DTX抗肿瘤活性。通过开环聚合及Michael加成合成共聚物,核磁共振波谱法表征其结构和分子量,酸碱滴定法测定碱解离常数(p Kb),荧光法测定临界胶束浓度(CMC)。采用薄膜水化法制备载DTX纳米胶束,激光粒度仪测定粒径和分布及稳定性, HPLC测定载药量、包封率及累积药物释放量。载药胶束粒径为10～100 nm,粒径分布较窄;制备的2种载药胶束PBAE1-DTX和PBAE2-DTX中DTX的载药量分别为(5.3±0.10)%和(4.9±0.05)%,包封率分别为(93.8±1.70)%和(87.2±4.10)%。建立小鼠Lewis肺癌模型,利用尾静脉注射给药方式...     

β-环糊精-聚乙二醇-α-常春藤皂苷自组装胶束的制备和抗肿瘤活性研究

目的研究β-环糊精-聚乙二醇-α-常春藤皂苷自组装胶束的制备、溶血性和体内外抗肿瘤活性。方法采用自组装方法制备了β-环糊精-聚乙二醇-α-常春藤皂苷的纳米胶束,考察了胶束在大鼠全血中的溶血性,研究了胶束在不同细胞上的体外活性,以荷HT29肿瘤细胞的小鼠为模型,研究了胶束的体内抗肿瘤活性。结果β-环糊精-聚乙二醇-α-常春藤皂苷在水性介质中能自组装成粒径较小的纳米胶束。该胶束能显著降低α-常春藤皂苷的溶血性;在体外细胞实验中,胶束的抗肿瘤活性明显低于α-常春藤皂苷,这可能与在体外环境中α-常春藤皂苷不能从胶束中释放出来有关。β-环糊精-聚乙二醇-α-常春藤皂苷自组装胶束在HT29荷瘤小鼠体内显示非常好的抗肿瘤活性。结论β-环糊精-聚乙二醇-α-常春藤皂苷自组装胶束为皂苷的减毒增效提供了一个非常好的研究模式。     

聚乙二醇接枝壳聚糖聚离子胶束的制备与体外表征研究

目的制备载有甘草酸二铵(DG)的聚乙二醇接枝壳聚糖(mPEG-CS)聚离子胶束,考察胶束的理化性质及体外释放机制.方法通过壳聚糖烷基化合成mPEG-CS,以三聚磷酸钠为引发剂,利用静电相互作用制备载有DG的mPEG-CS聚离子胶束.考察胶束的形态、粒径和zeta电位.用透析袋法研究载药胶束在不同介质中的释放动力学.结果制得胶束的平均粒径为(38.2±0.9)nm;包封率为(97.6±0.7)%;胶束的稳定性良好;胶束的释放与释放介质的种类、pH值和浓度有关.结论mPEG-CS是一种良好的制备聚离子胶束的高分子材料,聚离子胶束是离子型药物的良好载体.     

TPGS修饰的羧甲基壳聚糖-大黄酸聚合物胶束的制备与表征

目的合成TPGS修饰的羧甲基壳聚糖-大黄酸(TPGS-CR)偶联物,其能在水中自组装成聚合物胶束,为难溶性抗肿瘤药物递送提供一个新型载体材料。方法通过酰化反应,将TPGS与大黄酸(R)接枝于羧甲基壳聚糖(CMCS)上,经FT-IR与~1H-NMR验证其结构。通过UV法测R取代度,HPLC法测定TPGS的取代度,荧光分光光度法测定CMC值,DLS法测定胶束的粒径、分布及电位,综合各因素,确定最佳合成工艺,并通过TEM和AFM观察其形态特征。通过MTT法评估TPGS-CR偶联物对HepG-2细胞毒性。用透析法初步考察其载药性能。结果 TPGS与R通过酰胺键接枝于CMCS上,形成TPGS-CR偶联物,确定n(TPGS)∶n(CMCS)∶n(R)=0.75∶1∶1为最佳合成投料比,此时R的摩尔取代度为(5.73±0.23)%,TPGS摩尔取代度为(0.28±0.05)%,粒径为(117.1±24.9) nm,Zeta电位为负值,对HepG-2细胞毒性较小,载药性能较好。结论所设计合成的TPGS-CR偶联物能在水中自组装成具有壳-核结构的胶束,粒径较小,可作为递送难溶性药物的载体材料。     

pH/氧化还原双敏感聚合物的合成与siRNA复合胶束的制剂学性质

目的合成聚乙二醇-聚乳酸-聚组氨酸-聚乙烯亚胺(methoxy-poly(ethylene glycol)-polylactide-polyhistidine-ss-polyethylenimine,mPEG-PLA-PHis-ss-PEI)阳离子聚合物并制备siRNA胶束复合物,考察其体外制剂学性质。方法以聚乙二醇单甲醚、D,L-丙交酯、聚组氨酸和聚乙烯亚胺为起始原料,合成聚乙二醇-聚乳酸-聚组氨酸-聚乙烯亚胺,并应用核磁共振氢谱和凝胶渗透色谱进行表征;采用薄膜分散法制备mPEG-PLA-PHis-ss-PEI/siRNA胶束复合物(micelleplexes);动态激光散射法测定胶束复合物的粒径和Zeta电位值;透射电镜观察胶束复合物的形态;超速离心法测定胶束复合物中,小干扰RNA(small interference RNA,siRNA)的包封率;琼脂糖凝胶电泳阻滞法分别考察阳离子聚合物抵御阴离子置换能力、血清稳定性和胶束复合物在弱酸性和还原性条件下siRNA的触发释放行为。结果所制备的胶束复合物呈类球形,大小均一,分布均匀;当氮磷比(nitrogen/phosphoruse molar ratio,N/P)为20时,胶束复合物平均粒径为104.0 nm,Zeta电位值为30.1 mV,siRNA的包封率为(92.9±2.4)%;凝胶电泳阻滞实验结果显示,当N/P大于7时,胶束复合物具有良好的阴离子抵御能力及较强的血清稳定性;胶束复合物在较低N/P条件下具有pH敏感和还原敏感性。结论所制备的胶束复合物稳定性较好,可用于递送siRNA。     

紫杉醇pH敏感嵌段共聚物胶束的制备与体外评价

目的应用pH敏感聚组氨酸-聚乳酸-聚乙二醇(poly(L-histidine)-poly(D,L-lactide)-poly(ethylene glycol),PHis-PLA-mPEG)聚合物为载体材料,采用溶剂挥发法制备紫杉醇pH敏感嵌段共聚物胶束,并对其体外性质进行评价。方法采用芘荧光探针法测定PHis-PLA-mPEG聚合物的临界胶束浓度(critical micelle concentration,CMC);超速离心法测定紫杉醇共聚物胶束的包封率和载药量;分别利用动态光散射法和Zeta电位分析仪对胶束的粒径分布和表面电位进行测定;采用透析法测定载药胶束在不同pH条件下的体外释药行为。结果 PHis-PLA-mPEG临界胶束质量浓度为8.9 mg·L-1,胶束载药量质量分数为8%;包封率可达90%以上;载药胶束的平均粒径为150.2nm,PDI为0.097,粒度分布较窄,Zeta电位为-14.3 mV;载药胶束在弱酸性条件下,药物释放行为明显加快。结论 PHis-PLA-mPEG聚合物载体材料具有较好的pH敏感释药行为,其作为抗肿瘤药物的靶向传递系统具有较好的应用前景。     

Box-Behnken效应面法优化mPEG-PDLLA多烯紫杉醇/白藜芦醇载药胶束处方

目的通过优化手段筛选处方,制备同时包载多烯紫杉醇/白藜芦醇的单甲氧基聚乙二醇-聚丙交酯嵌段共聚物(poly(ethylene glycol)methoxy-poly(D,L-lactide)mPEG-PDLLA)胶束。方法采用薄膜分散法制备mPEG-PDLLA载药胶束,分别以成膜温度(X1)﹑水化温度(X2)、投药量(X3)为考察指标,以多烯他赛(docetaxel,DTX)的包封率(Y1,EE%)及载药质量分数(Y2,wLC%)、白藜芦醇(resveratrol,RES)的包封率(Y3,EE%)及载药质量分数(Y4,wLC%)为评价指标;采用3因素3水平Box-Behnken效应面设计法筛选载药胶束处方;并测定载药胶束的粒径和zeta电位。结果共聚物对2种药物的胶束包封率均大于98%,载药质量分数均大于16%。载药胶束的平均粒径为(17±3.2)nm;zeta电位为-18.0mV。结论采用Box-Behnken实验设计法优化处方所得到的数学模型预测性良好,可以用于多烯紫杉醇∕白藜芦醇载药胶束的处方优化。     

Design of naltrexone-loaded hydrolyzable crosslinked nanoparticles

A hydrolyzable crosslinker (N,O-dimethacryloylhydroxylamine (MANHOMA)) was synthesized by a modified method and was characterized using 1H-NMR, FTIR, and melting point determination. Naltrexone-loaded nanoparticles were prepared by copolymerization of poly(ethylene glycol)1000 monomethyl ether mono methacrylate (PEO-MA), methyl methacrylate (MMA) and N,O-dimethacryloylhydroxylamine (MANHOMA) in 0.4% poly(vinyl alcohol) aqueous solution. The nanoparticles were characterized by FTIR, particle size determination and transmission electron microscope (TEM). The TEM photomicrographs of the nanoparticles show a crosslinked core surrounded by a ring formed by the polyethylene glycol tail of PEO-MA. The loading efficiency of the nanoparticles and in vitro drug availability from the nanoparticles were investigated. The naltrexone-loaded hydrolyzable crosslinked nanoparticles were able to sustain the release of naltrexone for different periods of time, depending on the monomer feed composition.     

聚乙二醇化聚氰酯毫微粒作为salvicine载体：合成、制备和体外特性

目的：合成和鉴定聚乙二醇化聚氰酯共聚物,制备聚乙二醇化聚氰酯共聚物和聚氰酯聚合物的毫微粒,测定二种毫微粒的体外特性。方法：用^1H-NMR,^13C-NMR和FTIR测定聚乙二醇化聚氰酯共聚物的结构,用凝胶渗透色谱法测定共聚物的分子量,用乳化／蒸发法制备毫微粒。结果：^1H-NMR,^13C-NMR和FTIR光谱与聚乙二醇化聚氰酯共聚物的结构相符,合成共聚物的分子量是6680,用HPLC测定毫微粒的包封效率时,共聚物对salvicine的测定无干扰,聚乙二醇化聚氰酯共聚物毫微粒的包封率是92．6％,聚氰酯聚合物的包封效率是98．9％。二种毫微粒的粒径均为250nm左右。Zeta电位值受聚合物结构的影响,与聚氰酯毫微粒比较（－23．1mV),聚乙二醇化聚氰酯毫微粒显示低的Zeta电位值（－9.6mV)。毫微粒的体外释放显示一个开始的突释效应,然后缓慢释放达28天。结论：聚乙二醇化聚氰酯毫微粒可能是salvicine体内抗肿瘤作用的一个有效载体。     

Self-assembled hydrogel nanoparticles composed of dextran and poly(ethylene glycol) macromer

Biodegradable hydrogel nanoparticles were prepared from glycidyl methacrylate dextran (GMD) and dimethacrylate poly(ethylene glycol) (DMP). GMD was synthesized by coupling of glycidyl methacrylate to dextran in the presence of 4-(N,N-dimethylamino)pyridine (DMAP) using dimethylsulfoxide (DMSO) as an aprotic solvent. DMP was synthesized from poly(ethylene glycol) (PEG) and methacryloyl chloride. GMD/DMP (abbreviated as DP) hydrogel was prepared by radical polymerization of GMD and DMP using ammonium peroxydisulfate (APS) as an initiator and UV curing. DP hydrogel nanoparticles were obtained by diafiltration method using DMSO solution. The GMD and DMP were characterized by fourier transform infrared spectroscopy. Fluorescence probe technique was used to investigate the self-assembly of DP in water using pyrene as a hydrophobic probe. The critical association concentration (CAC) was determined to be 5.6 x 10(-2) g/l. The shape of DP hydrogel nanoparticles was spherical when observed by transmission electron microscope (TEM). The size range of DP hydrogel nanoparticles was about 20 approximately 50 nm. The hydrodynamic size of DP hydrogel nanoparticles was measured by photon correlation spectroscopy (PCS) and gradually increased with time in PBS (0.1 M, pH 7.4). Drug release study was performed using clonazepam (CNZ) as a hydrophobic model drug. In vitro release rate of CNZ from the DP hydrogel nanoparticles was dependent on the existence of dextranase and the pH of the release medium.     

端胺基聚乙二醇衍生物的合成研究

目的合成一种一端为胺基一端为羟基的异双官能团聚乙二醇衍生物(NH2-PEG-OH)。方法以聚乙二醇为原料,采用氧化银和碘化钾催化系统催化聚乙二醇单磺酰化,通过氨基化反应将磺酰基转化为胺基,TLC、IR、1H-NMR等方法对产物结构进行表征。结果制备的产物与目标分子的结构相吻合,产率64.63%。结论本合成方法选择性高、操作简便,可用来制备更多的异双官能团聚乙二醇衍生物。     

添加精氨酸的多西他赛聚合物胶束冻干粉的制备

目的:考察以精氨酸作为稳定剂的多西他赛嵌段聚合物胶束冻干粉的复溶稳定性与理化性质.方法:以聚乙二醇单甲醚-聚乳酸嵌段共聚物(mPEG-PLA)为载体制备多西他赛胶束冻干粉对照组制剂与实验组制剂(含精氨酸).通过动态光散射法(DLS)与高效液相色谱法(HPLC)测定了制剂复溶后胶束粒径与多西他赛含量随时间的变化.分别使用(HPLC)、原子力显微镜、动态光散射法和Zeta电位分析仪对包封率、胶束形态、粒径与分布、Zeta电位和pH进行了表征;采用透析法考察了载药聚合物胶束的体外释放.结果:室温条件下实验组制剂复溶后胶束溶液在考察的12h内保持稳定.结论:精氨酸的加入使实验组制剂稳定得到极大提高.     

Preparation and characterization of intestinal transporter-targeted polymeric micelles

The intestinal epithelium is the main barrier to the oral delivery of poorly water-soluble drugs. Based on the specific transporters expressed on the apical membrane of the intestinal epithelium, novel polymer micelles targeting to the organic cation transporter 2 (OCTN2) were constructed by combining carnitine conjugated poly(2-ethyl-2-oxazoline)-poly(D,L-lactide) (Car-PEOz-PLA) with monomethoxy poly(ethylene glycol)-poly(D,L-lactide) (mPEG-PLA). The structure of the synthesized Car-PEOz-PLA was confirmed by ¹H NMR, TLC and ammonium reineckate precipitation reaction, and the number-average molecular weight determined by GPC was 7260 g/mol with a low PDI of 1.44. Coumarin 6-loaded carnitine modified polymeric micelles prepared by film hydration method were characterized to have a nano-scaled size of about 31 nm in diameter, uniform spherical morphology, high drug loading content of 0.098%±0.03% and encapsulation efficiency of 92.67%±2.80%. Moreover, the carnitine-modified micelles exhibited the similar in vitro release behavior in SGF and SIF, and evidently enhanced intestinal absorption of poorly water-soluble agent. Therefore, the designed OCTN2-targeted micelles might have a promising potential for oral delivery of poorly water-soluble drugs.     

Improving physicochemical properties and doxorubicin cytotoxicity of novel polymeric micelles by poly(ε-caprolactone) segments

This study constructed a series of novel micelles based on star-shaped amphiphilic copolymers (sPEC/CDs), and aimed to confirm the important role poly(ε-caprolactone) (PCL) segments played to improve the various properties of micelles. sPEC/CDs, consisting of β-cyclodextrin (β-CD) as a core and monomethoxy poly(ethylene glycol) (mPEG) and PCL diblock copolymers as arms, were synthesized by arm-first method. The critical micelle concentrations (CMC) of sPEC/CDs were determined by fluorescence spectrophotometry using pyrene as a probe. 3-(4, 5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide and flow cytometry were used to detect drug cytotoxicity and cellular uptake of the doxorubicin-loaded micelles. Rhodamine-123 cellular accumulation was examined to evaluate the polymer action to P-glycoprotein. It was revealed that, once PCL segment was inserted between β-CD and mPEG, the CMC can be significantly decreased, the drug loading capability greatly improved, and the drug resistance of MCF-7/ADR cells effectively reversed. These findings suggest that sPEC/CDs own potential superiority for cancer therapy as drug carriers.     

Self-assembled nanoparticles based on galactosylated O-carboxymethyl chitosan-graft-stearic acid conjugates for delivery of doxorubicin

A novel polymer, i.e. galactosylated O-carboxymethyl chitosan-graft-stearic acid (Gal-OCMC-g-SA) was synthesized for liver targeting delivery of doxorubicin. The chemical structure was characterized by FT-IR, ¹H NMR and elemental analysis. Gal-OCMC-g-SA could self-assemble into nanoparticles with diameter of 160 nm by probe sonication in aqueous medium and exhibited a low critical aggregation concentration of 0.047 mg/mL. The DOX-loaded Gal-OCMC-g-SA (Gal-OCMC-g-SA/DOX) self-assembled nanoparticles were almost spherical in shape with an average diameter of less than 200 nm and zeta potential of around −10 mV. In vitro release revealed that the Gal-OCMC-g-SA/DOX nanoparticles exhibited a sustained and pH-dependent drug release manner. Furthermore, the hemolysis test demonstrated the good safety of Gal-OCMC-g-SA in blood-contacting applications. These results indicated that Gal-OCMC-g-SA/DOX nanoparticles were highly potential to be applied in cancer therapy.     

Biodegradable thermo-sensitive nanoparticles from poly(L-lactic acid)/poly(ethylene glycol) alternating multi-block copolymer for potential anti-cancer drug carrier.

In order to produce biodegradable thermo-sensitive nanoparticles, alternating multi-block copolymers (MBC) were synthesized by coupling dicarboxylated poly(ethylene glycol) (PEG; M(w) 2000) with poly(L-lactic acid) (PLLA)/PEG/PLLA triblock copolymers. Three different multi-block copolymers were synthesized by varying PLLA molecular weight (800 (MBC1), 1600 (MBC2), and 2800 (MBC3)). The MBC formed self-assembled nanoparticles with a unimodal size distribution during a dialysis process. The nanoparticles (NP) had a spherical shape with a size range of 90-330 nm in diameter and critical aggregation concentrations in a range of 5.6-12.6 microg/mL, depending on PLLA length in MBC. The thermo-sensitivity of MBC NP was monitored by the changes in particle size and interior structure as a function of temperature. The particle size slightly decreased as increasing temperature from 37 to 42 degrees C. The interior structure of the NP responded to temperature by altering microviscosity. The microviscosity, measured by the anisotropy (r value) of a fluorescence probe, of MBC1 NP significantly changed with increasing temperature (r = 0.187 at 25 degrees C and 0.216 at 42 degrees C), while MBC2 and MBC3 showed negligible changes in the microviscosity. This indicates that the temperature-dependent interior structure of the NP relied on the portion of PLLA in MBC. The thermo-sensitivity affected to the drug release behavior and cell cytotoxicity. At 42 degrees C, doxorubicin (DOX) loaded MBC1 NP showed enhanced cytotoxicity (approximately 20 times) against Lewis Lung Carcinoma (LLC) cells when compared to that at 37 degrees C.     

Cell interaction studies of PLA-MePEG nanoparticles

Poly(D,L-lactic acid)-methoxypoly(ethylene glycol) (PLA-MePEG) copolymers were synthesized by ring-opening polymerization of D,L-lactide in the presence of MePEG of different molecular weights and stannous octoate as the catalyst. The chemical composition of the diblock-copolymer PLA-MePEG was confirmed by 1H-NMR and the molecular weight and distribution were assessed by gel permeation chromatography. Nanoparticles containing Nile red as a fluorescent dye were prepared using poly(D,L-lactic acid) (PLA), blends of PLA and PLA-MePEG or PLA-MePEG alone. Incubation of nanoparticles with human blood monocytes was performed in serum or in PBS and the cell-associated fluorescence was analyzed by flow cytometry. In serum, a protective effect was obtained and the interaction of particles with mononuclear leukocytes decreased to 40%.