Using drug-excipient interactions for siRNA delivery

SiRNA is the trigger of RNA interference, a mechanism discovered in the late 1990s. To release the therapeutic potential of this versatile but large and fragile molecule, excipients are used which either interact by electrostatic interaction, passively encapsulate siRNA or are covalently attached to enable specific and safe delivery of the drug substance. Controlling the delicate balance between protective complexation and release of siRNA at the right point and time is done by understanding excipients-siRNA interactions. These can be lipids, polymers such as PEI, PLGA, Chitosans, Cyclodextrins, as well as aptamers and peptides. This review describes the mechanisms of interaction of the most commonly used siRNA delivery vehicles, and looks at the results of their clinical and preclinical studies.     

Dry powder aerosols of polyethylenimine (PEI)-based gene vectors mediate efficient gene delivery to the lung

Aerosol gene delivery holds great therapeutical potential for many inherited and acquired pulmonary diseases. The physical instability of aqueous suspensions of non-viral vector complexes is a major limitation for their successful application. In this study, we investigated dry powder aerosols as novel gene vector formulations for gene transfer in vitro and murine lungs in vivo. Lyophilization was used to produce dry powder cakes followed by powderization to produce dry powder aerosols. Different sugars, namely lactose, sucrose and trehalose, were tested as lyoprotectants for gene delivery complexes consisting of branched polyethylenimine 25 kDa and plasmid DNA. Biophysical particle characterization demonstrated that lyophilization and powderization in the presence of lyoprotectants were well tolerated. In vitro transfection efficiency remained unaffected by the choice of lyoprotectant and subsequent lyophilization and/or powderization. In vivo screening of powderized samples, by applying the powder with an insufflator, resulted in highest gene expression with lactose as lyoprotectant. Delivering a plasmid coding for murine erythropoietin together with lactose as lyoprotectant resulted in increased blood hematocrit values post application thereby demonstrating the potential of dry powder aerosol as a promising method for pulmonary gene delivery.     

裴正学教授“心脑同治”学术思想初探

探求裴正学教授“心脑同治”学术思想的内涵。临床应强调培补元气,调理气血;活血化瘀,疏通脉络;痰瘀同治,标本兼顾;辛香宣通,引经透络;虫类走窜,搜邪剔络;通阳散结,行气祛痰;取类比象,藤类入脉等方药的应用;尤其重视药物归经入脑和痰瘀同治及化瘀药物层次性选择问题,为中医药防治心脑血管病提供新的理论依据。     

Positive enhancement integral values in dynamic contrast enhanced magnetic resonance imaging of breast carcinoma: Ductal carcinoma in situ vs. invasive ductal carcinoma

Objectives

The aim of this study was to contribute to the standardization of the numeric positive enhancement integral (PEI) values in breast parenchyma, ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) and to evaluate the significance of the difference in PEI values between IDC and parenchyma, DCIS and parenchyma and IDC and DCIS.

Materials and Methods

In the prospective trial, we analyzed the dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) of 60 consecutive patients with histologically confirmed unilateral DCIS (n = 30) and IDC (n = 30) and defined the PEI values (range; mean ± SD) for the lesions and the breast parenchyma. Tumor-to-non-tumor (T/NT) ratios were calculated for DCIS and IDC and compared. PEI color maps (PEICM) were created. The differences in PEI values between IDC and parenchyma and between DCIS and parenchyma were tested according to t-test. Analysis of variance (ANOVA) was used to test the differences between the mean PEI values of parenchyma, DCIS and IDC.

Results

IDC showed highly statistically different PEI numeric values compared to breast parenchyma (748.7 ± 32.2 vs. 74.6 ± 17.0; p < 0.0001). The same applied to the differences in the group of patients with DCIS (428.0 ± 25.0 vs. 66.0 ± 10.6; p < 0.0001). The difference between IDC, DCIS and parenchyma were also considered highly statistically significant (p < 0.0001) and so were the T/NT ratios for IDC and DCIS (10.1 ± 2.4 vs. 6.6 ± 1.4; p < 0.0001).

Conclusions

PEI numeric values may contribute to differentiation between invasive and in situ breast carcinoma.     

裴正学教授治疗乳腺癌经验体会

裴正学教授擅长治疗各种疑难病症,尤其在乳腺癌的治疗方面积累了丰富的经验。裴老认为正虚为乳腺癌发病之关键,扶正固本为其治疗大法,临证以兰州方为基础方,配合西医手术、放化疗治疗此病,疗效显著。     

MCF-7细胞瞬时基因表达转染模式的建立

目的　建立一种高效、简便、低毒、价廉的瞬时基因表达细胞转染模式。　方法　选择人乳腺癌细胞 系MCF 7,采用Polyethylenimine(PEI)作为转染剂,对细胞密度、载体DNA量、PEI氮与DNA磷的比率(PEI N∶DNA P) 以及PEI DNA混合物与细胞共存的无血清培养时间等对转染效率的影响进行了比较分析。　结果　转染时的24 孔板每孔接种的细胞以2×105为宜,PEI N∶DNA P的最优比率不是固定的,它与转染时DNA的量有关,每孔转染1 μgDNA时其最优PEI N∶DNA P比率为33∶1左右,而每孔转染4μgDNA时其最优PEI N∶DNA P比率为9∶1左右。 另外,PEI DNA混合物与细胞共存的无血清培养时间至少需要3h,最佳时间为5～7h。　结论　利用转染剂PEI, 控制合适的细胞密度、DNA质量、PEI N∶DNA P比率和无血清培养的时间,可成为一种高效、简便、低毒、价廉的瞬时 基因表达的细胞转染模式。     

射频消融治疗与经皮无水酒精注射对小肝癌治疗疗效比较的系统评价

目的 评价射频消融治疗是否比经皮无水酒精注射治疗小肝癌具有更好的疗效。方法 计算机检索MEDLINE（1966—2005年）、EMBASE（1989—2005年）、Cochrane图书馆临床对照试验资料库（2005年第1期）、互联网上注册临床试验数据库、中国生物医学文献数据库（1980—2005年）,文献语种不限。纳入比较射频消融治疗和经皮无水酒精注射治疗小肝癌的随机对照试验,对纳入研究的方法学及质量进行评价,并应用RevMan4．2软件进行统计分析。结果共纳入4个随机对照试验。Meta分析结果表明,射频消融治疗较经皮无水酒精注射能提高对〉2cm小肝癌的3年生存率、1年和3年无瘤生存率并减少1年和3年局部复发率;而对直径≤2cm的小肝癌,射频消融治疗与经皮无水酒精注射治疗效果相似,无显著差异。结论射频消融治疗小肝癌的总体疗效优于经皮无水酒精注射治疗,尤其对于肿瘤直径〉2cm的小肝癌;且两种方法副作用均小,安全可靠。上述结论对于指导临床治疗小肝癌的方法选择具有很好的意义。     

PEI增强G250 DNA疫苗和重组肽疫苗的prime-boost免疫策略研究

目的 :探讨PEI作为免疫佐剂对G250抗原肽基因PVAX1/C-G250肽-C免疫保护效果的增强作用及联合应用CAIX蛋白疫苗进行PRIME—BOOST免疫程序免疫增强效果。方法:用Eco R I、Xho I和Eco R I、Sal I分别双酶切PVAX1及既往构建的p ET28a(+)/C-G250肽-C质粒。利用DNA重组技术构建重组质粒PVAX1/C-G250肽-C,酶切分析鉴定。大量提取质粒并分光光度计测质粒含量。将32只雌性昆明小鼠随机分为(A)裸DNA组,(B)DNA-PEI复合物组,(C)DNA-PEI+蛋白疫苗组,(D)空白对照组。按0,10,20,30天程序经股四头肌注射免疫。C组在第20天及第30天进行蛋白冲击。初次免疫前和第40天鼠尾取血,ELISA法检测抗体滴度。流式细胞术测淋巴细胞亚群CD4+和CD8+。结果:酶切及基因测序鉴定证实G250抗原肽c DNA正确插入PVAX1/C-G250肽-C真核表达的重组质粒中。昆明小鼠经4次免疫后,3个实验组都产生了特异性的体液和细胞免疫反应,B组的抗体滴度1:1.28×104及CD4+、CD8+达26.12%和12.60%,明显高于A组的1:3.2×103和CD4+,CD8+占到19.32%和10.74%。而蛋白冲击组的1:5.12×104和CD4+,CD8+占到41.96%和15.14%,明显高于B组(P0.05)。结论:成功构建重组质粒PVAX1/C-G250肽-C。该DNA疫苗与PEI和G250蛋白疫苗联合使用后产生极强的免疫原性,诱导产生了高滴度、高特异性抗体及细胞免疫反应。证实G250DNA疫苗联合PEI使用并进行蛋白疫苗冲击的免疫策略可产生强大的免疫保护作用。为恶性肿瘤的术后辅助治疗提供新的思路和方法。     

Targeted-gene silencing of BRAF to interrupt BRAF/MEK/ERK pathway synergized photothermal therapeutics for melanoma using a novel FA-GNR-siBRAF nanosystem

Melanoma is significantly associated with mutant BRAF gene, a suitable target for siRNA-based anti-melanoma therapy. However, a tumor-specific delivery system is a major hurdle for clinical applications. Here, we developed a novel nano-carrier, FA-GNR-siBRAF for safe topical application, which consists of folic acid (FA) as the tumor-targeting moiety, golden nanorods (GNR) providing photothermal capability to kill tumor cells under laser irradiation, and siRNA specifically silencing BRAF (siBRAF). The in vitro and in vivo results revealed that FA-GNR-siBRAF displayed high transfection rates, and subsequently induced remarkable gene knockdown of BRAF, resulting in suppression of melanoma growth due to the interruption of the MEK/ERK pathway. Combinatorial photothermal effects and BRAF knockdown by FA-GNR-siBRAF effectively killed tumor cells through apoptosis, with enhanced efficiency than individual treatments. Therefore, the FA-GNR-siBRAF simultaneously induced BRAF gene silencing and photothermal effects which achieved synergistic efficacy in the treatment of melanoma, paving a new path for developing clinical treatment methods for melanoma.