叶酸受体靶向高分子相变超声造影剂的制备及体外寻靶实验研究

目的制备一种叶酸受体靶向的相变超声造影剂,观察其体外细胞靶向效果及超声显影情况。方法采用单乳化法制备包裹液态氟碳的高分子纳米微球,通过碳二亚胺法连接叶酸配体制备叶酸受体靶向的相变超声造影剂,以人卵巢癌SKOV3细胞验证其体外细胞靶向性能,以高强度聚焦超声(HIFU)体外辐照实验观察其超声显影情况。结果所制备叶酸受体靶向高分子相变超声造影剂平均粒径为(229.13±13.46)nm,体外细胞寻靶实验显示靶向造影剂与SKOV3细胞大量结合,而普通造影剂组与游离叶酸干预组未见明显特异性结合。造影剂溶液经一定功率HIFU辐照后,超声显影效果较辐照前明显增强。结论成功制备了叶酸受体靶向包裹液态氟碳的高分子造影剂,对高表达叶酸受体的SKOV3细胞具有良好靶向性,并具备HIFU辐照后增强超声显影的特性,有望成为卵巢癌靶向显影与治疗的理想分子探针。     

制备叶酸受体靶向载阿霉素/黑色素多功能造影剂及体外超声/光声显像

目的制备叶酸受体靶向载阿霉素/黑色素的多功能造影剂,评估其基本特性、体外细胞靶向能力、超声和光声显像效果以及对人乳腺癌MDA-MB-231细胞增殖活力的影响。方法采用单乳化法和冷冻干燥减压充入C3F8气体的方法制备叶酸受体靶向载阿霉素/黑色素多功能造影剂,以马尔文激光仪检测其平均粒径及表面电位,应用紫外-可见光分光光度法测量阿霉素载药量,于凝胶模型中观察其体外超声、光声增强显影能力,以人乳腺癌MDA-MB-231细胞验证其体外细胞靶向能力和细胞增殖活力的影响。结果叶酸受体靶向载阿霉素/黑色素脂质多功能造影剂平均粒径为(659.60±27.56)nm,Zeta电位为(-38.90±4.00)mV,阿霉素载药率为85.72μg/mg。叶酸受体靶向造影剂大量聚集在MDA-MB-231细胞表面,具有较好的体外超声、光声显像能力,对MDA-MB-231细胞增殖有显著抑制作用。结论叶酸受体靶向载阿霉素/黑色素多功能造影剂是集靶向治疗、超声和光声多模态显像于一体的多功能造影剂,有望成为乳腺癌精准显像和治疗的理想分子探针。     

光声/超声实时双模态显影纳米粒制备及示踪实验

目的研制一种包裹纳米炭和液态氟碳的纳米粒(CNPs),并验证其光声/超声实时双模态示踪能力。方法采用双乳化法制备CNPs。于光镜及透射电镜下观察并通过激光粒径仪检测CNPs的基本性质;以激光仪辐照CNPs,观察其液气相变情况;于激光共聚焦显微镜下观察荧光标记的CNPs被巨噬细胞吞噬的情况;激光辐照后观察CNPs体外增强光声/超声实时双模态显影情况;进行在体实验观察CNPs增强光声/超声实时双模态显影示踪VX2荷瘤兔腘窝转移淋巴结的效果。结果成功制备CNPs,其平均粒径为(483.32±45.09)nm,Zeta电位为(-26.30±5.02)mV;且经激光辐照后,可发生液气相变;与巨噬细胞共孵育后,CNPs大量被巨噬细胞吞噬;CNPs在体外、体内均具有良好的光声显影与超声造影效果,且CNPs浓度越高,平均光声信号值及平均灰度值越大。结论 CNPs可在激光辐照后发生液气相变,具备光声成像与超声造影的实时双模态显影能力,并可在活体实验中示踪兔恶性肿瘤转移淋巴结。     

载多烯紫杉醇及酞菁锌并靶向乳腺癌多功能分子探针体外光声显像特性及寻靶能力

目的观察载多烯紫杉醇及酞菁锌并靶向乳腺癌多功能分子探针的体外光声显像特性及寻靶能力。方法采用双乳化法及碳二亚胺法制备载多烯紫杉醇及酞菁锌并连接RDG靶向肽的多功能分子探针。检测探针的一般物理特性、多烯紫杉醇及酞菁锌包封率及载药量、与RGD连接情况及体外寻靶能力,观察其体外光声显像情况、体外释放特性及体外抑制乳腺癌细胞的能力。结果成功制备靶向载多烯紫杉醇及酞菁锌的多功能分子探针,平均粒径(266.00±65.85)nm,表面电位(-29.20±6.27)mV;多烯紫杉醇包封率及载药量分别为(88.00±0.32)%、(34.92±0.02)μg/mg,酞菁锌包封率及载药量分别为(97.25±0.22)%、(30.87±0.11)μg/mg;靶向载多烯紫杉醇及酞菁锌纳米粒具有一定缓释效应,且呈明显光声信号,并随酞菁锌含量增加而增强。流式细胞仪检测RGD肽与靶向载多烯紫杉醇及酞菁锌纳米粒连接率为89.19%。激光辐照靶向载多烯紫杉醇及酞菁锌纳米粒后,乳腺癌细胞凋亡率明显增高。结论载多烯紫杉醇及酞菁锌并靶向乳腺癌多功能分子探针呈明显光声信号,并具备乳腺癌细胞靶向能力,可通过光声介导抑制乳腺癌细胞增殖。     

膀胱癌BIU-87细胞靶向超声纳米脂质微泡造影剂的制备及体外超声成像

目的制备具有膀胱癌BIU-87细胞靶向结合的超声纳米脂质微泡造影剂,并观察体外靶向能力、检测其对细胞增殖的影响和体外超声成像效果。方法通过机械振动法制备纳米脂质微泡,用生物素-亲和素桥接法将NYZL1连接到纳米脂质微泡的表面制备靶向纳米脂质微泡造影剂,利用倒置显微镜观察其一般特性,Zate检测仪检测粒径大小和表面电荷,使用倒置显微镜来观察其体外靶向效果,用MTT法检测其不同浓度对细胞增殖的影响,使用超声诊断仪观察体外显影效果。结果膀胱癌BIU-87细胞靶向超声纳米脂质微泡呈圆形,分布均匀,无聚集;在体外靶向实验中,靶向纳米脂质微泡造影剂能高效特异性的结合在膀胱癌BIU-87细胞表面并沿细胞表面规则排列;不同浓度下两种微泡对细胞增殖无明显变化(P0.05);体外超声成像实验中,靶向纳米微泡呈点状细密高回声。结论本实验成功制备膀胱癌BIU-87细胞超声纳米脂质微泡造影剂,能够在体外与膀胱癌BIU-87细胞特异性地靶向结合,两种微泡对细胞生长无明显影响且体外超声显像效果良好。     

载超顺磁性氧化铁高分子液态氟碳纳米粒的制备及体外显像

目的制备一种能同时用于超声造影及MR成像的多模态造影剂,观察其体外成像效果。方法采用双乳化法合成载超顺磁性氧化铁（SPIO）纳米颗粒及全氟己烷（PFH）的高分子微球（s-PFH／PLGA）,检测其一般特性及光声信号。对不同浓度的s—PFH／PLGA水囊模型进行超声显影,以JC200聚焦超声肿瘤治疗系统辐照后观察回声强度变化;对不同铁含量的s—PFH／PLGA进行MR成像。结果透射电镜下s—PFH／PLGA呈球形,SPIO颗粒均匀分布在外壳上,平均粒径（738．9±158．4）nm,平均电位（-15．9士6．9）mV,并检测到明显光声信号。体外超声显像中s—PFH／PLGA呈点状高回声,HIFU辐照后回声强度增强。s-PFH／PLGA在T2wI中呈负增强显像;随着铁含量增高,MRI信号呈降低趋势。结论成功制备的载SPIO及PFH多模态造影剂具有体外超声、MR显影功能。     

包裹液态氟碳的高分子超声造影剂的制备及体外显影实验

目的制备一种新型超声造影剂——液态氟碳高分子微球,观察其体外物理特性及超声、CT显影效果。方法采用乳化-蒸发法制备包裹液态氟碳的高分子微球;以光镜、扫描电镜观察微球形态、分布,以激光粒度和电位分析仪检测微球粒径、电位;DiI着色后,于荧光显微镜下观察微球结构和荧光强度;行体外超声和CT,观察其显影效果。结果所制备的液态氟碳高分子微球外观为乳白色混悬液,形态规则,呈球包球形;平均粒径为（413±32）nm,平均电位为（-2.42±5.71）mV;荧光染色后微球的乳酸/羟基乙酸共聚物（PLGA）外壳呈环形红色荧光,而内部包绕的液态氟碳不发光、呈黑色;体外超声与CT显影效果好。结论成功制备的液态氟碳高分子微球粒径小,稳定性好,体外可同时增强超声和CT,有望成为一种新型的多功能超声造影剂。     

制备靶向相变型载硫化铋纳米粒并用于体外CT/超声显像

目的制备叶酸靶向相变型载硫化铋(Bi_2S_3)纳米粒(FBS-PFH-NPs)并用于体外细胞靶向及CT/超声显像。方法采用旋转蒸发法和声振法制备FBS-PFH-NPs,检测其基本性质;以宫颈癌Hela细胞验证FBS-PFH-NPs体外寻靶能力;观察60、90、120、150、180 W功率HIFU辐照后FBS-PFH-NPs回声强度和温度变化,以及纳米粒中Bi_2S_3浓度为1.0、2.0、3.0、4.0、5.0 mg/ml时FBS-PFH-NPs体外CT及超声显像效果。结果光镜下FBS-PFH-NPs呈球形,平均粒径(458.50±69.22)nm;Bi_2S_3均匀分布于其外壳,浓度为1.0 mg/ml。FBS-PFH-NPs大量结合于Hela细胞周围。HIFU辐照后,FBS-PFH-NPs发生液气相变,且随功率增高,FBS-PFH-NPs回声强度及温度均逐渐增高(F=110.09、440.69,P均0.01)。随纳米粒中Bi_2S_3浓度增高,FBS-PFH-NPs的CT值及回声强度均逐渐增高(F=146.14、16.74,P均0.01)。结论 FBS-PFH-NPs兼具靶向Hela细胞及CT/超声双模态显像能力。     

包裹液态氟碳高分子纳米球相变及体外超声显影

目的制备一种新型纳米级超声造影剂——液态氟碳（PFP）高分子纳米球,观察其物理特性、体外热致相变、声致相变及体外超声显影效果。方法采用乳化一蒸发法（单乳化法）制备包裹PFP的MPEG-PLGA纳米球（P-NP）,以光镜观察纳米球形态分布,以纳米粒度及Zeta电位分析仪检测纳米球粒径和电位;在显微镜上放置加热板实时观察P-NP的相变情况;于体外应用低强度聚焦超声（LIFU）仪辐照后,观察其超声显影效果。结果所制备的包裹PFP的高分子纳米球外观为乳白色混悬液,形态规则,呈球包球形;平均粒径为（393．2±40．7）nm,平均电位为（-5．23±8．69）mV;加热板显示的温度约为42．6℃时,光镜下显示纳米球开始转变为微气泡,且随着温度增高,所产生的气泡逐渐增多;体外行LIFU辐照后,在超声基波和谐波模式下可观察到显影明显增强。结论成功制备了PFP高分子纳米球,其粒径小、稳定性好,体外经LIFU仪辐照后可增强超声显影,有望成为一种新型超声造影剂。     

DR5mAb-载替加氟高分子材料微球的构建及体外靶向肝癌能力

目的构建一种DR5mAb-载替加氟主动靶向载药高分子微球,并鉴定其理化性质和体外寻靶能力。方法采用双乳化法制备替加氟载药微球,以碳二亚胺法和生物素-亲和素法将DR5mAb与载药微球连接,构建DR5mAb-载替加氟主动靶向载药高分子微球。检测载药微球的一般性质;用流式细胞仪检测抗体连接率,并对比碳二亚胺法和生物素-亲和素法的连接率;以人肝癌细胞株SMMC-7721和正常肝细胞株L02为实验对象验证其体外细胞靶向性。结果生物素亲和素法的载药微球抗体连接率高于碳二亚胺法(P0.05);靶向载药微球-7721组的细胞表面及周围有大量微球聚集,非靶向载药微球-7721组和靶向载药微球-L02组的细胞表面及周围均无明显微球聚集。结论 DR5mAb-载替加氟主动靶向载药高分子微球可与肝癌细胞SMCC-7721主动靶向结合,有望为肝癌靶向治疗提供一种新的手段和思路。     

Photoacoustic and Fluorescence Image-Guided Surgery Using a Multifunctional Targeted Nanoprobe

Purpose

A complete surgical excision with negative tumor margins is the single most important factor in the prediction of long-term survival for most cancer patients with solid tumors. We hypothesized that image-guided surgery using nanoparticle-enhanced photoacoustic and fluorescence imaging could significantly reduce the rate of local recurrence.

Methods

A murine model of invasive mammary carcinoma was utilized. Three experimental groups were included: (1) control; (2) tumor-bearing mice injected with non-targeted nanoprobe; and (3) tumor-bearing mice injected with targeted nanoprobe. The surgeon removed the primary tumor following the guidance of photoacoustic imaging (PAI), then inspected the surgical wound and removed the suspicious tissue using intraoperative near-infrared (NIR) fluorescence imaging. The mice were followed with bioluminescence imaging weekly to quantify local recurrence.

Results

Nanoprobe-enhanced photoacoustic contrast enabled PAI to map the volumetric tumor margins up to a depth of 31 mm. The targeted nanoparticles provided significantly greater enhancement than non-targeted nanoparticles. Seven mice in the group injected with the targeted nanoprobes underwent additional resections based upon NIR fluorescence imaging. Pathological analysis confirmed residual cancer cells in the re-resected specimens in 5/7 mice. Image-guided resection resulted in a significant reduction in local recurrence; 8.7 and 33.3 % of the mice in the targeted and control groups suffered recurrence, respectively.

Conclusions

These results suggest that photoacoustic and NIR intraoperative imaging can effectively assist a surgeon to locate primary tumors and to identify residual disease in real-time. This technology has promise to overcome current clinical challenges that result in the need for second surgical procedures.     

链霉亲和素化载紫杉醇相变型PLGA纳米粒的制备及体外超声显影

目的制备链霉亲和素化载紫杉醇相变型PLGA纳米粒(PTX-PLGA-SA/PFPs),并观察其体外低强度聚焦超声(LIFU)致相变后超声增强显影特性。方法采用单乳化法(O/W)制备载紫杉醇相变型PLGA纳米粒,高效液相色谱法检测紫杉醇包封率;碳二亚胺法连接链霉亲和素(SA),共聚焦激光显微镜观察二者连接情况,流式细胞术检测二者链接率;体外LIFU致相变观察超声增强显影情况。结果制备的纳米粒粒径为(322.2±85.6)nm,表面电位(-5.66±3.46)mV。紫杉醇的包封率及载药量分别为(71.56±6.51)%、(6.57±0.61)%,与链霉亲和素的连接率为(97.16±1.20)%。LIFU功率7.5 W作用3min时可明显增强该纳米粒在体外的B-mode及造影模式下的超声显影。结论成功制备了PTX-PLGA-SA/PFPs纳米粒,其紫杉醇包封率高、链霉亲和素连接率高,体外声致相变后可显著增强超声显影。     

An engineered anti-CA19-9 cys-diabody for positron emission tomography imaging of pancreatic cancer and targeting of polymerized liposomal nanoparticles

Background

Antibody-based therapeutics is a rapidly growing field. Small engineered antibody fragments demonstrate similar antigen affinity compared with the parental antibody but have a shorter serum half-life and possess the ability to be conjugated to nanoparticles. The goal of this study was to engineer an anti-carbohydrate antigen 19-9 (CA19-9) cys-diabody fragment in hopes of targeting nanoparticles to pancreatic cancer.

Methods

The anti-CA19-9 cys-diabody was created by engineering a C-terminal cysteine residue into the DNA single-chain Fv construct of the anti-CA19-9 diabody and expressed in NS0 cells. Maleimide chemistry was used to conjugate the cys-diabody to polymerized liposomal nanoparticles (PLNs) through the cysteine residues. Flow cytometry was used to evaluate targeting of cys-diabody and cys-diabody–PLN conjugate to human pancreatic cancer cell lines. The cys-diabody was radiolabeled with a positron emitter (¹²⁴I) and evaluated in a mouse model of CA19-9–positive and CA19-9–negative xenografts with micro–positron emission tomography/micro–computed tomography at successive time intervals after injection. Percentage of injected dose per gram of radioactivity was measured in blood and tumor to provide objective confirmation of the micro–positron emission tomographic images.

Results

Tumor xenograft imaging of the anti-CA19-9 cys-diabody demonstrated an average tumor-to-blood ratio of 3.0 and positive-to-negative tumor ratio of 7.4. Successful conjugation of the cys-diabody to PLNs was indicated by flow cytometry showing specific binding of cys-diabody–PLN conjugate to human pancreatic cancer cells in vitro.

Conclusions

Our results show that the anti-CA19-9 cys-diabody targets pancreatic cancer providing specific molecular imaging in tumor xenograft models. Furthermore, the cys-diabody–PLN conjugate demonstrates target-specific binding of human pancreatic cancer cells with the potential to deliver targeted treatment.     

PTK787/ZK222584 Combined with Interferon Alpha and 5-Fluorouracil Synergistically Inhibits VEGF Signaling Pathway in Hepatocellular Carcinoma

Background

The prognosis of patients with hepatocellular carcinoma (HCC) and portal vein tumor thrombus remains poor. We previously reported the beneficial effects of interferon alpha (IFN) and 5-fluorouracil (5-FU) combination therapy for these patients. We showed that the mechanism of therapy was regulation of vascular endothelial growth factor (VEGF). Here, we combined IFN/5-FU therapy with the VEGF receptor–selective inhibitor PTK787/ZK222584 (PTK/ZK) and examined the antitumor effects and the mechanism of action.

Methods

We studied two HCC cell lines, PLC/PRF/5 and HuH7, and a human umbilical vein endothelial cell line, HUVEC. We studied the effects of IFN/5-FU with or without PTK/ZK in growth inhibition assays, immunohistochemistry, Western blot analysis, and immunocytochemistry.

Results

In a HuH7 xenograft model, the combination of PTK/ZK and IFN/5-FU significantly inhibited proliferation, induced apoptosis, decreased microvessel density, reduced the number of tumor cells that expressed VEGF receptor 2 (VEGFR-2), and repressed the phosphorylation of Akt in vivo. In HCC cells and HUVECs in vitro, IFN/5-FU plus PTK/ZK repressed the expression of VEGFR-2 and repressed the phosphorylation of VEGFR, Akt, Erk, and p38MAPK.

Conclusions

VEGF signaling inhibition enhanced the antitumor effects of IFN/5-FU therapy on HCC cells and endothelial cells via Erk, Akt, and p38MAPK pathways.

     

两种相变型多功能纳米粒的制备及体外特性比较

目的制备包裹吲哚菁绿(ICG)及不同相变材料[全氟正戊烷(PFP)或全氟己烷(PFH)]的两种相变型多功能纳米粒,比较其理化性质、稳定性及体外相变特性。方法采用改良的双乳化法制备包裹ICG及液态PFP或PFH的乳酸-羟基乙酸(PLGA)纳米粒(分别称为IPNPs、IHNPs),比较两者的一般特性、稳定性及热致相变、光致相变、声致相变和光声成像能力,采用MTT法检测光致相变所需能量的细胞毒性。结果IPNPs及IHNPs粒径分别为(490.53±36.96)nm及(512.23±38.52)nm,电位分别为(-14.40±1.40)mV及(-13.97±1.61)mV,二者差异无统计学意义(P均0.05);二者形态、吸收光谱也均无明显差异,于37℃储存24h均较稳定;IPNPs热致相变、光致相变及声致相变所需能量均低于IHNPs,且IPNPs光声及超声成像增强能力高于IHNPs。经能使IPNPs及IHNPs发生相变的激光能量辐照后,MH7A细胞存活率分别为(95.34±7.96)%,(54.92±6.11)%,二者差异有统计学意义(P0.05)。结论 IPNPs是更适于诊疗一体化应用的多功能纳米粒。