Ceramide Regulates Anti-Tumor Mechanisms of Erianin in Androgen-Sensitive and Castration-Resistant Prostate Cancers.

Prostate cancer is the second most prevalent malignancy worldwide. In the early stages, the development of prostate cancer is dependent on androgens. Over time with androgen deprivation therapy, 20% of prostate cancers progress to a castration-resistant form. Novel treatments for prostate cancers are still urgently needed. Erianin is a plant-derived bibenzyl compound. We report herein that erianin exhibits anti-tumor effects in androgen-sensitive and castration-resistant prostate cancer cells through different mechanisms. Erianin induces endoplasmic reticulum stress-associated apoptosis in androgen-sensitive prostate cancer cells. It also triggers pro-survival autophagic responses, as inhibition of autophagy predisposes to apoptosis. In contrast, erianin fails to induce apoptosis in castration-resistant prostate cancer cells. Instead, it results in cell cycle arrest at the M phase. Mechanistically, C16 ceramide dictates differential responses of androgen-sensitive and castration-resistant prostate cancer cells to erianin. Erianin elevates C16 ceramide level in androgen-sensitive but not castration-resistant prostate cancer cells. Overexpression of ceramide synthase 5 that specifically produces C16 ceramide enables erianin to induce apoptosis in castration-resistant prostate cancer cells. Our study provides both experimental evidence and mechanistic data showing that erianin is a potential treatment option for prostate cancers.

Co-delivery of paclitaxel and anti-VEGF siRNA by tripeptide lipid nanoparticle to enhance the anti-tumor activity for lung cancer therapy.

The combination of chemotherapeutic drug paclitaxel (PTX) and VEGF siRNA could inhibit cancer development with synergistic efficacy. However, efficient and safe delivery systems with high encapsulation efficiency of PTX and a long-time release of drugs are urgently needed. In this study, novel nanoparticles (PTX/siRNA/FALS) were constructed by using tripeptide lipid (L), sucrose laurate (S), and folate-PEG2000-DSPE (FA) to co-deliver PTX and siRNA. The cancer cell targeting nanoparticle carrier (PTX/siRNA/FALS) showed anticipated PTX encapsulation efficiency, siRNA retardation ability, improved cell uptake and sustained and controlled drug release. It led to significant anti-tumor activity in vitro and in vivo by efficient inhibition of VEGF expression and induction of cancer cell apoptosis. Importantly, the biocompatibility of the carriers and low dosage of PTX required for effective therapy greatly reduced the toxicity to mice. The targeting nanoparticles show potential as an effective co-delivery platform for RNAi and chemotherapy drugs, aiming to improve the efficacy of cancer therapy.

Stimuli-Responsive Polysaccharide Enveloped Liposome for Targeting and Penetrating Delivery of survivin-shRNA into Breast Tumor.

Silencing the inhibitor of apoptosis (IAP) by RNAi is a promising method for tumor therapy. One of the major challenges lies in how to sequentially overcome the system barriers in the course of the tumor targeting delivery, especially in the tumor accumulation and penetration. Herein we developed a novel stimuli-responsive polysaccharide enveloped liposome carrier, which was constructed by layer-by-layer depositing redox-sensitive amphiphilic chitosan (CS) and hyaluronic acid (HA) onto the liposome and then loading IAP inhibitor survivin-shRNA gene and permeation promoter hyaluronidase (HAase) sequentially. The as-prepared HA/HAase/CS/liposome/shRNA (HCLR) nanocarrier was verified to be stable in blood circulation due to the negative charged HA shield. The tumor targeting recognition and the enhanced tumor accumulation of HCLR were visualized by fluorescence resonance energy transfer (FRET) and in vivo fluorescence biodistribution. The deshielding of HA and the protonizing of CS in slightly acidic tumor extracellular pH environment (pHe, 6.8-6.5) were demonstrated by ζ potential change from -23.1 to 29.9 mV. The pHe-responsive HAase release was confirmed in the tumor extracellular mimicking environments, and the intratumoral biodistribution showed that the tumor penetration of HCLR was improved. The cell uptake of HCLR in pHe environment was significantly enhanced compared with that in physiological pH environment. The increased shRNA release of HCLR was approved in 10 mM glutathione (GSH) and tumor cells. Surprisingly, HCLR suppressed the tumor growth markedly through survivin silencing and meanwhile maintained low toxicity to mice. This study indicates that the novel polysaccharide enveloped HCLR is promising in clinical translation, thanks to the stimuli-triggered tumor accumulation, tumor penetration, cell uptake, and intracellular gene release.

Correlation of the cytotoxic effects of cationic lipids with their headgroups.

As effective non-viral vectors of gene therapy, cationic lipids still have the problem of toxicity, which has become one of the main bottlenecks for their applications. The toxicity of cationic lipids is strongly connected to the headgroup structures. In this article, we studied the cytotoxicity of two cationic lipids with a quaternary ammonium headgroup (CDA14) and a tri-peptide headgroup (CDO14), respectively, and with the same linker bond and hydrophobic domain. The IC50 values of CDA14 and CDO14 against NCI-H460 cells were 109.4 µg mL-1 and 340.5 µg mL-1, respectively. To determine the effects of headgroup structures of cationic lipids on cytotoxicity, apoptosis related pathways were investigated. As the lipids with a quaternary ammonium headgroup could induce more apoptotic cells than the ones with a peptide headgroup, the enzymatic activity of caspase-9 and caspase-3 increased obviously, whereas the mitochondrial membrane potential (MMP) decreased. At the same time, the reactive oxygen species (ROS) levels also increased and the cell cycle was arrested at the S phase. The results showed that the toxicity of the cationic lipid had a close relationship with its headgroup structures, and the cytotoxic mechanism was mainly via the caspase activation dependent signaling pathway and mitochondrial dysfunction. Through this study, we hope to provide the scientific basis for exploiting safer and more efficient cationic lipids for gene delivery.

A review on cationic lipids with different linkers for gene delivery.

Cationic lipids have become known as one of the most versatile tools for the delivery of DNA, RNA and many other therapeutic molecules, and are especially attractive because they can be easily designed, synthesized and characterized. Most of cationic lipids share the common structure of cationic head groups and hydrophobic portions with linker bonds between both domains. The linker bond is an important determinant of the chemical stability and biodegradability of cationic lipid, and further governs its transfection efficiency and cytotoxicity. Based on the structures of linker bonds, they can be grouped into many types, such as ether, ester, amide, carbamate, disulfide, urea, acylhydrazone, phosphate, and other unusual types (carnitine, vinyl ether, ketal, glutamic acid, aspartic acid, malonic acid diamide and dihydroxybenzene). This review summarizes some research results concerning the nature (such as the structure and orientation of linker groups) and density (such as the spacing and the number of linker groups) of linker bond for improving the chemical stability, biodegradability, transfection efficiency and cytotoxicity of cationic lipid to overcome the critical barriers of in vitro and in vivo transfection.

Dual stimuli-responsive saccharide core based nanocarrier for efficient Birc5-shRNA delivery.

Stimuli-responsive delivery systems show great promise in meeting the requirements of several delivery stages to achieve satisfactory gene transfection. This report describes a novel stimuli-responsive nanocarrier consisting of a dual stimuli-responsive chitosan (SCS) core and a polyethyleneimine (PEI) and polyethylene glycol (PEG) corona. The SCS core was constructed by cross-linking bio-safe chitosan saccharide via pH-sensitive hydrazone bonds and redox-sensitive disulfide bonds, which conferred the nanocarrier with a dual stimuli-responsive property. PEG and low-molecular-weight PEI were further immobilized on the core to endow the synthesized SCS-g-PEI-g-PEG with improved gene condensation, prolonged circulation and enhanced lysosome escape. In vitro and in vivo Birc5-shRNA delivery results revealed that significantly enhanced gene expression and tumor inhibition were achieved by SCS-g-PEI-g-PEG, compared with the non-stimuli-responsive counterpart CS-g-PEI-g-PEG. Moreover, the SCS-g-PEI-g-PEG nanocarrier was biocompatible as shown in both in vitro and in vivo toxicity evaluations. This study may provide a promising strategy for constructing an efficient and bio-safe delivery system for gene therapy.

Cationic lioposomes with folic acid as targeting ligand for gene delivery.

In our previous Letter, we have carried out the synthesis of a novel DDCTMA cationic lipid which was formulated with DOPE for gene delivery. Herein, we used folic acid (FA) as targeting ligand and cholesterol (Chol) as helper lipid instead of DOPE for enhancing the stability of the liposomes. These liposomes were characterized by dynamic laser scattering (DLS), transmission electron microscopy (TEM) and agarose gel electrophoresis assays of pDNA binding affinity. The lipoplexes were prepared by using different weight ratios of DDCTMA/Chol (1:1, 2:1, 3:1, 4:1) liposomes and different concentrations of FA (50-200µg/mL) combining with pDNA. The transfection efficiencies of the lipoplexes were evaluated using pGFP-N2 and pGL3 plasmid DNA against NCI-H460 cells in vitro. Among them, the optimum gene transfection efficiency with DDCTMA/Chol (3:1)/FA (100µg/mL) was obtained. The results showed that FA could improve the gene transfection efficiencies of DDCTMA/Chol cationic liposome. Our results also convincingly demonstrated FA (100µg/mL)-coated DDCTMA/Chol (3:1) cationic liposome could serve as a promising candidate for the gene delivery.

Scale adaptive compressive tracking.

Recently, the compressive tracking (CT) method (Zhang et al. in Proceedings of European conference on computer vision, pp 864-877, 2012) has attracted much attention due to its high efficiency, but it cannot well deal with the scale changing objects due to its constant tracking box. To address this issue, in this paper we propose a scale adaptive CT approach, which adaptively adjusts the scale of tracking box with the size variation of the objects. Our method significantly improves CT in three aspects: Firstly, the scale of tracking box is adaptively adjusted according to the size of the objects. Secondly, in the CT method, all the compressive features are supposed independent and equal contribution to the classifier. Actually, different compressive features have different confidence coefficients. In our proposed method, the confidence coefficients of features are computed and used to achieve different contribution to the classifier. Finally, in the CT method, the learning parameter λ is constant, which will result in large tracking drift on the occasion of object occlusion or large scale appearance variation. In our proposed method, a variable learning parameter λ is adopted, which can be adjusted according to the object appearance variation rate. Extensive experiments on the CVPR2013 tracking benchmark demonstrate the superior performance of the proposed method compared to state-of-the-art tracking algorithms.

Sucrose ester based cationic liposomes as effective non-viral gene vectors for gene delivery.

As sucrose esters (SEs) are natural and biodegradable excipients with excellent drug dissolution and drug absorption/permeation in controlled release systems, we firstly incorporated SE into liposomes for gene delivery in this article. A peptide-based lipid (CDO14), Gemini-based quaternary ammonium-based lipid (CTA14), and mono-head quaternary ammonium lipid (CPA14), and SE as helper lipid, were prepared into liposomes which could enhance the interactions between liposomes and pDNA. Most importantly, the liposomes with helper lipid SE showed higher transfection and lower cytotoxicity than those without SE in Hela and A549 cells. It was also found that the transfection efficiency increased with the increase of SE content. The selected liposome, CDO14/SE, was able to deliver siRNA against luciferase for silencing gene in lung tumors of mice, with little in vivo toxicity. The results convincingly demonstrated SEs could be highly desirable candidates for gene delivery systems.

Conjugates of small targeting molecules to non-viral vectors for the mediation of siRNA.

UNLABELLED: To use siRNA (small interfering RNA) for gene therapy, a gene delivery system is often necessary to overcome several challenging requirements including rapid excretion, low stability in blood serum, non-specific accumulation in tissues, poor cellular uptake and inefficient intracellular release. Active and/or passive targeting should help the delivery system to reach the desired tissue or cell, to be internalized, and to deliver siRNA to the cytoplasm so that siRNA can inhibit protein synthesis. This review covers conjugates of small targeting molecules and non-viral delivery systems for the mediation of siRNA, with a focus on their transfection properties in order to help the development of new and efficient siRNA delivery systems, as the therapeutic solutions of tomorrow. STATEMENT OF SIGNIFICANCE: The delivery of siRNA into cells or tissues remains to be a challenge for its applications, an alternative strategy for siRNA delivery systems is direct conjugation of non-viral vectors with targeting moieties for cellular delivery. In comparison to macromolecules, small targeting molecules have attracted great attention due to their many potential advantages including significant simplicity and ease of production, good repeatability and biodegradability. This review will focus on the most recent advances in the delivery of siRNA using conjugates of small targeting molecules and non-viral delivery systems. Based the editor's suggestions, we hope the revised manuscript could provide more profound understanding to the conjugates of targeting molecules to vectors for mediation of siRNA.

Carbamate-linked cationic lipids with different hydrocarbon chains for gene delivery.

A series of carbamate-linked cationic lipids containing saturated or unsaturated hydrocarbon chains and quaternary ammonium head were designed and synthesized. After recrystallization, carbamate-linked cationic lipids with high purity (over 95%) were obtained. The structures of these lipids were proved by IR spectrum, HR-ESI-MS, HPLC, (1)H NMR and (13)C NMR. The liposomes were prepared by using these cationic lipids and neutral lipid DOPE. Particle size and zeta-potential were studied to show that they were suitable for gene transfection. The DNA-bonding ability of C12:0, C14:0 and C18:1 cationic liposomes was much better than others. The results of transfection showed that hydrophobic chains of these lipids have great effects on their transfection activity. The lipids bearing C12:0, C14:0 saturated chains or C18:1 unsaturated chain showed relatively higher transfection efficiency and lower cytotoxicity. So these cationic lipids could be used as non-viral gene carriers for further studies.

Grafting chitosan with polyethylenimine in an ionic liquid for efficient gene delivery.

Modifying chitosan (CS) with polyethylenimine (PEI) grafts is an effective way to improve its gene transfection performance. However, it is still a challenge to conduct the grafting with fine control and high efficiency, particularly for the modification of water-insoluble CS. Herein, a novel method to graft CS with PEI (1.8 kDa, PEI-1.8) was developed by using ionic liquid 1-butyl-3-methyl imidazolium acetate ([BMIM]Ac) as a reaction solvent, water-insoluble CS as a reaction substrate and 1,1-carbonyldiimidazole (CDI) as a linking agent. The grafting reaction was greatly accelerated and the reaction time was largely shortened to 4 h by taking advantages of the good solubility of CS, the enhanced nucleophilicity of amino groups and the preferential stability of the activated complexes in the ionic liquid. The chitosan-graft-polyethylenimine (CS-g-PEI) products were characterized by 1H NMR, FTIR and GPC. PEI-1.8 was quantitatively grafted to CS through urea linkages, and the grafting degree (GD) was conveniently tuned by varying the molar ratios of PEI-1.8 to D-glucosamine units of CS in the range of 9.0 × 10(-3) to 9.0 × 10(-2). Compared with CS, the synthesized CS-g-PEI copolymers showed higher pDNA-binding affinity, which increased with the GD as shown in Agarose gel electrophoresis. The dynamic light scattering (DLS) experiment demonstrated that the CS-g-PEI/pDNA polyplexes had suitable particle sizes and proper ζ-potentials for cell transfection. The CS-g-PEI copolymer with a medium GD of 4.5% conferred the best gene transfection, with the efficiency 44 times of CS and 38 times of PEI-1.8 in HEp-2 cells. The cytotoxicity of CS-g-PEI was tested and found nearly as low as that of CS and much lower than that of PEI.

Tri-peptide cationic lipids for gene delivery.

Several novel tri-peptide cationic lipids were designed and synthesized for delivering DNA and siRNA. They have tri-lysine and tri-ornithine as head groups, carbamate group as linker and 12 and 14 carbon atom alkyl groups as tails. These tri-peptide cationic lipids were prepared into cationic liposomes for the study of the physicochemical properties and gene delivery. Their particle size, Zeta potential and DNA-binding were characterized to show that they were suitable for gene transfection. The further results indicate that these lipids can transfer DNA and siRNA very efficiently into NCI-H460 and Hep-2 tumor cells. The selected lipid, CDO14, was able to deliver combined siRNAs against c-Myc and VEGF for silencing distinct oncogenic pathways in lung tumors of mice, with little in vitro and in vivo toxicity.

Novel gemini cationic lipids with carbamate groups for gene delivery.

To obtain efficient non-viral vectors, a series of Gemini cationic lipids with carbamate linkers between headgroups and hydrophobic tails were synthesized. They have the hydrocarbon chains of 12, 14, 16 and 18 carbon atoms as tails, designated as G12, G14, G16 and G18, respectively. These Gemini cationic lipids were prepared into cationic liposomes for the study of the physicochemical properties and gene delivery. The DNA-bonding ability of these Gemini cationic liposomes was much better than their mono-head counterparts (designated as M12, M14, M16 and M18, respectively). In the same series of liposomes, bonding ability declined with an increase in tail length. They were tested for their gene-transferring capabilities in Hep-2 and A549 cells. They showed higher transfection efficiency than their mono-head counterparts and were comparable or superior in transfection efficiency and cytotoxicity to the commercial liposomes, DOTAP and Lipofectamine 2000. Our results convincingly demonstrate that the gene-transferring capabilities of these cationic lipids depended on hydrocarbon chain length. Gene transfection efficiency was maximal at a chain length of 14, as G14 can silence about 80 % of luciferase in A549 cells. Cell uptake results indicate that Gemini lipid delivery systems could be internalised by cells very efficiently. Thus, the Gemini cationic lipids could be used as synthetic non-viral gene delivery carriers for further study.

Transmembrane routes of cationic liposome-mediated gene delivery using human throat epidermis cancer cells.

For studying the mechanism of cationic liposome-mediated transmembrane routes for gene delivery, various inhibitors of endocytosis were used to treat human throat epidermis cancer cells, Hep-2, before transfection with Lipofectamine 2000/pGFP-N2 or Lipofectamine 2000/pGL3. To eliminate the effect of inhibitor toxicity on transfection, the RLU/survival rate was used to represent the transfection efficiency. Chlorpromazine and wortmannin, clathrin inhibitors, decreased transfection efficiency by 44 % (100 µM) and 31 % (100 nM), respectively. At the same time, genistein, a caveolin inhibitor, decreased it by 30 % (200 µM). Thus combined transmembrane routes through the clathrin and caveolae-mediated pathways were major mechanisms of cell uptake for the cationic liposome-mediated gene delivery. After entering the cells, microtubules played an important role on gene delivery as vinblastine, a microtubulin inhibitor, could reduce transfection efficiency by 41 % (200 nM).

Structure-activity relationship of carbamate-linked cationic lipids bearing hydroxyethyl headgroup for gene delivery.

A novel series of carbamate-linked cationic lipids containing hydroxyl headgroup were synthesized and included in formulations for transfection assays. The DNA-lipid complexes were characterized for their ability to bind DNA, their size, ζ-potential and cytotoxicity. Compared with our previously reported cationic transfection lipid DDCDMA lacking the hydroxyl group and the commercially available, these cationic liposomes exhibited relatively higher transfection efficiency.

The headgroup evolution of cationic lipids for gene delivery.

Cationic lipids are one of the most widely used nonviral vectors for gene delivery and are especially attractive because they can be easily synthesized and extensively characterized. Additionally, they can best facilitate the elucidation of structure-activity relationships by modifying each of their constituent domains. The polar hydrophilic headgroups enable the condensation of nucleic acids by electrostatic interactions with the negatively charged phosphate groups of the genes, and further govern transfection efficiency. The headgroups of cationic lipids play a crucial role for gene delivery; they can be quaternary ammoniums, amines, aminoacids or peptides, guanidiniums, heterocyclic headgroups, and some unusual headgroups. This review summarizes recent research results concerning the nature (such as the structure and shape of cationic headgroup) and density (such as the number and the spacing of cationic headgroup) of head functional groups for improving the design of efficient cationic lipids to overcome the critical barriers of in vitro and in vivo transfection.

[Research on cationic vector-mediated RNAi].

In order to study the efficiency of small interfering RNA (siRNA) transfer mediated by cationic liposome, we used luciferase siRNA to evaluate the gene silencing activity in the Hep-2 cells, which were stably transduced with a luciferase gene. The pDNA transfection was studied, and siRNA arrearage assay was conducted to determine the capability of cationic liposome with siRNA. Different concentrations of siRNA was used to silence luciferase gene' activity, and then the result was examined by microplate reader. Cell viability was analyzed after transfection by MTT assay. The results suggested that Lipofectamine 2000 could transfer the pDNA efficiently, and have strong binding capacity with siRNA. The silencing efficiency of luciferase was obtained with low concentration of siRNA. The cell viability was influenced by RNA interference (RNAi) very slightly, but the cell survival rate decreased with the increase of siRNA concentrations. It was well concluded that by optimizing the experimental conditions, cationic liposome can transfer low concentration siRNA to silence target gene's activity efficiently.

Synthesis and biological activity of carbamate-linked cationic lipids for gene delivery in vitro.

We have introduced a convenient synthesis method for carbamate-linked cationic lipids. Two cationic lipids N-[1-(2,3-didodecylcarbamoyloxy)propyl]-N,N,N-trimethylammonium iodide (DDCTMA) and N-[1-(2,3-didodecyl carbamoyloxy)propyl]-N-ethyl-N,N-dimethylammonium iodide (DDCEDMA), with identical length of hydrocarbon chains, alternative quaternary ammonium heads, carbamate linkages between hydrocarbon chains and quaternary ammonium heads, were synthesized for liposome-mediated gene delivery. Liposomes composed of DDCEDMA and DOPE in 1:1 ratio exhibited a lower zeta potential as compared to those made of pure DDCEDMA alone, which influences their DNA-binding ability. pGFP-N2 plasmid was transferred by cationic liposomes formed from the above cationic lipids into Hela and Hep-2 cells, and the transfection efficiency of some of cationic liposomes was superior or parallel to that of two commercial transfection agents, Lipofectamine2000 and DOTAP. Combined with the results of the agarose gel electrophoresis and transfection experiment, the DNA-binding ability of cationic lipids was too strong to release DNA from complex in the transfection, which could lead to relative low transfection efficiency and high cytotoxicity.

Chitosan enhanced gene delivery of cationic liposome via non-covalent conjugation.

Two new types of stable ternary complexes were formed by mixing chitosan with DOTAP/pDNA lipoplex and DOTAP with chitosan/pDNA polyplex via non-covalent conjugation for the efficient delivery of plasmid DNA. They were characterized by atomic force microscopy, gel retarding, and dynamic light scattering. The DOTAP/CTS/pDNA complexes were in compacted spheroids and irregular lump of larger aggregates in structure, while the short rod- and toroid-like and donut shapes were found in CTS/DOTAP/pDNA complexes. The transfection efficiency of the lipopolyplexes showed higher GFP gene expression than DOTAP/pDNA and CTS/pDNA controls in Hep-2 and Hela cells, and luciferase gene expression 2-3-fold than DOTAP/pDNA control and 70-120-fold than CTS/pDNA control in Hep-2 cells. The intracellular trafficking was examined by confocal laser scanning microscopy. Rapid pDNA delivery to the nucleus enchanced by chitosan was achieved after 4 h transfection.