Discovery of a new class of cell-penetrating peptides by novel phage display platform.

The primary hurdles for small interference RNA (siRNA) in clinical use are targeted and cytosolic delivery. To overcome both challenges, we have established a novel platform based on phage display, called NNJA. In this approach, a lysosomal cathepsin substrate is engineered within the flexible loops of PIII, that is displaying a unique random sequence at its N-terminus. NNJA library selection targeting cell-expressed targets should yield specific peptides localized in the cytoplasm. That is because phage internalization and subsequent localization to lysosome, upon peptide binding to the cell expressed target, will result in cleavage of PIII, rendering phage non-infective. Such phage will be eliminated from the selected pool and only peptide-phage that escapes lysosomes will advance to the next round. Proof of concept studies with the NNJA library demonstrated cytosolic localization of selected peptide-phage and peptide-siRNA, confirmed through confocal microscopy. More importantly, conjugation of siHPRT to monomeric or multimeric NNJA peptides resulted in significant reduction in HPRT mRNA in various cell types without significant cytotoxicity. Sequence similarity and clustering analysis from NGS dataset provide insights into sequence composition facilitating cell penetration. NNJA platform offers a highly efficient peptide discovery engine for targeted delivery of oligonucleotides to cytosol.

Membrane-Fusing Vehicles for Re-Sensitizing Transporter-Mediated Multiple-Drug Resistance in Cancer.

Reversing the multiple drug resistance (MDR) arising from the overexpression of the efflux transporters often fails mainly due to the high toxicity or the poor water solubility of the inhibitors of these transporters. Here, we demonstrate the delivery of an inhibitor targeting three ABC transporters (ABCB1, ABCC1 and ABCG2) directly to the cell membrane using membrane-fusing vehicles (MFVs). Three different transfected MDCK II cell lines, along with parental cells, were used to investigate the inhibitory effect of cyclosporine A (CsA) in solution versus direct delivery to the cell membrane. CsA-loaded MFVs successfully reversed MDR for all three investigated efflux transporters at significantly lower concentrations compared with CsA in solution. Results showed a 15-fold decrease in the IC50 value for ABCB1, a 7-fold decrease for ABCC1 and an 11-fold decrease for ABCG2. We observed binding site specificity for ABCB1 and ABCG2 transporters. Lower concentrations of empty MFVs along with CsA contribute to the inhibition of Hoechst 33342 efflux. However, higher concentrations of CsA along with the high amount of MFVs activated transport via the H-binding site. This supports the conclusion that MFVs can be useful beyond their role as delivery systems and also help to elucidate differences between these transporters and their binding sites.

Direct cytoplasmic delivery of RNAi therapeutics through a non-lysosomal pathway for enhanced gene therapy.

The first approved RNAi therapeutics, ONPATTRO, in 2017 moves the concept of RNA interference (RNAi) therapy from research to clinical reality, raising the hopes for the treatment of currently incurable diseases. However, RNAi therapeutics are still facing two main challenges-susceptibility to enzymatic degradation and low ability to escape from endo/lysosome into the cytoplasm. Therefore, we developed disulfide-based nanospheres (DBNPs) as universal vehicles to achieve efficient RNA delivery to address these problems. Notably, the DBNPs possess unique and desirable features, including improved resistance to nuclease degradation, direct cytoplasmic delivery through thiol-mediated cellular uptake, and cytosolic environment-responsive release, greatly enhancing the bioavailability of RNA therapeutics. Additionally, DBNPs are superior in terms of overcoming formidable physiological barriers, including vascular barriers and impermeable tumor tissues. Owning to these advantages, the DBNPs exhibit efficient gene silencing effect when delivering either small interfering RNA (siRNA) or microRNA in various cell lines and generate remarkable growth inhibition in the zebrafish and mouse model of pancreatic tumors as compared to traditional delivery vectors, such as PEI. Therefore, DBNPs have potential application prospect in RNAi therapy both in vitro and in vivo. STATEMENT OF SIGNIFICANCE: RNA interference (RNAi) therapeutics could target and alter any disease-related mRNA translation, thus have great potential in clinical application. Delivery efficiency of RNA modalities into cell cytoplasm is the main problem that currently limit RNAi therapeutics to release their full potential. Most of the known delivery materials suffer from the endo/lysosomal entrapment and enzymatic degradation during endocytosis-dependent uptake, resulting unsatisfied efficiency of the cytoplasmic release. Here, we developed disulfide-based nanospheres could directly transfer RNA modalities into the cytoplasm and significantly enhance the delivery efficiency, thus holding great potential in RNAi therapy.

Cytoplasmic Delivery of an Antibiotic, Trimethoprim, with a Simple Bidentate Catechol Analog as a Siderophore Mimetic.

Concerns about antibiotic-resistant Gram-negative pathogens are escalating, and accordingly siderophore-based intracellular antibiotic delivery is attracting more attention as an effective means to overcome these infections. Despite the successful clinical translation of this strategy, the delivery potential of siderophores has been limited to periplasm targeting, and this has appreciably restricted the repertoire of applicable antibiotics. To overcome this shortcoming of the current technology, this study focused on investigating the capability of simple bidentate catechol analogs to function as vehicles for cytoplasmic antibiotic delivery. Specifically, by employing trimethoprim, an inhibitor of dihydrofolate reductase located in the cytoplasm, as a model antibiotic, a chemical library of chelator-antibiotic conjugates featuring four different catechol analogs was prepared. Then, their various pharmacological properties and antimicrobial activities were evaluated. Analysis of these characterization data led to the identification of the active conjugates exhibiting notable iron- and trimethoprim-dependent potency against Escherichia coli. Further characterization of these hit molecules using E. coli mutant strains revealed that 2,3-dihydroxybenzoate could effectively deliver several corresponding conjugates to the cytoplasm by exploiting the siderophore uptake machineries present across the outer and inner membranes, originally designated for the native siderophore of E. coli, enterobactin. Considering the synthetic simplicity, such a catechol analog could have appreciable usage in potentiating cytoplasm-active antibiotics against recalcitrant Gram-negative pathogens.

Endosomal Escape of Bioactives Deployed via Nanocarriers: Insights Into the Design of Polymeric Micelles.

Cytoplasmic delivery of bioactives requires the use of strategies such as active transport, electroporation, or the use of nanocarriers such as polymeric nanoparticles, liposomes, micelles, and dendrimers. It is essential to deliver bioactive molecules in the cytoplasm to achieve targeted effects by enabling organelle targeting. One of the biggest bottlenecks in the successful cytoplasmic delivery of bioactives through nanocarriers is their sequestration in the endosomes that leads to the degradation of drugs by progressing to lysosomes. In this review, we discussed mechanisms by which nanocarriers are endocytosed, the mechanisms of endosomal escape, and more importantly, the strategies that can be and have been employed for their escape from the endosomes are summarized. Like other nanocarriers, polymeric micelles can be designed for endosomal escape, however, a careful control is needed in their design to balance between the possible toxicity and endosomal escape efficiency. Keeping this in view, polyion complex micelles, and polymers that have the ability to escape the endosome, are fully discussed. Finally, we provided some perspectives for designing the polymeric micelles for efficient cytoplasmic delivery of bioactive agents through endosomal escape.

Mucoadhesive Nanocarriers as a Promising Strategy to Enhance Intracellular Delivery against Oral Cavity Carcinoma.

Oral cancer, particularly squamous cell carcinoma (SCC), has posed a grave challenge to global health due to its high incidence, metastasis, and mortality rates. Despite numerous studies and favorable improvements in the therapeutic strategies over the past few decades, the prognosis of this disease remains dismal. Moreover, several drawbacks are associated with the conventional treatment; including permanent disfigurement and physical impairment that are attributed to surgical intervention, and systemic toxicity that results from aggressive radio- or chemotherapies, which impacts patients' prognosis and post-treatment quality of life. The highly vascularized, non-keratinized oral mucosa appears as a potential route for cytotoxic drug administration in treating oral cancer. It acts as a non-invasive portal for drug entry targeting the local oral lesions of the early stages of cancer and the systemic metastasis sites of advanced cancer. The absorption of the poorly aqueous-soluble anti-cancer drugs can be enhanced due to the increased permeability of the ulcerous mucosa lining in the disease state and by bypassing the hepatic first-pass metabolism. However, some challenges in oral transmucosal drug delivery include the drugs' taste, the limited surface area of the membrane lining the oral cavity, and flushing and enzymatic degradation by saliva. Therefore, mucoadhesive nanocarriers have emerged as promising platforms for controlled, targeted drug delivery in the oral cavity. The surface functionalization of nanocarriers with various moieties allows for drug targeting, bioavailability enhancement, and biodistribution at the site of action, while the mucoadhesive feature prolongs the drug's residence time for preferential accumulation to optimize the therapeutic effect and reduce systemic toxicity. This review has been focused to highlight the potential of various nanocarriers (e.g., nanoparticles, nanoemulsions, nanocapsules, and liposomes) in conferring targeting, solubility and bioavailability enhancement of actives and mucoadhesive properties as novel tumor-targeted drug delivery approaches in oral cancer treatment.

Interest of extracellular vesicles in regards to lipid nanoparticle based systems for intracellular protein delivery.

Compared to chemicals that continue to dominate the overall pharmaceutical market, protein therapeutics offer the advantages of higher specificity, greater activity, and reduced toxicity. While nearly all existing therapeutic proteins were developed against soluble or extracellular targets, the ability for proteins to enter cells and target intracellular compartments can significantly broaden their utility for a myriad of exiting targets. Given their physical, chemical, biological instability that could induce adverse effects, and their limited ability to cross cell membranes, delivery systems are required to fully reveal their biological potential. In this context, as natural protein nanocarriers, extracellular vesicles (EVs) hold great promise. Nevertheless, if not present naturally, bringing an interest protein into EV is not an easy task. In this review, we will explore methods used to load extrinsic protein into EVs and compare these natural vectors to their close synthetic counterparts, liposomes/lipid nanoparticles, to induce intracellular protein delivery.

Photo-responsive prodrug nanoparticles for efficient cytoplasmic delivery and synergistic photodynamic-chemotherapy of metastatic triple-negative breast cancer.

Triple-negative breast cancer (TNBC) have been considered as the most malignant subtype of breast cancer with leading incidence and mortality among females. Herein, photo-responsive prodrug nanoparticles (AlP/CPT-NPs) were designed with efficient cytoplasmic delivery of anti-cancer agent for cooperative photodynamic-chemotherapy. AlP/CPT-NPs were prepared using photosensitizer Al(III) phthalocyanine chloride disulfonic acid (AlP) and ROS-activatable camptothecin prodrug (CPT-PD). AlP/CPT-NPs could induce intracellular 1O2 generation upon light exposure, which not only initiate immediate disassembly of AlP/CPT-NPs but also promote cytoplasmic delivery of CPT through 1O2-mediated lysosomal rupture. The released intracellular CPT could be translocated into nuclei in only 5 min post-irradiation. Consequently, AlP/CPT-NPs efficiently suppressed the tumor growth and metastasis of TNBC in a spatiotemporally controlled manner, providing a promising option for effective treatment of metastatic TNBC. STATEMENT OF SIGNIFICANCE: Breast cancer is a complex disease with leading incidence among females, in which triple-negative breast cancer (TNBC) is considered as the most malignant subtype with increased risk of resistance, recurrence and metastasis. Herein, we designed photo-responsive prodrug nanoparticles (AlP/CPT-NPs) for synergistic treatment of metastatic TNBC. Upon 660 nm light exposure, the 1O2 generated by AlP/CPT-NPs could initiate immediate disassembly of AlP/CPT-NPs and further promote cytoplasmic delivery of the therapeutic payloads (camptothecin, CPT). The prepared AlP/CPT-NPs induced potent in vivo phototherapeutic damage through photodynamic-chemotherapy, resulting in complete tumor ablation with metastasis suppression.

Cell membrane fusing liposomes for cytoplasmic delivery in brain endothelial cells.

Direct cytoplasmic delivery is essential for susceptible molecules as proteins and some nucleic acids to improve their therapeutic efficacy in cells. Using liposomes for their delivery proved challenging due to known uptake by endocytosis followed by partial or complete lysosomal breakdown. Thus, "fusogenic" liposomes (FL) composed of the neutral lipid dioleoylphosphatidylethanolamine (DOPE) combined with the cationic lipid 1, 2-dioleoyl-3-trimethylammoniumpropane (DOTAP) were tested in different ratios for their cell membrane fusion ability and their cytoplasmic delivery was compared to "pH-sensitive" liposomes in murine brain endothelial cells (bEnd.3). They were loaded with cargos of different molecular sizes (calcein/ enhanced green fluorescent-protein (EGFP)/ EGFP coding plasmid) and their intracellular delivery was quantitatively and qualitatively analyzed. FL composed of equimolar ratios of DOPE and DOTAP showed the most efficient cytoplasmic delivery of all cargos by fusing with the cell membranes within the first 15 min of addition. Their EGFP plasmid delivery to cells was quantified to be 58.2 ±â€¯9.5 % of the total EGFP load and calcein delivery was measured in buffer to be 64.1 ±â€¯4.0 % of the total calcein load, and reduced in blood to 26.1 ±â€¯0.6 %. Thus our tested FL allowed a fast and abundant cytoplasmic delivery of cargos independent of their molecular sizes while avoiding endocytosis, although they also underwent fast fusion with erythrocytes. Seemingly, these carriers could be used as a powerful delivery tool for in-vitro purposes.

Characteristics of unique endocytosis induced by weak current for cytoplasmic drug delivery.

We previously reported that a weak current (WC, 0.3-0.5 mA/cm2) applied to cells can induce endocytosis to promote cytoplasmic delivery of hydrophilic macromolecules (MW: <70,000), such as dextran and siRNA, which leak from WC-induced endosomes into the cytoplasm (Hasan et al., 2016). In this study, we evaluated the characteristics of WC-mediated endocytosis for application of the technology to cytoplasmic delivery of macromolecular medicines. WC induced significantly higher cellular uptake of exogenous DNA fragments compared to untreated cells; the amount increased in a time-dependent manner, indicating that endocytosis was induced after WC. Moreover, following WC treatment of cells in the presence of an antibody (MW: 150,000) with the lysosomotropic agent chloroquine, the antibody was able to bind to its intracellular target. Thus, high molecular weight protein medicines delivered by WC-mediated endocytosis were functional in the cytoplasm. Transmission electron microscopy of cells treated by WC in the presence of gold nanoparticles covered with polyethylene glycol showed that the WC-induced endosomes exhibited an elliptical shape. In the WC-induced endosomes, ceramide, which makes pore structures in the membrane, was localized. Together, these results suggest that WC can induce unique endocytosis and that macromolecular medicines leak from endosomes through a ceramide pore.

Efficient Cytoplasmic Delivery of Antisense Probes Assisted by Cyclized-Peptide-Mediated Photoinduced Endosomal Escape.

Intracellular delivery and endosomal release of antisense oligonucleotides remain a significant challenge in the development of gene-targeted therapeutics. Previously, noncovalently cyclized TAT peptide (Cyc-TAT), in which the final ring-closing step is accomplished by hybridization of two short complementary γPNA segments, has been proven more efficient than its linear analogues at entering cells. As Cyc-TAT also readily accommodates a binding site, that is, an overhanging γPNA sequence, for codelivery of functional nucleic acid probes into cells, we were able to demonstrate that the overhang-Cyc-TAT penetrated into A549 cells when carrying an anti-telomerase γPNA that specifically reduced telomerase activity by over 97 %. Herein, we report that the cyclized TAT(FAM) can escape endosomes much more efficiently than the linear TAT(FAM) after LED illumination (490 nm). Based on this observation, the endosomal release of overhang-Cyc-TAT(FAM)/anti-telomerase γPNA complex can be greatly enhanced by photoactivation, thus shortening cell treatment time from 60 to 3 h, while keeping the same high efficiency in inhibiting telomerase activity inside A549 cells.

Functionalized extracellular vesicles as advanced therapeutic nanodelivery systems.

Extracellular vesicles (EVs) are membrane enclosed vesicles that are shed by almost all cell types, and play a fundamental role in cell-to-cell communication. The discovery that EVs are capable of functionally transporting nucleic acid- and protein-based cargoes between cells, rapidly promotes the idea of employing them as drug delivery systems. These endogenous vesicles indeed hold tremendous promise for therapeutic delivery. However, issues associated with exogenously administered EVs, including rapid clearance by the immune system, apparent lack of targeting cell specificity, and insufficient cytoplasmic delivery efficiency, may limit their therapeutic applicability. In this review, we discuss recent research avenues in EV-based therapeutic nanodelivery systems. Furthermore, we narrow our focus on the development of modification strategies to enhance the delivery properties of EVs, and elaborate on how to rationally harness these functionalized vesicles for therapeutic delivery.

Light-Responsive Nanoparticles for Highly Efficient Cytoplasmic Delivery of Anticancer Agents.

Stimuli-responsive nanostructures have shown great promise for intracellular delivery of anticancer compounds. A critical challenge remains in the exploration of stimuli-responsive nanoparticles for fast cytoplasmic delivery. Herein, near-infrared (NIR) light-responsive nanoparticles were rationally designed to generate highly efficient cytoplasmic delivery of anticancer agents for synergistic thermo-chemotherapy. The drug-loaded polymeric nanoparticles of selenium-inserted copolymer (I/D-Se-NPs) were rapidly dissociated in several minutes through reactive oxygen species (ROS)-mediated selenium oxidation upon NIR light exposure, and this irreversible dissociation of I/D-Se-NPs upon such a short irradiation promoted continuous drug release. Moreover, I/D-Se-NPs facilitated cytoplasmic drug translocation through ROS-triggered lysosomal disruption and thus resulted in highly preferable distribution to the nucleus even in 5 min postirradiation, which was further integrated with light-triggered hyperthermia for achieving synergistic tumor ablation without tumor regrowth.

Chitooligosaccharides Modified Reduction-Sensitive Liposomes: Enhanced Cytoplasmic Drug Delivery and Osteosarcomas-Tumor Inhibition in Animal Models.

PURPOSE: To investigate the potential of a reduction-sensitive and fusogenic liposomes, enabled by surface-coating with chotooligosaccharides (COS) via a disulfide linker, for tumor-targeted cytoplasmic drug delivery. METHODS: COS (MW2000-5000) were chemically tethered onto the liposomes through a disulfide linker (-SS-) to cholesterol (Chol). Doxorubicin (DOX) was actively loaded in the liposomes. Their reduction-sensitivities, cellular uptake, cytotoxicity, pharmacokinetics and antitumor efficacy were investigated. RESULTS: The Chol-SS-COS/DOX liposomes (100 nm) had zeta potential of 33.9 mV and high drug loading (13% w/w). The liposomes were stable with minimal drug leakage under physiological conditions but destabilized in the presence of reducing agents, dithiothreitol (DTT) or glutathione (GSH) at 10 mM, the cytosolic level. MTT assay revealed that the cationic Chol-SS-COS/DOX liposomes had higher cytotoxicity to MG63-osteosarcoma cells than non-reduction sensitive liposome (Chol-COS/DOX). Flow cytometry and confocal microscopy revealed that Chol-SS-COS/DOX internalized more efficiently than Chol-COS/DOX with more content to cytoplasm whereas Chol-COS/DOX located around the cell membrane. Chol-SS-COS/DOX preferentially internalized into MG63 cancer cell over LO2 normal liver cells. In rats both liposomes produced a prolonged half-life of DOX by 4 - 5.5 fold (p < 0.001) compared with the DOX solution. Chol-SS-COS/DOX exhibited strong inhibitory effect on tumor growth in MG63 cell-bearing nude mice (n = 6), and extended animal survival rate. CONCLUSIONS: Reduction-responsive Chol-SS-COS liposomes may be an excellent platform for cytoplasmic delivery of anticancer drugs. Conjugation of liposomes with COS enhanced tumor cell uptake, antitumor effect and survival rate in animal models.

The novel functional nucleic acid iRed effectively regulates target genes following cytoplasmic delivery by faint electric treatment.

An intelligent shRNA expression device (iRed) contains the minimum essential components needed for shRNA production in cells, and could be a novel tool to regulate target genes. However, general delivery carriers consisting of cationic polymers/lipids could impede function of a newly generated shRNA via electrostatic interaction in the cytoplasm. Recently, we found that faint electric treatment (fET) of cells enhanced delivery of siRNA and functional nucleic acids into the cytoplasm in the absence of delivery carriers. Here, we examined fET of cells stably expressing luciferase in the presence of iRed encoding anti-luciferase shRNA. Transfection of lipofectamine 2000 (LFN)/iRed lipoplexes showed an RNAi effect, but fET-mediated iRed transfection did not, likely because of the endosomal localization of iRed after delivery. However, fET in the presence of lysosomotropic agent chloroquine significantly improved the RNAi effect of iRed/fET to levels that were higher than those for the LFN/iRed lipoplexes. Furthermore, the amount of lipid droplets in adipocytes significantly decreased following fET with iRed against resistin in the presence of chloroquine. Thus, iRed could be a useful tool to regulate target genes following fET-mediated cytoplasmic delivery with endosomal escape devices.

Direct Cytoplasmic Delivery and Nuclear Targeting Delivery of HPMA-MT Conjugates in a Microtubules Dependent Fashion.

As the hearts of tumor cells, the nucleus is the ultimate target of many chemotherapeutic agents and genes. However, nuclear drug delivery is always hampered by multiple intracellular obstacles, such as low efficiency of lysosome escape and insufficient nuclear trafficking. Herein, an N-(2-hydroxypropyl) methacrylamide (HPMA) polymer-based drug delivery system was designed, which could achieve direct cytoplasmic delivery by a nonendocytic pathway and transport into the nucleus in a microtubules dependent fashion. A special targeting peptide (MT), derived from an endogenic parathyroid hormone-related protein, was conjugated to the polymer backbone, which could accumulate into the nucleus a by microtubule-mediated pathway. The in vitro studies found that low temperature and NaN3 could not influence the cell internalization of the conjugates. Besides, no obvious overlay of the conjugates with lysosome demonstrated that the polymer conjugates could enter the tumor cell cytoplasm by a nonendocytic pathway, thus avoiding the drug degradation in the lysosome. Furthermore, after suppression of the microtubule dynamics with microtubule stabilizing docetaxel (DTX) and destabilizing nocodazole (Noc), the nuclear accumulation of polymeric conjugates was significantly inhibited. Living cells fluorescence recovery after photobleaching study found that the nuclear import rate of conjugates was 2-fold faster compared with the DTX and Noc treated groups. These results demonstrated that the conjugates transported into the nucleus in a microtubules dependent way. Therefore, in addition to direct cytoplasmic delivery, our peptide conjugated polymeric platform could simultaneously mediate nuclear drug accumulation, which may open a new path for further intracellular genes/peptides delivery.

Faint electric treatment-induced rapid and efficient delivery of extraneous hydrophilic molecules into the cytoplasm.

Effective delivery of extraneous molecules into the cytoplasm of the target cells is important for several drug therapies. Previously, we showed effective in vivo transdermal delivery of naked siRNA into skin cells induced by faint electric treatment (ET) iontophoresis, and significant suppression of target mRNA levels (Kigasawa et al., Int. J. Pharm., 2010). This result indicates that electricity promoted the delivery of siRNA into cytoplasm. In the present study, we analyzed the intracellular delivery of naked anti-luciferase siRNA by faint ET, and found that the luciferase activity of cells expressing luciferase was reduced by in vitro ET like in vivo iontophoresis. Cellular uptake of fluorescent-label siRNA was increased by ET, while low temperature exposure, macropinocytosis inhibitor amiloride and caveolae-mediated endocytosis inhibitor filipin significantly prevented siRNA uptake. These results indicate that the cellular uptake mechanism involved endocytosis. In addition, voltage sensitive fluorescent dye DiBAC4 (3) penetration was increased by ET, and the transient receptor potential channel inhibitor SKF96365 reduced siRNA uptake, suggesting that faint ET reduced membrane potentials by changing intracellular ion levels. Moreover, to analyze cytoplasmic delivery, we used in-stem molecular beacon (ISMB), which fluoresces upon binding to target mRNA in the cytoplasm. Surprisingly, cytoplasmic ISMB fluorescence appeared rapidly and homogeneously after ET, indicating that cytoplasmic delivery is markedly enhanced by ET. In conclusion, we demonstrated for the first time that faint ET can enhance cellular uptake and cytoplasmic delivery of extraneous molecules.

Method for Confirming Cytoplasmic Delivery of RNA Aptamers.

RNA aptamers are single-stranded RNA oligos that represent a powerful emerging technology with potential for treating numerous diseases. More recently, cell-targeted RNA aptamers have been developed for delivering RNA interference (RNAi) modulators (siRNAs and miRNAs) to specific diseased cells (e.g., cancer cells or HIV infected cells) in vitro and in vivo. However, despite initial promising reports, the broad application of this aptamer delivery technology awaits the development of methods that can verify and confirm delivery of aptamers to the cytoplasm of target cells where the RNAi machinery resides. We recently developed a functional assay (RIP assay) to confirm cellular uptake and subsequent cytoplasmic release of an RNA aptamer which binds to a cell surface receptor expressed on prostate cancer cells (PSMA). To assess cytoplasmic delivery, the aptamer was chemically conjugated to saporin, a ribosome inactivating protein toxin that is toxic to cells only when delivered to the cytoplasm (where it inhibits the ribosome) by a cell-targeting ligand (e.g., aptamer). Here, we describe the chemistry used to conjugate the aptamer to saporin and discuss a gel-based method to verify conjugation efficiency. We also detail an in vitro functional assay to confirm that the aptamer retains function following conjugation to saporin and describe a cellular assay to measure aptamer-mediated saporin-induced cytotoxicity.

Enhanced Cytoplasmic Delivery of RAGE siRNA Using Bioreducible Polyethylenimine-based Nanocarriers for Myocardial Gene Therapy.

This study aims to develop bioreducible polyethylenimine (rPEI)/siRNA polyplexes with high stability, high transfection efficiency, and low cytotoxicity for efficient cytoplasmic siRNA delivery. rPEI successfully incorporated siRNA into stable and compact nanocomplexes, and the disulfide linkages in rPEI/siRNA were cleaved under reductive environments, resulting in efficient intracellular translocation and siRNA release. In this study, receptor for advanced glycation end-products (RAGE) was selected as a therapeutic target gene because it is associated with inflammatory responses in ischemia/reperfusion injury. rPEI/siRAGE exhibited high target gene silencing and low cytotoxicity in cardiomyocytes, and the treatment of rPEI/siRAGE reduced the myocardial infarction size.