PAMAM Dendrimers Mediate siRNA Delivery to Target Hsp27 and Produce Potent Antiproliferative Effects on Prostate Cancer Cells

RNA interference (RNAi) holds great promise for the treatment of inherited and acquired diseases, provided that safe and efficient delivery systems are available. Herein we report that structurally flexible triethanolamine (TEA) core PAMAM dendrimers are able to deliver an Hsp27 siRNA effectively into prostate cancer (PC‐3) cells by forming stable nanoparticles with siRNA, protecting the siRNA nanoparticles from enzymatic degradation, and enhancing cellular uptake of siRNA. The Hsp27 siRNA resulted in potent and specific gene silencing of heat‐shock protein 27, an attractive therapeutic target in castrate‐resistant prostate cancer. Silencing of the hsp27 gene led to induction of caspase‐3/7‐dependent apoptosis and inhibition of PC‐3 cell growth in vitro. In addition, the siRNA–dendrimer complexes are non‐cytotoxic under the conditions used for siRNA delivery. Altogether, TEA core PAMAM dendrimer‐mediated siRNA delivery, in combination with RNAi that specifically targets Hsp27, may constitute a promising approach for combating castrate‐resistant prostate cancer, for which there is no efficacious treatment.     

Gene Therapy in HIV‐Infected Cells to Decrease Viral Impact by Using an Alternative Delivery Method

The ability of dendrimer 2G‐[Si{O(CH₂)₂N(Me)₂⁺(CH₂)₂NMe₃⁺(I^?)₂}]₈ (NN16) to transfect a wide range of cell types, as well as the possible biomedical application in direct or indirect inhibition of HIV replication, was investigated. Cells implicated in HIV infection such as primary peripheral blood mononuclear cells (PBMC) and immortalized suspension cells (lymphocytes), primary macrophages and dendritic cells, and immortalized adherent cells (astrocytes and trophoblasts) were analyzed. Dendrimer toxicity was evaluated by mitochondrial activity, cell membrane rupture, release of lactate dehydrogenase, erythrocyte hemolysis, and the effect on global gene expression profiles using whole‐genome human microarrays. Cellular uptake of genetic material was determined using flow cytometry and confocal microscopy. Transfection efficiency and gene knockdown was investigated using dendrimer‐delivered antisense oligonucleotides and small interfering RNA (siRNA). Very little cytotoxicity was detected in a variety of cells relevant to HIV infection and erythrocytes after NN16 dendrimer treatment. Imaging of cellular uptake showed high transfection efficiency of genetic material in all cells tested. Interestingly, NN16 further enhanced the reduction of HIV protein 24 antigen release by antisense oligonucleotides due to improved transfection efficiency. Finally, the dendrimer complexed with siRNA exhibited therapeutic potential by specifically inhibiting cyclooxygenase‐2 gene expression in HIV‐infected nervous system cells. NN16 dendrimers demonstrated the ability to transfect genetic material into a vast array of cells relevant to HIV pathology, combining high efficacy with low toxicity. These results suggest that NN16 dendrimers have the potential to be used as a versatile non‐viral vector for gene therapy against HIV infection.     

Efficient Transfection of siRNA by Peptide Dendrimer–Lipid Conjugates

Efficient delivery of small interfering RNA (siRNA) into cells is the basis of target‐gene‐specific silencing and, ultimately, gene therapy. However, current transfection reagents are relatively inefficient, and very few studies provide the sort of systematic understanding based on structure–activity relationships that would provide rationales for their improvement. This work established peptide dendrimers (administered with cationic lipids) as siRNA transfection reagents and recorded structure–activity relationships that highlighted the importance of positive charge distribution in the two outer layers and a hydrophobic core as key features for efficient performance. These dendrimer‐based transfection reagents work as well as highly optimised commercial reagents, yet show less toxicity and fewer off‐target effects. Additionally, the degrees of freedom in the synthetic procedure will allow the placing of decisive recognition features to enhance and fine‐tune transfection and cell specificity in the future.     

Antitumor efficacy of doxorubicin encapsulated within PEGylated poly(amidoamine) dendrimers

We report here a general approach to using poly(amidoamine) (PAMAM) dendrimers modified with polyethylene glycol (PEG) as a platform to encapsulate an anticancer drug doxorubicin (DOX) for in vitro cancer therapy applications. In this approach, PEGylated PAMAM dendrimers were synthesized by conjugating monomethoxypolyethylene glycol with carboxylic acid end group (mPEG‐COOH) onto the surface of generation 5 amine‐terminated PAMAM dendrimer (G5.NH₂), followed by acetylation of the remaining dendrimer terminal amines. By varying the molar ratios of mPEG‐COOH/G5.NH₂, G5.NHAc‐mPEG_n (n = 5, 10, 20, and 40, respectively) with different PEGylation degrees were obtained. We show that the PEGylated dendrimers are able to encapsulate DOX with approximately similar loading capacity regardless of the PEGylation degree. The formed dendrimer/DOX complexes are water soluble and stable. In vitro release studies show that DOX complexed with the PEGylated dendrimers can be released in a sustained manner. Further cell viability assay in conjunction with cell morphology observation demonstrates that the G5.NHAc‐mPEG_n/DOX complexes display effective antitumor activity, and the DOX molecules encapsulated within complexes can be internalized into the cell nucleus, similar to the free DOX drug. Findings from this study suggest that PEGylated dendrimers may be used as a general drug carrier to encapsulate various hydrophobic drugs for different therapeutic applications. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40358.     

Functionalized graphene oxide mediated nucleic acid delivery

We report a simple preparation of linear polyethylenimine-grafted graphene oxide (LP-GO) conjugates and their efficacy to transfer nucleic acids into the mammalian cells. Graphene oxide (GO), with epoxy functions on its surface, was reacted with different amounts of linear polyethylenimine (lPEI), a non-toxic polymer, to obtain three different positively charged LP-GO conjugates (LP-GO-1 to LP-GO-3), capable of interacting with negatively charged nucleic acids (gel retardation assay) and transporting them efficiently into the cells. The results show that these conjugates not only exhibited considerably higher transfection efficiency but also possessed even better cell viability than lPEI. LP-GO-2, the best system in terms of transfection efficiency, showed improved buffering capacity compared to lPEI and provided sufficient stability to bound DNA against DNase I. Further, LP-GO-2 was used for the sequential delivery of GFP specific siRNA, which resulted in ∼70% suppression of the target gene expression. Intracellular trafficking using fluorescence microscopy revealed that LP-GO-2 conjugate delivered pDNA in the nucleus within 1 h of exposure. The results indicate the prospect of using these conjugates as efficient carriers of nucleic acids for future gene therapy applications.     

Nucleic acid delivery: the missing pieces of the puzzle?

The delivery of genes or RNA interference (RNAi) agents can increase or decrease the expression of virtually any protein in a cell, and this process opens the path for cures to most diseases that afflict humans. However, the high molecular weight, anionic nature, and instability of nucleic acids in the presence of enzymes pose major obstacles to their delivery and frustrates their use as human therapies. This Account describes current ideas about the mechanisms in nonviral nucleic acid delivery and how lipidic and polymeric carriers can overcome some of the critical barriers to delivery. Over the last 20 years, researchers have developed a multitude of polymeric and lipidic vectors, but only a small fraction of these have progressed into clinical trials. None of these vectors has received FDA approval, which indicates that the current vectors do not yet have suitable properties for effective in vivo nucleic acid delivery. Nucleic acid delivery is a multistep process and inefficiencies at any stage result in a dramatic decrease in gene delivery or gene silencing. However, the majority of studies investigating synthetic vectors focus solely on optimization of endosomal escape. A small number of studies address how to improve uptake via targeted delivery, and an even smaller fraction examine the intracellular fate of the delivery systems and nucleic acid cargo. The internalization of genes into the cell nucleus remains an inefficient and mysterious process. In the case of DNA delivery, strategies are needed to increase and accelerate the migration of DNA through the cytoplasm and transport it through the nuclear membrane. siRNA delivery involves fewer barriers. siRNA is more readily released from the carrier and more resistant to enzymatic degradation, and its target is in the cytoplasm; hence, siRNA delivery systems are becoming a clinical reality. With regard to siRNA therapy, the exact cytoplasmic location of RNA-induced silencing complex (RISC) formation and activity is unknown, which makes specific targeting of the RISC for more efficient delivery difficult. Furthermore, we would like to identify the factors that favor the binding of siRNA to Ago-2. If we could understand how the half-life of siRNA and Ago-2/siRNA complex in the cytoplasm can be modulated without interfering with RISC functions that are essential for normal cell activity, we could increase siRNA delivery efficiency. In this Account, we review the current synthetic vectors and propose alternative strategies in a few cases. We also suggest how certain cellular mechanisms might be exploited to improve gene transfection and silencing. Finally, we discuss whether some carriers that deliver the siRNA to cells could also repackage the siRNA into exosomes. The exosomes would then transport the siRNA into a subsequent population of cells that manifest the siRNA effect. This piggy-back mechanism may be responsible for reported deep tissue siRNA effects using certain carriers.     

An Influence of Modification with Phosphoryl Guanidine Combined with a 2′-O-Methyl or 2′-Fluoro Group on the Small-Interfering-RNA Effect

Small interfering RNA (siRNA) is the most important tool for the manipulation of mRNA expression and needs protection from intracellular nucleases when delivered into the cell. In this work, we examined the effects of siRNA modification with the phosphoryl guanidine (PG) group, which, as shown earlier, makes oligodeoxynucleotides resistant to snake venom phosphodiesterase. We obtained a set of siRNAs containing combined modifications PG/2′-O-methyl (2′-OMe) or PG/2′-fluoro (2′-F); biophysical and biochemical properties were characterized for each duplex. We used the UV-melting approach to estimate the thermostability of the duplexes and RNAse A degradation assays to determine their stability. The ability to induce silencing was tested in cultured cells stably expressing green fluorescent protein. The introduction of the PG group as a rule decreased the thermodynamic stability of siRNA. At the same time, the siRNAs carrying PG groups showed increased resistance to RNase A. A gene silencing experiment indicated that the PG-modified siRNA retained its activity if the modifications were introduced into the passenger strand.     

Incorporation of Sulfonamide Moiety into Biguanide Scaffold Results in Apoptosis Induction and Cell Cycle Arrest in MCF-7 Breast Cancer Cells

Metformin, apart from its glucose-lowering properties, has also been found to demonstrate anti-cancer properties. Anti-cancer efficacy of metformin depends on its uptake in cancer cells, which is mediated by plasma membrane monoamine transporters (PMAT) and organic cation transporters (OCTs). This study presents an analysis of transporter mediated cellular uptake of ten sulfonamide-based derivatives of metformin in two breast cancer cell lines (MCF-7 and MDA-MB-231). Effects of these compounds on cancer cell growth inhibition were also determined. All examined sulfonamide-based analogues of metformin were characterized by greater cellular uptake in both MCF-7 and MDA-MB-231 cells, and stronger cytotoxic properties than those of metformin. Effective intracellular transport of the examined compounds in MCF-7 cells was accompanied by high cytotoxic activity. For instance, compound 2 with meta-methyl group in the benzene ring inhibited MCF-7 growth at micromolar range (IC₅₀ = 87.7 ± 1.18 µmol/L). Further studies showed that cytotoxicity of sulfonamide-based derivatives of metformin partially results from their ability to induce apoptosis in MCF-7 and MDA-MB-231 cells and arrest cell cycle in the G0/G1 phase. In addition, these compounds were found to inhibit cellular migration in wound healing assay. Importantly, the tested biguanides are more effective in MCF-7 cells at relatively lower concentrations than in MDA-MB-231 cells, which proves that the effectiveness of transporter-mediated accumulation in MCF-7 cells is related to biological effects, including MCF-7 cell growth inhibition, apoptosis induction and cell cycle arrest. In summary, this study supports the hypothesis that effective transporter-mediated cellular uptake of a chemical molecule determines its cytotoxic properties. These results warrant a further investigation of biguanides as putative anti-cancer agents.     

S1P Lyase siRNA Dampens Malignancy of DLD-1 Colorectal Cancer Cells

Sphingosine-1-phosphate lyase 1 (S1P lyase or SGPL1) is an essential sphingosine-1-phosphate-degrading enzyme. Its manipulation favors onset and progression of colorectal cancer and others in vivo. Thus, SGPL1 is an important modulator of cancer initiation. However, in established cancer, the impact of retrospective SGPL1 modulation is elusive. Herein, we analyzed how SGPL1 siRNA affects malignancy of the human colorectal cancer cells DLD-1 and found that in parallel to the reduction of SGPL1 expression levels, migration, invasion, and differentiation status changed. Diminished SGPL1 expression was accompanied with reduced cell migration and cell invasion in scratch assays and transwell assays, whereas metabolic activity and proliferation was not altered. Decreased migration was attended by increased cell–cell-adhesion through upregulation of E-cadherin and formation of cadherin-actin complexes. Spreading cell islets showed lower vimentin abundance in border cells. Furthermore, SGPL1 siRNA treatment induced expression of epithelial cell differentiation markers, such as intestinal alkaline phosphatase and cytokeratin 20. Hence, interference with SGPL1 expression augmented a partial redifferentiation of colorectal cancer cells toward normal colon epithelial cells. Our investigation showed that SGPL1 siRNA influenced tumorigenic activity of established colorectal cancer cells. We therefore suggest SGPL1 as a target for lowering malignant potential of already existing cancer.     

Simvastatin Downregulates Cofilin and Stathmin to Inhibit Skeletal Muscle Cells Migration

Statins are the most effective therapeutic agents for reducing cholesterol synthesis. Given their widespread use, many adverse effects from statins have been reported; of these, musculoskeletal complications occurred in 15% of patients after receiving statins for 6 months, and simvastatin was the most commonly administered statin among these cases. This study investigated the negative effects of simvastatin on skeletal muscle cells. We performed RNA sequencing analysis to determine gene expression in simvastatin-treated cells. Cell proliferation and migration were examined through cell cycle analysis and the transwell filter migration assay, respectively. Cytoskeleton rearrangement was examined through F-actin and tubulin staining. Western blot analysis was performed to determine the expression of cell cycle-regulated and cytoskeleton-related proteins. Transfection of small interfering RNAs (siRNAs) was performed to validate the role of cofilin and stathmin in the simvastatin-mediated inhibition of cell migration. The results revealed that simvastatin inhibited the proliferation and migration of skeletal muscle cells and affected the rearrangement of F-actin and tubulin. Simvastatin reduced the expression of cofilin and stathmin. The knockdown of both cofilin and stathmin by specific siRNA synergistically impaired cell migration. In conclusion, our results indicated that simvastatin inhibited skeletal muscle cell migration by reducing the expressions of cofilin and stathmin.     

聚酰胺-胺树状分子锡配合物催化性能研究

通过固相合成的方法将聚酰胺-胺树状大分子PAMAM担载于大孔硅胶上,并对其外围分别用对羟基苯甲醛、2,4-二羟基苯甲醛和邻羟基苯甲醛进行修饰,再与SnCl2.2H2O反应形成三类共计9种不同代数树状高分子锡配合物。将该类配合物用作质量分数30%双氧水氧化酮的Baeyer-Villiger(B-V)反应的催化剂,考察其催化活性,结果表明,在该类配合物作用下,2-金刚烷酮、环戊酮、环己酮、4-甲基环己酮、4-叔丁基环己酮、3-甲基-2-戊酮和4-甲基-2-戊酮都可以发生B-V氧化反应而转化为相应的酯和内酯,底物的转化率(75%～99%)和产物选择性(95%～100%)都较高。比较了不同载体(氯球、纤维素、壳聚糖、硅胶)、不同配体的金属锡配合物对B-V催氧化反应的催化效果,研究发现,载体、配体和金属担载量对配合物的催化活性均有不同程度的影响。其中,硅胶为最好的载体,而邻羟基苯甲醛为最好的配体。     

Mechanism of Anticancer Action of Novel Imidazole Platinum(II) Complex Conjugated with G2 PAMAM-OH Dendrimer in Breast Cancer Cells

Transition metal coordination compounds play an important role in the treatment of neoplastic diseases. However, due to their low selectivity and bioavailability, as well as the frequently occurring phenomenon of drug resistance, new chemical compounds that could overcome these phenomena are still being sought. The solution seems to be the synthesis of new metal complexes conjugated with drug carriers, e.g., dendrimers. Numerous literature data have shown that dendrimers improve the bioavailability of the obtained metal complexes, solving the problem of their poor solubility and stability in an aqueous environment and also breaking down inborn and acquired drug resistance. Therefore, the aim of this study was to synthesize a novel imidazole platinum(II) complex conjugated with and without the second-generation PAMAM dendrimer (PtMet2–PAMAM and PtMet2, respectively) and to evaluate its antitumor activity. Cell viability studies indicated that PtMet2–PAMAM exhibited higher cytotoxic activity than PtMet2 in MCF-7 and MDA-MB-231 breast cancer cells at relatively low concentrations. Moreover, our results indicated that PtMet2–PAMAM exerted antiproliferative effects in a zebrafish embryo model. Treatment with PtMet2–PAMAM substantially increased apoptosis in a dose-dependent manner via caspase-9 (intrinsic pathway) and caspase-8 (extrinsic pathway) activation along with pro-apoptotic protein expression modulation. Additionally, we showed that apoptosis can be induced by activating POX, which induces ROS production. Furthermore, our results also clearly showed that the tested compounds trigger autophagy through p38 pathway activation and increase Beclin-1, LC3, AMPK, and mTOR inhibition. The high pro-apoptotic activity and the ability to activate autophagy by the imidazole platinum(II) complex conjugated with a dendrimer may be due to its demonstrated ability to reverse multidrug resistance (MDR) and thereby increase cellular accumulation in breast cancer cells.     

Modeling the Interaction between Dendrimers and Nucleic Acids: a Molecular Perspective through Hierarchical Scales

Cationic dendrimers are promising nanocarriers for gene delivery thanks to their ability to establish strong interactions with oppositely charged strands of DNA and siRNA and to promote their aggregation. The binding between dendrimers and nucleic acids is typically a complex process that involves various types of interactions at different scales. To design efficient dendrimer candidates for DNA and siRNA binding it is necessary to have a detailed understanding of their interactions with oligonucleotides in the solvent. Molecular simulation can support experimental work, providing a privileged point of view on the aggregation process. This Minireview discusses recent computational efforts to unravel dendrimer–oligonucleotide binding, and proposes a perspective of the multiscale aggregation process based on hierarchy and on the transformations of the interacting “molecular units” following intermolecular interactions.     

Poly(amidoamine) Dendrimers as Nanocarriers for 5-Fluorouracil: Effectiveness of Complex Formation and Cytotoxicity Studies

Two generations of positively charged poly(amidoamine) dendrimers (PAMAMs) were selected for study as potential carriers for the anticancer drug 5-fluorouracil (5FU), a drug primarily used in the treatment of colorectal cancer. Analytical techniques, such as UV-Vis spectrophotometry, NMR Spectroscopy and Laser Doppler Velocimetry (LDV), have shown that the most critical factor determining the formation of a PAMAM–5FU complex is the starting components’ protonation degree. The tests confirmed the system’s ability to attach about 20 5FU molecules per one dendrimer molecule for the G4PAMAM dendrimer and about 25 molecules for the G6PAMAM dendrimer, which gives a system yield of 16% for the fourth generation and 5% for sixth generation dendrimers. Additionally, using the QCM-D method, the adsorption efficiency and the number of drug molecules immobilized in the dendrimer structure were determined. A new aspect in our study was the determination of the change in zeta potential (ζ) induced by the immobilization of 5FU molecules on the dendrimer’s outer shell and the importance of this effect in the direct contact of the carrier with cells. Cytotoxicity tests (resazurin reduction and MTS tests) showed no toxicity of dendrimers against mouse fibroblast cells (L929) and a significant decrease in cell viability in the case of four human malignant cell lines: malignant melanoma (A375), glioblastoma (SNB-19), prostate cancer (Du-145) and colon adenocarcinoma (HT-29) during incubation with PAMAM–5FU complexes. The purpose of our work was to investigate the correlation between the physicochemical properties of the carrier and active substance and the system efficiency and optimizing conditions for the formation of an efficient system based on PAMAM dendrimers as nanocarriers for 5-fluorouracil. An additional aspect was to identify potential application properties of the complexes, as demonstrated by cytotoxicity tests.     

Investigations on surface modified dendrimers: enzymatic hydrolysis and uptake into MCF-7 breast cancer cells

A series of G(0) and G(1) generation 1,3,5-tris(3-aminopropyl)benzene dendrimers surface modified with the amino acids phenylalanine (Phe), methionine (Met), aspartic acid (Asp), and diaminopropionic acid (Dap) was investigated with regard to their stability against enzymatic hydrolysis with the model enzymes papain, chymotrypsin, trypsin, and pepsin. Additionally, the cytosol of MCF-7 cells was used to get an insight into the possible degradation in intracellular medium. The cytosol, chymotrypsin, and papain had similar activities on the tested dendrimers and efficiently cleaved methionine and phenylalanine from the surface of the dendrimers. The cellular uptake of the dendrimers into the MCF-7 cells depended on the surface modification (Phe>Met unmodified>Dap=Asp) and the generations (G(0)>G(1)).     

RI基因siRNA干扰质粒的构建及其对人膀胱癌细胞迁移和侵袭能力的影响

目的构建核糖核酸酶抑制因子(RI)基因siRNA干扰质粒,并检测其对人膀胱癌细胞BIU-87迁移和侵袭能力的影响。方法设计并构建针对RI基因mRNA的2个特异性siRNA表达载体和1个无同源性的阴性对照载体,经酶切和DNA测序鉴定后,转染BIU-87细胞,通过RT-PCR、Western blot和免疫细胞化学法检测干扰的有效性。光镜及HE染色观察细胞形态的改变,黏附试验、划痕试验和Transwell法检测细胞黏附、迁移和侵袭能力的变化,激光共聚焦显微镜观察细胞骨架微丝结构。结果酶切和测序结果证明干扰质粒构建正确;转染特异性siRNA表达载体的BIU-87细胞RI蛋白的表达水平明显降低,对照组未发生明显改变;转染干扰质粒的细胞堆叠严重,细胞黏附能力显著降低,迁移和侵袭能力显著提高,细胞骨架微丝聚集,伸展出片状伪足。结论已成功构建RI基因siRNA干扰质粒,其可显著提高膀胱癌细胞的迁移和侵袭能力,说明RI可作为抑制肿瘤迁移和侵袭的有效靶点。     

人CD147基因siRNA真核表达质粒的构建及其对HeLa细胞中CD147基因表达的影响

目的构建针对人CD147基因的siRNA真核表达质粒,并观察其对HeLa细胞中CD147基因表达的影响。方法根据GenBank中登录的人CD147基因序列,设计并合成针对CD147基因的特异性小干扰片段,并将其定向克隆至带有卡那霉素抗性和增强型绿色荧光蛋白的真核表达载体pGenesil-1中,对重组质粒进行酶切分析和DNA序列测定。用脂质体将重组质粒转染至HeLa细胞中,观察其对HeLa细胞CD147基因mRNA及蛋白表达水平的影响。结果酶切鉴定和测序结果表明,3个表达短发夹RNA的质粒及其阴性对照质粒构建正确。3个siRNA真核表达质粒对HeLa细胞CD147基因mRNA转录水平的抑制率分别为32.5%、66.8%和59.1%;对CD147蛋白表达水平的抑制率分别为28.3%、63.5%和56.2%。结论已成功构建针对人CD147基因的siRNA真核表达质粒,其对HeLa细胞CD147基因的表达具有明显的抑制作用,为进一步研究CD147基因的功能奠定了基础。     

Cationic and Neutral N‐Heterocyclic Carbene Gold(I) Complexes: Cytotoxicity,NCI‐60 Screening,Cellular Uptake,Inhibition of Mammalian Thioredoxin Reductase,and Reactive Oxygen Species Formation

A structurally diverse library of 14 gold(I) cationic bis(NHC) and neutral mono(NHC) complexes (NHC: N‐heterocyclic carbene) was synthesized and characterized in this work. Four of them were new cationic gold(I) complexes containing functionalized NHCs, and their X‐ray crystal structures are presented herein. All of the complexes were investigated for their anticancer activities in four cancer cell lines, including a cisplatin‐resistant variant, and a noncancerous cell line. Seven of the cationic gold(I) complexes were found to display high and specific cytotoxic activities toward cancer cells. Two of them were even able to overcome cisplatin resistance. Two highly potent cationic complexes ( 11 and 15 ) were also submitted to the NCI‐60 cancer panel for further cytotoxicity evaluation. Complex 15 showed a surprisingly high potency toward leukemia among the nine examined cancer subtypes, particularly toward the CCRF‐CEM leukemia cell line with a concentration for 50 % inhibition of growth down to 79.4 nm . In addition, cationic complex 13 , which demonstrated a remarkable cytotoxicity against hepatocellular carcinoma, was selected to obtain insight into the mechanistic aspects in HepG2 cells. Cellular uptake measurements were indicative of good bioavailability. By various biochemical assays, this complex was found to effectively inhibit thioredoxin reductase and its cytotoxicity toward HepG2 cells was found to be reactive oxygen species dependent.     

siRNA介导NF-κB p65沉默联合5-Fu诱导胃癌细胞SGC7901凋亡

目的运用siRNA抑制胃癌细胞株SGC7901中NF-κB p65基因的表达,探讨其对细胞增殖和凋亡的影响,并分析该基因对化疗药物5-氟尿嘧啶(5-Fu)耐药性的作用。方法将SGC7901细胞分为空白对照组、脂质体组、5-Fu组、siRNA组和联合组,除空白对照组外,各组分别给予相应的药物。Western blot法检测各组细胞NF-κBp65蛋白的表达;Annexin V-PI法检测细胞凋亡;MTT法检测各组细胞的增殖水平。结果NF-κBp65蛋白在空白对照组和脂质体组中高表达,在5-Fu组、siRNA组和联合组中低表达,其中以联合组表达量最低;各组细胞早期凋亡率分别为1.40%±0.20%、2.36%±0.58%、6.68%±0.34%、6.60%±0.64%和21.62%±1.12%,其中以联合组最高;细胞的增殖活性随着siRNA浓度的增加和作用时间的延长而下降。结论siRNA能有效抑制NF-κBp65基因的表达,从而抑制SGC7901细胞增殖,促进其凋亡,并增强肿瘤细胞对化疗药物的敏感性。     

Enhanced growth inhibition of hepatic multicellular tumor spheroids by lactosylated poly(ethylene glycol)-siRNA conjugate formulated in PEGylated polyplexes

PEGylated polyplexes (lac-PEGylated polyplexes) composed of poly(L-lysine) and lactosylated poly(ethylene glycol)-small interfering RNA conjugate, which inhibits the RecQL1 gene product, were revealed to show an appreciable growth inhibition of multicellular HuH-7 spheroids (human hepatocarcinoma cell lines) for up to 21 days (IC(50)=6 nM); this system used as an in vitro three-dimensional (3D) model mimicking the in vivo biology of tumors. The PEGylated polyplexes thus prepared had a size of approximately 110 nm with clustered lactose moieties on their periphery as targeting ligands for the asialoglycoprotein-receptor-expressing HuH-7 cells. In contrast, OligofectAMINE/siRNA (cationic lipoplex) was observed to have almost no growth-inhibitory effect against HuH-7 spheroids, even though the lipoplex showed a stronger growth-inhibitory effect than the lac-PEGylated polyplexes on conventional monolayer-cultured HuH-7 cells. The FITC-tagged conjugate in the lac-PEGylated polyplexes showed smooth penetration into the HuH-7 spheroids compared with that in the lipoplexes, as observed by confocal fluorescence-scanning microscopy. This indicates that the small size of approximately 100 nm and the reduced nonspecific interaction due to the nonionic and hydrophilic lactosylated PEG layer contributes to the smooth penetration of the PEGylated polyplexes into the spheroid interior, eventually facilitating their uptake into the cells composing the spheroids. Cellular apoptosis indicating programmed cell death was also observed in the HuH-7 spheroids treated with the PEGylated polyplexes, revealing that the observed growth inhibition was indeed induced by the RNAi of the RecQL1 siRNA. These data suggest that the smart PEGylated polyplexes can indeed penetrate into the multiple cell layers of 3D tumor masses in vivo, exerting therapeutic effects through the RNAi.