Design and evaluation of new pH-sensitive amphiphilic cationic lipids for siRNA delivery.

Synthetic small interfering RNA (siRNA) has become the basis of a new generation of gene-silencing cancer therapeutics. However, successful implementation of this novel therapy relies on the ability to effectively deliver siRNA into target cells and to prevent degradation of siRNA in lysosomes after endocytosis. In this study, our goal was to design and optimize new amphiphilic cationic lipid carriers that exhibit selective pH-sensitive endosomal membrane disruptive capabilities to allow for the efficient release of their siRNA payload into the cytosol. The pH sensitive siRNA carriers consist of three domains (cationic head, hydrophobic tail, amino acid-based linker). A library of eight lipid carriers were synthesized using solid phase chemistry, and then studied to determine the role of (1) the number of protonable amines and overall pKa of the cationic head group, (2) the degree of unsaturation of the hydrophobic tail, and (3) the presence of histidine residues in the amino acid linker for transfection and silencing efficacy. In vitro screening evaluation of the new carriers demonstrated at least 80% knockdown of a GFP reporter in CHO cells after 72h. The carriers ECO and ECLn performed the best in a luciferase knockdown study in HT29 human colon cancer cells, which were found to be more difficult to transfect. They significantly reduced expression of this reporter to 22.7±3.31% and 23.5±5.11% after 72h post-transfection, better than Lipofectamine RNAiMax. Both ECO and ECLn carriers caused minimal cytotoxicity, preserving relative cell viabilities at 87.3±2.72% and 88.9±6.84%, respectively. A series of hemolysis assays at various pHs revealed that increasing the number of amines in the protonable head group, and removing the histidine residue from the linker, both resulted in improved membrane disruptive activity at the endosomal pH of 6.5. Meanwhile, the cellular uptake into HT29 cancer cells was improved, not only by increasing the amines of the head group, but also by increasing the degree of unsaturation in the lipid tails. Due to flexibility of the synthetic procedure, the delivery system could be modified further for different applications. The success of ECO and ECLn for in vitro siRNA delivery potentially makes them promising candidates for future in vivo studies.

Integrin targeted delivery of gene therapeutics.

Integrins have become key targets for molecular imaging and for selective delivery of anti-cancer agents. Here we review recent work concerning the targeted delivery of antisense and siRNA oligonucleotides via integrins. A variety of approaches have been used to link oligonucleotides to ligands capable of binding integrins with high specificity and affinity. This includes direct chemical conjugation, incorporating oligonucleotides into lipoplexes, and use of various polymeric nanocarriers including dendrimers. The ligand-oligonucleotide conjugate or complex associates selectively with the integrin, followed by internalization into endosomes and trafficking through subcellular compartments. Escape of antisense or siRNA from the endosome to the cytosol and nucleus may come about through endogenous trafficking mechanisms, or because of membrane disrupting capabilities built into the conjugate or complex. Thus a variety of useful strategies are available for using integrins to enhance the pharmacological efficacy of therapeutic oligonucleotides.

Targeted albumin-based nanoparticles for delivery of amphipathic drugs.

We report the preparation and physical and biological characterization of human serum albumin-based micelles of approximately 30 nm diameter for the delivery of amphipathic drugs, represented by doxorubicin. The micelles were surface conjugated with cyclic RGD peptides to guide selective delivery to cells expressing the α(v)ß(3) integrin. Multiple poly(ethylene glycol)s (PEGs) with molecular weight of 3400 Da were used to form a hydrophilic outer layer, with the inner core formed by albumin conjugated with doxorubicin via disulfide bonds. Additional doxorubicin was physically adsorbed into this core to attain a high drug loading capacity, where each albumin was associated with about 50 doxorubicin molecules. The formed micelles were stable in serum but continuously released doxorubicin when incubated with free thiols at concentrations mimicking the intracellular environment. When incubated with human melanoma cells (M21+) that express the α(v)ß(3) integrin, higher uptake and longer retention of doxorubicin was observed with the RGD-targeted micelles than in the case of untargeted control micelles or free doxorubicin. Consequently, the RGD-targeted micelles manifested cytotoxicity at lower doses of drug than control micelles or free drug.

Synthesis and evaluation of nanoglobule-cystamine-(Gd-DO3A), a biodegradable nanosized magnetic resonance contrast agent for dynamic contrast-enhanced magnetic resonance urography.

Dynamic contrast-enhanced magnetic resonance imaging has been recently shown to be effective for diagnostic urography. High-resolution urographic images can be acquired with T1 contrast agents for the kidney and urinary tract with minimal noise in the abdomen. Currently, clinical contrast agents are low molecular weight agents and can rapidly extravasate from blood circulation, leading to slow contrast agent elimination through kidney and consequently providing limited contrast enhancement in urinary tract. In this study, a new biodegradable macromolecular contrast agent, nanoglobule-G4-cystamine-(Gd-DO3A), was prepared by conjugating Gd-DO3A chelates on the surface of a generation 4 nanoglobule, poly-L-lysine octa(3-aminopropyl)silsesquioxane dendrimer, via a disulfide spacer, where the carrier had a precisely defined nanosize that is far smaller than the renal filtration threshold. The in vivo contrast enhancement and dynamic imaging of the urinary tract of the agent was evaluated in nude mice using a low molecular weight agent Gd(DTPA-BMA) as a control. The agent eliminated rapidly from blood circulation and accumulated more abundantly in urinary tract than Gd(DTPA-BMA). The fast elimination kinetics is ideal for functional evaluation of the kidneys. The morphology of the kidneys and urinary tract was better visualized by the biodegradable nanoglobular contrast agent than Gd(DTPA-BMA). The agent also resulted in low liver contrast enhancement, indicating low nonspecific tissue deposition. These features render the G4 nanoglobule-cystamine-(Gd-DO3A) conjugate a promising contrast agent for magnetic resonance urography.

New amphiphilic carriers forming pH-sensitive nanoparticles for nucleic acid delivery.

Amphiphilic lipids are promising for efficient intracellular delivery of nucleic acids. In this study, two new amphiphilic carriers, EKHCO and EHHKCO, were designed and synthesized as multifunctional carriers for efficient intracellular delivery of nucleic acids. The critical micelle concentrations of EKHCO and EHHKCO were 9.50 and 6.87 microM, respectively. Dynamic light scattering showed that the surfactants complexed with plasmid DNA and siRNA to form stable nanoparticles at the concentrations below their critical micelle concentrations. The nanoparticles of the surfactants with pDNA and siRNA exhibited pH-sensitive hemolysis against rat red blood cells when the pH decreased from 7.4 to 5.5, the endosomal-lysosomal pH. The nanoparticles of EHHKCO showed more concentration-dependent pH sensitivity than those of EKHCO. The EHHKCO and EKHCO nanoparticles of both pNDA and siRNA exhibited low cytotoxicity of at physiological pH. Both EKHCO and EHHKCO resulted in high intracellular uptake of pDNA and siRNA. EKHCO and EHHKCO resulted in relatively lower luciferase expression efficiency in U87 cells than DOTAP but produced a much higher percentage of GFP expression in the transfected cells than DOTAP. Both EKHCO and EHHKCO mediated much higher gene silencing efficiency of luciferase and green fluorescence protein (GFP) than DOTAP. The surfactants were more effective for intracellular siRNA delivery than intracellular delivery of plasmid DNA. The pH-sensitive amphiphilic carriers are promising multifunctional carriers for intracellular delivery of nucleic acids.

A family of bioreducible poly(disulfide amine)s for gene delivery.

A family of bioreducible poly(disulfide amine)s, which differ in the length of polymethylene spacer [-(CH(2))(n)-] in the main chain and the side chain, has been synthesized. These bioreducible poly(disulfide amine)s exhibit local environment specific degradability and are associated with lower cytotoxicity than branched poly(ethylenimine) (bPEI, 25 kDa). These cationic polymers also show higher buffering capacity and protonation degree than bPEI, facilitating the endosomal escape of carried genetic materials. The transfection efficiency of these agents is oligomethylene length dependent. Poly(cystaminebisacrylamide-spermine) [poly(CBA-SP)], poly(cystaminebisacrylamide-bis(3-aminopropyl)-1,3-propanediamine) [poly(CBA-APPD)], and poly(cyxtaminebisacrylamide-bis(3-aminopropyl)-ethylenediamine) [ploy(CBA-APED)] with longer propylene [-(CH(2))(3)-] side spacer, demonstrate higher transfection efficacy than the counterpart poly(cystaminebisacrylamide-bis(2-aminoethyl)-1,3-propanediamine) [poly(CBA-AEPD)] and poly(cystaminebisacrylamide-triethylenetetramine) [poly(CBA-TETA)], which have shorter ethylene [-(CH(2))(2)-] side spacer. The poly(CBA-SP), poly(CBA-APPD), poly(CBA-APED) with the main chain spacer of -(CH(2))(4)-, -(CH(2))(3)-, -(CH(2))(2)- demonstrate similar transfection efficiency, indicating the length of polymer main chain spacer has less influence on transfection efficiency. However, with the same short ethylene [-(CH(2))(2)-] side spacer, poly(CBA-AEPD), with the longer main chain oligomethylene units [-(CH(2))(3)-], showed relatively higher transfection efficiency than poly(CBA-TETA), having shorter main chain oligomethylene units [-(CH(2))(2)-]. Of these polymeric carriers, poly(CBA-SP) demonstrated the highest transfection in the C2C12 cell line, while poly(CBA-APED) showed the highest transfection in the HeLa cell line. All of these agents showed greater transfection activity than commercialized bPEI 25 kDa. The poly(disulfide amine)s are promising safe and efficient non-viral vectors for gene delivery.

Targeted systemic delivery of a therapeutic siRNA with a multifunctional carrier controls tumor proliferation in mice.

In this study, novel peptide-targeted delivery systems were developed for systemic and targeted delivery of therapeutic siRNA based on a multifunctional carrier, (1-aminoethyl)iminobis[N-(oleicylcysteinylhistinyl-1-aminoethyl)propionamide] (EHCO), which showed pH-sensitive amphiphilic cell membrane disruption. EHCO formed stable nanoparticles with siRNA. Targeted siRNA delivery systems were readily formed by surface modification of the nanoparticles. PEGylation of the siRNA/EHCO nanoparticles significantly reduced nonspecific cell uptake. The incorporation of a bombesin peptide or RGD peptide via a PEG spacer resulted in receptor-mediated cellular uptake and high gene silencing efficiency in U87 cells. Fluorescence confocal microscopic studies demonstrated that EHCO/siRNA nanoparticles and PEG modified EHCO/siRNA nanoparticles were able to facilitate endosomal escape of the siRNA delivery systems. Systemic administration of a therapeutic anti-HIF-1alpha siRNA with the peptide-targeted delivery systems resulted in significant tumor growth inhibition than a nontargeted delivery system or free siRNA via intravenous injection in nude mice bearing human glioma U87 xenografts. The results indicate a great promise of the multifunctional carrier EHCO for systemic and targeted delivery of therapeutic siRNA to treat human diseases with RNAi.

A peptide-targeted delivery system with pH-sensitive amphiphilic cell membrane disruption for efficient receptor-mediated siRNA delivery.

The efficient delivery of therapeutic siRNA into cells of interest is a critical challenge to broad application of RNAi. In this study, we developed a peptide-targeted delivery system for highly efficient receptor-mediated cellular siRNA delivery. The targeted delivery system was readily prepared by in situ functionalization of a polymerizable pH-sensitive amphiphilic surfactant, N-(1-aminoethyl)iminobis[N-(oleicyl-cysteinyl-histinyl-1-aminoethyl)propionamide] (EHCO) and self-assembly with siRNA. The intrinsic pH-sensitive amphiphilicity of EHCO at pH 5-6 was able to induce cell membrane disruption at endosomal pH and facilitate endosomal escape of the siRNA nanoparticles after internalization. The siRNA/EHCO nanoparticles and PEGylated siRNA/EHCO nanoparticles were not cytotoxic as compared to PEI/siRNA or TransFast/siRNA nanoparticles. siRNA/EHCO nanoparticles resulted in higher siRNA delivery efficiency than PEI and TransFast. The PEGylation of the siRNA/EHCO nanoparticles significantly reduced non-specific cell uptake. The incorporation of a bombesin peptide via a PEG spacer resulted in specific cellular uptake and high gene silencing efficiency in CHO-d1EGFP cells with overexpression of bombesin receptors. Receptor-mediated endocytosis and pH-sensitive amphiphilic endosomal escape are the advantageous features of the targeted siRNA delivery system for highly efficient cell-specific siRNA delivery. This novel targeted delivery system holds a great promise for systemic and targeted delivery of therapeutic siRNA.

Kv11.1 channel subunit composition includes MinK and varies developmentally in mouse cardiac muscle.

The Kv11.1 (also ERG1) K(+) channel underlies cardiac I(Kr), a current that contributes to repolarization in mammalian heart. In mice, I(Kr) current density decreases with development and studies suggest that changes in the structure and/or properties of the heteromultimeric I(Kr)/Kv11.1 channel are responsible. Here, using immunohistochemistry, we report that total Kv11.1 alpha subunit protein is more abundant in neonatal heart and is distributed throughout both adult and neonatal ventricles with greater abundance in epicardia. Immunoblots reveal that the alpha subunit alternative splice variant, Kv11.1a, is more abundant in adult heart while the Kv11.1b variant is more abundant in neonatal heart. Additionally, MinK channel subunit protein is shown to co-assemble with Kv11.1 protein and is more abundant in neonatal heart. In summary, Kv11.1/I(Kr) channel composition varies developmentally and the higher I(Kr) current density in neonatal heart is likely attributable to higher abundance of Kv11.1/I(Kr) channels, more specifically, the Kv11.1b splice variant.

Novel biodegradable poly(disulfide amine)s for gene delivery with high efficiency and low cytotoxicity.

Novel biodegradable poly(disulfide amine)s with defined structure, high transfection efficiency, and low cytotoxicity were designed and synthesized as nonviral gene delivery carriers. Michael addition between N, N'-cystaminebisacrylamide (CBA) and three N-Boc protected diamines ( N-Boc-1,2-diaminoethane, N-Boc-1,4-diaminobutane, and N-Boc-1,6-diaminohexane) followed by N-Boc deprotection under acidic condition resulted in final cationic polymers with disulfide bonds, tertiary amine groups in main chains, and pendant primary amine groups in side chains. Polymer structures were confirmed by 1H NMR, and their molecular weights were in the range 3.3-4.7 kDa with narrow polydispersity (1.12-1.17) as determined by size exclusion chromatography (SEC). Acid-base titration assay showed that the poly(disulfide amine)s possessed superior buffering capacity to branched PEI 25 kDa in the pH range 7.4-5.1, which may facilitate the escape of DNA from the endosomal compartment. Gel retardation assay demonstrated that significant polyplex dissociation was observed in the presence of 5.0 mM DTT within 1 h, suggesting rapid DNA release in the reduction condition such as cytoplasm due to the cleavage of disulfide bonds. Genetic transfections mediated by these poly(disulfide amine)s were side-chain spacer length dependent. The poly(disulfide amine) with a hexaethylene spacer, poly(CBA-DAH), had comparable transfection efficiency to bPEI 25 kDa in the tested cell lines, i.e., 293T cells, Hela cells, and NIH3T3 cells. This same poly(disulfide amine) mediated 7-fold higher luciferase expression than bPEI 25 kDa in C2C12 cells (mouse myoblast cell line), a cell line difficult to transfect with many cationic polymers. Furthermore, MTT assay indicated that all three poly(disulfide amine)s/pDNA polyplexes were significantly less toxic than bPEI/pDNA complexes.

In Vivo evaluation of a PAMAM-cystamine-(Gd-DO3A) conjugate as a biodegradable macromolecular MRI contrast agent.

Macromolecular Gd(III) chelates are superior magnetic resonance imaging (MRI) contrast agents for blood pool and tumor imaging. However, their clinical development is limited by the safety concerns related to the slow excretion and long-term gadolinium tissue accumulation. A generation 6 PAMAM Gd(III) chelate conjugate with a cleavable disulfide spacer, PAMAM-G6-cystamine-(Gd-DO3A), was prepared as a biodegradable macromolecular MRI contrast agent with rapid excretion from the body. T(1) and T(2) relaxivities of the contrast agent were 11.6 and 13.3 mM(-1)sec(-1) at 3T, respectively. Blood pool and tumor contrast enhancement of the agent were evaluated in female nude mice bearing MDA-MB-231 human breast carcinoma xenografts with a nondegradable conjugate PAMAM-G6-(Gd-DO3A) as a control. PAMAM-G6-cystamine-(Gd-DO3A) resulted in significant contrast enhancement in the blood for about 5 mins, and Gd-DO3A was released from the conjugate and rapidly excreted via renal filtration after the disulfide spacer was cleaved. The nondegradable control had much longer blood circulation and excreted more slowly from the body. PAMAM-G6-cystamine-(Gd-DO3A) also resulted in more prominent tumor contrast enhancement than the control. However, PAMAM-G6-cystamine-(Gd-DO3A) demonstrated high toxicity due to the intrinsic toxicity of PAMAM dendrimers. In conclusion, although PAMAM-G6-cystamine-(Gd-DO3A) showed some advantages compared with the nondegradable control, PAMAM dendrimers are not suitable carriers for biodegradable macromolecular MRI contrast agents, due to their high toxicity.