Release Of siRNA Research Articles

Double Controlled Release of Therapeutic RNA Modules through Injectable DNA-RNA Hybrid Hydrogel.

Advances in the DNA nanotechnology have enabled the fabrication of DNA-based hydrogels with precisely controlled structures and tunable mechanical and biological properties. Compared to DNA hydrogel, preparation of RNA-based hydrogel remains challenging due to the inherent instability of naked RNA. To overcome these limitations, we fabricated a DNA-RNA hybrid hydrogel via stepwise dual enzymatic polymerization. Multimeric short hairpin RNAs (shRNAs) were hybridized with functional DNA aptamers for targeting and mechanical properties of the hydrogel. The obtained DNA-RNA hybrid hydrogel was ultrasoft, robust, and injectable hence reconfigurable into any confined structures. As a model system, the hydrogel was able to mimic microtubule structures under physiological conditions and designed to release the functional small interfering RNA (siRNA)-aptamer complex (SAC) sequentially. In addition, we encoded restriction enzyme-responsive sites in DNA-RNA hybrid hydrogel to boost the release of SAC. This novel strategy provides an excellent platform for systematic RNA delivery through double-controlled release, SAC release from hydrogel, and subsequent release of siRNA from the SAC, which has promising potential in RNA therapy.

Silencing of MEF2D by siRNA Loaded Selenium Nanoparticles for Ovarian Cancer Therapy.

BackgroundDelivery of therapeutic small interfering RNA (siRNA) via functionalized nanoparticles holds great promise for cancer therapy. However, developing a safe and efficient delivery carrier of siRNA is a challenging issue.MethodsRGDfC peptide was used to modify the surface of selenium nanoparticles (SeNPs) to synthesize a biocompatible siRNA delivery vehicle (R-SeNPs), and MEF2D-siRNA was loaded onto R-SeNPs to prepare a functionalized selenium nanoparticle R-Se@MEF2D-siRNA. The chemical properties of R-SeNPs were characterized, and the anticancer efficacy as well as related mechanisms of R-Se@MEF2D-siRNA were further explored.ResultsR-Se@MEF2D-siRNA was significantly taken up by SKOV3 cells and could enter SKOV3 cells mainly in the clathrin-associated endocytosis way. The result of in vitro siRNA release demonstrated that R-Se@MEF2D-siRNA could release MEF2D-siRNA quicker in a microenvironment simulating a lysosomal environment in tumor cells compared to a normal physiological environment. The results of qRT-PCR assay proved that R-Se@MEF2D-siRNA could effectively silence the expression of the MEF2D gene in SKOV3 cells. R-Se@MEF2D-siRNA remarkably suppressed the proliferation of SKOV3 cells and further triggered its apoptosis. In addition, R-Se@MEF2D-siRNA had the capability to disrupt mitochondrial membrane potential (MMP) in SKOV3 cells and resulted in the overproduction of reactive oxygen species (ROS), indicating that mitochondrial dysfunction and ROS generation played an important role in the apoptosis of SKOV3 cells induced by R-Se@MEF2D-siRNA. In vivo, R-Se@MEF2D-siRNA also exhibited excellent antitumor activity mainly through decreasing tumor cells proliferation and triggering their apoptosis in tumor-bearing nude mice.ConclusionR-Se@MEF2D-siRNA provides an alternative strategy for ovarian cancer treatment in the clinic.

Few-Layer Bismuthene for Checkpoint Knockdown Enhanced Cancer Immunotherapy with Rapid Clearance and Sequentially Triggered One-for-All Strategy.

As a conceptually attractive strategy, the use of immune checkpoint blockade antibodies to treat cancer is limited due to the restrained tumor-infiltrating lymphocytes (TILs), poor accumulation and penetration of antibodies, and deficient checkpoint blockade in malignancies. In this study, we describe a pH and mild photothermal sequentially triggered PD-L1 siRNA release nanosystem, based on monoelemental bismuthene, as a one-for-all strategy to realize enhanced tumor mild photothermal immunotherapy. Under manually controlled NIR irradiation, the bismuthene-based nanosystem simultaneously induces a tumor-enhanced pathological permeability and retention (EPPR) effect, increases TIL recruitment, and triggers programmed siRNA release, thereby amplifying anti-PD-L1 immunotherapy. In addition, the nanosystem's rapid removal through intestinal and renal clearance mitigates toxicity risk associated with long-term retention. In vivo antitumor experiments demonstrate that this bismuthene-based nanosystem is a promising and effective approach for "cold" tumor management.

Calcium-siRNA Nanocomplexes Optimized by Bovine Serum Albumin Coating Can Achieve Convenient and Efficient siRNA Delivery for Periodontitis Therapy.

PurposeReducing toxicity, immunogenicity, and costs of small interfering RNAs (siRNA) carrier materials are key goals for RNA interference (RNAi) technology transition from bench to bed. Recently, calcium ions (Ca2+) have garnered attention as a novel, alternative material for delivering siRNA to cells. However, the tolerance for Ca2+ concentration varies in different cell types, which has limited its applications in vivo. Bovine serum albumin (BSA) can bind to Ca2+ through chelation. Moreover, BSA is a favorable coating material for nanoparticles owing to its excellent biocompatibility. Therefore, we hypothesized that coating Ca2+-siRNA with BSA helps buffer Ca2+ toxicity in vivo.MethodsBSA-Ca2+-siRNA nanoparticles were prepared, and the size, shape, encapsulation, and release efficiency were characterized using atomic force microscopy, scanning electronic microcopy, and gel electrophoresis. Binding nanoparticles were evaluated using attenuated total reflection-Fourier-transform infrared spectroscopy. The cellular uptake, intracellular release, cytotoxicity, and gene knockdown of nanoparticles were evaluated in periodontal ligament stem cells (PDLSCs) using laser-scanning confocal microscope, flow cytometry, and real-time quantitative polymerase chain reaction.ResultsBSA and Ca2+-siRNA could form a stable nano-scale complex (~140 nm in diameter). The nanocomplexes could maintain siRNA release for more than 1 week in neutral phosphate-buffered saline (PBS) and could induce accelerated degradation in acidic PBS (pH 5.0). The nanoparticles were taken up by the cells, primarily through macropinocytosis, and were then released intracellularly through the acidification of endosomes/lysosomes. Importantly, the BSA-Ca2+ carrier had high transfection efficiency and biocompatibility both in vitro and in vivo. To demonstrate the therapeutic potential of our BSA coating-optimized Ca2+-siRNA technology, we showed that BSA-Ca2+-siWWP1 complexes strongly enhanced the osteogenic differentiation of inflammatory PDLSCs.ConclusionBSA-Ca2+ could potentially be used for siRNA delivery, which is not only highly efficient and cost-effective but also biocompatible to host tissues owing to the BSA coating.

Antiangiogenic and Antitumor Therapy for Retinoblastoma with Hypoxia-Inducible Factor-1α siRNA and Celastrol Co-Delivery Nanomicelles.

Retinoblastoma (RB) makes up about 3% of all childhood malignancies. Chemotherapy is commonly applied for RB treatment, while the clinical effectiveness varies significantly due to the cancer therapeutic resistances. Hypoxic tumor microenvironment, a hallmark of all tumors, is strongly associated with malignant progress and therapeutic resistances. The hypoxia mainly promotes the angiogenesis by upregulating pro-angiogenetic pathways. In this work, polymeric micelles are used as the carrier to deliver celastrol and siRNA to RB cells for achieving synergistic anti-tumor and antiangiogenesis effects. The micelle vectors have shown effective cellular internalization and release of loaded-celastrol and HIF-1 siRNA. The co-delivery system specifically and synergistically inhibits the expression of HIF-1α and VEGF in RB cells, suppresses the HIF-1α /VEGF/VEGFR signaling pathway, and impedes the proliferation, migration, and invasion of vascular endothelial cells. The polymer micellar carrier that co-delivers HIF-1α siRNA and celastrol is used for antiangiogenic and antitumor therapy of RB. Altogether, the results show that our polymeric micelle delivery system can be used to overcome barriers of drug resistance induced by angiogenesis and develop new drug/siRNA combinatory therapies.

Abstract 2868: A novel fusogenic peptide for delivery of bioactive siRNAs targeting CSNK2A1 in ovarian cancer cells in vitro

Abstract Introduction: Ovarian cancer recurrence and survival rates have remained stagnant over time with current therapeutics displaying low efficacy and often rendered ineffective by degradation. This work presents novel fusogenic peptides that will electrostatically complex with siRNAs and cause a pH-responsive insertion into the endosomal membrane, resulting in destabilization and breakdown allowing for release of siRNAs into the cytoplasm of ovarian cancer cells. Delivery of bioactive siRNAs targeting the CSNK2A1 gene in ovarian cancer cells is hypothesized to reduce cell viability, migration, and invasion. Experimental Procedures: Peptide formulations were complexed with non-targeting siRNAs (siNT) and CSNK2A1 siRNAs (siCSNK2A1) at various N:P ratios for all procedures. Analysis of complex formation was completed through gel-shift assays and dynamic light scattering. After characterization of the complexes, each formulation without siRNA was delivered to OVCAR-3 and CAOV-3 cells for cytotoxicity analysis measured via MTS assay. Observation of cellular uptake and endosomal escape was completed through fluorescent imaging after delivering each peptide formulation complexed with Cy3-labeled siNT (Cy3-siNT) and performing early endosome antigen-1 staining. To confirm bioactivity of targeted siRNAs, complexes with siCSNK2A1 were delivered and gene silencing was analyzed via western blotting. Results: Complexation of siRNAs with each formulation was achieved at N:P ratios of 40:1 and greater. Each of the three peptide formulations were able to form uniform nanocomplexes, at 195 nm, 111 nm, and 59 nm with 8.25 mV, -14.56 mV, and -18.42 mV surface charges respectively, and did not display vehicle cytotoxicity at an 80:1 ratio. Each peptide also mediated siRNA uptake into ovarian cancer cells, with fluorescent siRNA signal increasing with increasing N:P ratios. Of note, we observed that Cy3-siRNA appeared separate from the endosome, indicative of endosomal escape, which was seen in several peptide formulations with both CAOV-3 and OVCAR-3 cell lines. Confirmation of bioactive delivery of siCSNK2A1 is shown through western blot imaging, demonstrating knockdown of CSNK2A1 in comparison to cells treated with siNT alone or left untreated. Conclusion: Our novel fusogenic peptides have shown the ability to complex with and deliver siRNAs into ovarian cancer cells. Each peptide mediates endosomal escape activity, with varying degrees of effectiveness, and silencing of CSNK2A1 as measured by western blot. Further study into downstream effects of CSNK2A1 is ongoing. Future work includes optimization of a single fusogenic sequence for optimal delivery of siRNAs and development of a novel targeting peptide to increase cancer targeting and uptake efficiency. Acknowledgements: This research was supported in part by the National Science Foundation EPSCoR Program under NSF Award # OIA-1655740. Citation Format: Timothy Samec, Jessica Boulos, Serena Gilmore, Angela Alexander-Bryant. A novel fusogenic peptide for delivery of bioactive siRNAs targeting CSNK2A1 in ovarian cancer cells in vitro [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2868.

Collaborative assembly-mediated siRNA delivery for relieving inflammation-induced insulin resistance

Obesity plays a primary causative role in insulin resistance and hyperglycemia that contributes to type 2 diabetes. Excess lipid storage in the liver renders activation of the resident macrophages and chronic secretion of inflammatory mediators, therefore causing or aggravating insulin resistance. Herein, we develop collaborative assemblies using a “one-pot” synthesis method for macrophage-specific delivery of small interfering RNAs (siRNAs) that target the inflammatory proteins. Ternary nanocomplex (NC) composed of the siRNA molecule, a synthetic thiol-bearing methacrylated hyaluronic acid (sm-HA) and protamine forms through an electrostatic-driven physical assembly, which is chemically crosslinked to acquire the collaboratively assembled nanocapsule (cNC) concurrently. The obtained cNC displays significantly higher stability than NC. Functional moieties as flexible assembly units can be easily equipped on cNC for long circulation, active targeting, or controlled siRNA release. cNC-F decorated with folic acid, a macrophage-targeting ligand promotes the siRNA accumulation in the activated macrophages in the liver of the obese mouse model. cNC-F loaded with siRNA targeting inflammatory indicators efficiently control the macrophage inflammatory response by reducing the expression of the inflammatory proteins (> 40% reduction) and ameliorating the insulin resistance symptoms of the obese mice.

“Building-block crosslinking” micelles for enhancing cellular transfection of biocompatible polycations

Incorporating functional ligands and biodegradable bonds into biocompatible low-molecular-weight (LMW) polymers, such as 1.8 kDa poly(ethylenimine) (PEI 1.8k), is a common strategy to improve the properties of LMW polymers including biosafety and delivery efficacy. This study demonstrates the hypothesis that introducing different functional ligands and linked reductive disulfides in PEI 1.8k will achieve superior siRNA transfection efficiency. By incorporating PEI-X (X represents cholesterol (Ch), heptafluorobutyric anhydride (HFBA, F) and 4-carboxyphenyl-boronic acid (PBA)) functional ligands into PEI 1.8k and subsequently crosslinking with each other via disulfide bond links, reductive-responsive PEI-X-SS-X-PEI copolymers were constructed to enhance the cellular transfection via the synergistic effect of the high affinity of Ch, F and PBA to cell membranes and the disulfide reduction triggered intracellular disassembly of micelles and subsequent siRNA release. Extraordinarily, ternary Ch-SS-F-SS-PBA micelles exhibited the strongest siRNA transfection efficiencies in in vitro cell experiments and in vivo animal experiments due to the coordination of enhanced serum stability, promoted cell uptake and endosomal escape, and cell targeting ability. This strategy of constructed multifunctional polymer here we called “building-block crosslinking” showed a simple and smart way to synthesize new materials. Also this strategy of constructing ligands-directed reduction-sensitive micelles improves the transfection efficiency of LMW PEI and provides a valuable insight to develop novel gene delivery systems.

Degradable double hydrophilic block copolymers and tripartite polyionic complex micelles thereof for small interfering ribonucleic acids (siRNA) delivery

Polymer vectors for gene therapy have been largely investigated as an alternative to viral vectors. In particular, double hydrophilic block copolymers (DHBCs) have shown potential in this domain, but to date studies mainly focus on non-degradable copolymers, which may be a restriction for further development. To overcome this limitation, we synthesized a DHBC (PEG43-b-PCL12(COOH)6.5) composed of a poly(ethylene glycol) (PEG) non-ionic and bioeliminable block and a degradable carboxylic acid-functionalized poly(ε-caprolactone) (PCL) block. The potential of this DHBC as an original vector for small interfering ribonucleic acids (siRNA) to formulate tripartite polyionic complex (PIC) micelles with poly(lysine) (PLL) was evaluated. We first studied the impact of the charge ratio (R) on the size and the zeta potential of the resulting micelles. With a charge ratio R = 1, one formulation with optimized physico-chemical properties showed the ability to complex 75% of siRNA. We showed a stability of the micelles at pH 7.4 and a disruption at pH 5, which allowed a pH-triggered siRNA release and proved the pH-stimuli responsive character of the tripartite micelles. In addition, the tripartite PIC micelles were shown to be non-cytotoxic below 40 µg/mL. The potential of these siRNA vectors was further evaluated in vitro: it was found that the tripartite PIC micelles allowed siRNA internalization to be 3 times higher than PLL polyplexes in murine mesenchymal stem cells, and were able to transfect human breast cancer cells. Overall, this set of data pre-validates the use of degradable DHBC as non-viral vectors for the encapsulation and the controlled release of siRNA, which may therefore constitute a sound alternative to non-degradable and/or cytotoxic polycationic vectors.

Systematic Evaluation of Protein-Based Nanoparticles for Stable Delivery of Small Interfering RNA.

Developing a delivery vehicle to protect siRNA from degradation is a significant challenge. To solve this challenge, researchers attempted to use protein-based nanoparticles to deliver siRNA with limited to moderate success. However, a systematic investigation of comparing the ability of different protein-based nanoparticles as vehicles to deliver siRNA stably within cells is still unavailable. Therefore, in this study we synthesized a library of both non-targeted (proteinsiRNA) nanoparticles (NPs) and targeted (antibody conjugated protein-siRNA) NPs and evaluated ability to stably deliver siRNA in to cells to silence the gene of interest. We investigated nanoparticles of casein, bovine serum albumin, and gelatin for the delivery of siRNA. We synthesized and characterized a total of 12 nanoconjugates; in these conjugates, we either encapsulated, electrostatically attached, or covalently conjugated siRNA. We evaluated the efficiency of attaching siRNA to nanoconjugates, stability, and cellular delivery. The ability of siRNA to silence the protein of interest in cancer cells was also investigated. Among non-targeted conjugates, BSA matrix imparted relatively high stability to siRNA when encapsulated. Among targeted nanoconjugates, gelatin nanoparticles rendered high stability to siRNA upon covalent conjugation to the surface. On comparing with both targeted and non-targeted NPs for release of siRNA within cells, antibody-gelatin-siRNA conjugate exhibited high release and functional activity (down-regulation of target protein levels) within the cells as confirmed by both fluorescence imaging and Western blotting. In summary, our investigations show that targeted gelatin nanoparticles and non-targeted BSA nanoparticles possess high stability and excellent gene suppression capabilities and warrants further studies. We can extend the results from this study to develop stable siRNA delivery vehicles to specifically silence the protein of interest.

Small interfering RNA-loaded chitosan hydrochloride/carboxymethyl chitosan nanoparticles for ultrasound-triggered release to hamper colorectal cancer growth in vitro

Development of nontoxic, targetable and potent small interfering RNAs (siRNA) delivery systems remains a predominant challenge for clinical application of siRNA therapy. The nanoparticles of carboxymethyl chitosan (CMC) and labeled fluorescein isothiocyanate (FITC)-chitosan hydrochloride (CHC) were fabricated as carriers for ultrasound-triggered drug delivery to treat colon cancer. The results showed the (FITC-CHC)-CMC nanoparticles could effectively encapsulate anti-β-catenin siRNA through ionic gelation self-assembly to improve the stability of siRNA. The cumulative release ratio of siRNA from crosslinked (FITC-CHC)-CMC nanoparticles was merely 11.08% in pH 2.2 solution within 120 min, whereas about 70.07% of the loaded siRNA was released within 120 min in pH 5.5 solution after an 8-min ultrasonic treatment. It indicated that the (FITC-CHC)-CMC based pH-sensitive delivery system could fulfill a controlled release of siRNA through responding to external stimulus (ultrasound) under favorable pH condition. Fluorescence microscopy measurements clearly visualized the entry of fluorescently-labeled siRNA into HT-29 cells. Following the transfection of anti-β-catenin siRNA for 48 h, the β-catenin protein expression of the colon cancer cells was reduced to about 40.10%, indicating effective reduction of the protein that promotes colon cancer proliferation. Our results demonstrated that the siRNA-(FITC-CHC)-CMC delivery system hold substantial potential for RNAi therapeutical applications in diseased cells.

Single siRNA Nanocapsules for Effective siRNA Brain Delivery and Glioblastoma Treatment.

Small interfering RNA (siRNA) has been considered as a highly promising therapeutic agent for human cancer treatment including glioblastoma (GBM), which is a fatal disease without effective therapy methods. However, siRNA-based GBM therapy is seriously hampered by a number of challenges in siRNA brain delivery including poor stability, short blood circulation, low blood-brain barrier (BBB) penetration, and tumor accumulation, as well as inefficient siRNA intracellular release. Herein, an Angiopep-2 (Ang) functionalized intracellular-environment-responsive siRNA nanocapsule (Ang-NCss (siRNA)) is successfully developed as a safe and efficient RNAi agent to boost siRNA-based GBM therapy. The experimental results demonstrate that the developed Ang-NCss (siRNA) displays long circulation in plasma, efficient BBB penetration capability, and GBM accumulation and retention, as well as responsive intracellular siRNA release due to the unique design of small size (25 nm) with polymeric shell for siRNA protection, Ang functionalization for BBB crossing and GBM targeting, and disulfide bond as a linker for intracellular-environment-responsive siRNA release. Such superior properties of Ang-NCss (siRNA) result in outstanding growth inhibition of orthotopic U87MG xenografts without causing adverse effects, achieving remarkably improved survival benefits. The developed siRNA nanocapsules provide a new strategy for RNAi therapy of GBM and beyond.

Modification of KL4 Peptide Revealed the Importance of Alpha-Helical Structure for Efficient siRNA Delivery.

A safe and effective delivery system is considered a key to the success of nucleic acid therapeutics. It has been reported that pulmonary surfactants or their components could facilitate the uptake of small interfering RNA (siRNA) into the lung epithelial cells. Previously, our group investigated the use of KL4 peptide, a synthetic cationic peptide that simulates the structural properties of surfactant protein B (SP-B), as siRNA delivery vector. Although KL4 peptide exhibits good in vitro siRNA transfection efficiency on lung epithelial cells, its therapeutic potential is limited by its poor aqueous solubility due to the presence of a high proportion of hydrophobic leucine residues. In this study, we aim to address the solubility issue, designing five different modified peptides by replacing the hydrophobic leucine with alanine or valine, and assess their potential as siRNA delivery vectors. While the modified peptides retain the overall cationic property, their siRNA binding is also affected and their transfection efficiency is inferior to the parent KL4 peptide. A closer examination of the conformation of these peptides by circular dichroism shows that substitution of leucine residues leads to the change of the secondary structure from α-helical content to either β-sheet or more disordered, β-turn conformations. Relatively conservative amino acid substitutions, in terms of hydrophobicity bulk, lead to substantial conformational alteration, heavily impacting siRNA binding and release, cellular uptake, and transfection efficiency. Although the peptide modification strategy employed in this study was unsuccessful in developing an improved version of KL4 peptide for siRNA delivery, it highlights the importance of the α-helical conformation for efficient siRNA transfection, providing useful insights for future development of peptide-based RNA delivery system.

Imaging small molecule-induced endosomal escape of siRNA

Small interfering RNAs (siRNAs) are a new class of promising therapeutic molecules that can be used for sequence-specific downregulation of disease-causing genes. However, endosomal entrapment of siRNA is a key hurdle for most delivery strategies, limiting the therapeutic effect. Here, we use live-cell microscopy and cytosolic galectin-9 as a sensor of membrane damage, to probe fundamental properties of endosomal escape of cholesterol-conjugated siRNA induced by endosome-disrupting compounds. We demonstrate efficient release of ligand-conjugated siRNA from vesicles damaged by small molecules, enhancing target knockdown up to ∼47-fold in tumor cells. Still, mismatch between siRNA-containing and drug-targeted endolysosomal compartments limits siRNA activity improvement. We also show widespread endosomal damage in macroscopic tumor spheroids after small molecule treatment, substantially improving siRNA delivery and knockdown throughout the spheroid. We believe the strategy to characterize endosomal escape presented here will be widely applicable, facilitating efforts to improve delivery of siRNA and other nucleic acid-based therapeutics.

Species differences in cellular processing of siRNA‐Lipid complex

Lipid‐based nanocarriers provide a means to deliver nucleic acid therapeutics such as short interfering RNA (siRNA); a major challenge is the low success rate in preclinical‐to‐clinical translation. siRNA gene knockdown depends on its ability to reach the intracellular target RISC (RNA Induced Silencing Complex, degrades the targeted mRNA), and is hence determined by the kinetics of multiple cellular processes (see below). The present project used experimental and computational studies to investigate the species differences in the processing of siRNA‐lipid complex (Lipoplex).Experiments were performed using human breast MCF7 tumor cells transfected with Rab5, a marker for early endosomes (EE). Cells were treated with Lipoplex (with fluorescence‐labeled siRNA and lipids), in the presence of fetal bovine serum, mouse plasma, or rat plasma. Pilot studies showed that mouse and human plasma had similar effects on Lipoplex destabilization, cellular uptake and gene knockdown. Time lapse live cell confocal microscopy was used to monitor Lipoplex cellular binding, endocytosis, and siRNA release, every 10 min for 2 h. The results were analyzed using quantitative image analysis for the amounts of siRNA and lipids in individual regions of interest. Over 95% of intracellular siRNA+lipid signals in the first 10 min were co‐localized with Rab5, confirming Lipoplex was endocytosed into EE. Some intracellular siRNA+lipid signals were no longer associated with Rab5 after 30 min, indicating Lipoplex progression through the endocytic pathway. Of the Rab5‐co‐localizaing siRNA+lipid signals, the siRNA‐to‐lipid ratio (Ratio) decreased with time (up to 3‐fold between 10 to 90 min), whereas Ratio in the signals not co‐localizing with Rab5 was up to 4‐fold lower. These findings indicate continuous release of free siRNA from Lipoplex while in EE. A quantitative pharmacology or QP model was developed to describe the first order kinetics of Lipoplex transfer between extracellular fluid/cell membrane/intracellular endocytic organelles and siRNA release into the cytosol. The model assumptions were (a) extracellular Lipoplex destabilization due to interactions with plasma/serum proteins, (b) rapid degradation of free siRNA released from Lipoplex, (c) two pools of membrane‐associated Lipoplex (internalized and not internalized), and (d) Lipoplex degradation/elimination in late endosomes or lysosomes. Fitting the QP model to experimental results yielded the rate constants.We found significant species differences, i.e., 2‐ to >15‐fold variations in rate constants for Lipoplex binding to cell membrane, internalization, release of siRNA into cytosol, and elimination. These results suggest a need of accounting for species differences in cellular processing of Lipoplex during animal‐to‐human translation. This study further demonstrates the utility of QP in difficult‐to‐measure subcellular kinetic processes.Support or Funding InformationSupported in part by research grants (RO1CA158300, RO1CA163015, RO1EB015253) from NCI and NIBIB, and by Mosier Chair of Pharmaceutical Sciences.

Polyarginine-Mediated siRNA Delivery: A Mechanistic Study of Intracellular Trafficking of PCL-R15/siRNA Nanoplexes.

As a cell-penetrating peptide, polyarginine is widely used in drug delivery systems based on its membrane permeation ability. Previously, we developed the mPEG-PLA-b-polyarginine(R15) triblock copolymer, which exhibited a high siRNA delivery efficiency both in vitro and in vivo. As a continued effort, here the amphiphilic diblock polymer PCL-R15 was synthesized as a simplified model to further elucidate the structure-activity relationship of arginine-based amphiphilic polymers as siRNA delivery systems, and the cellular trafficking mechanisms of the PCL-R15/siRNA nanoplexes were investigated to understand the interaction patterns between the nanoplexes and cells. Compared to the R15/siRNA complexes, the introduction of PCL moiety was found to result in the stronger interactions with cells and the enhanced transfection efficiency after the formation of condensed nanoplexes. Caveolae-mediated endocytosis and clathrin-mediated endocytosis were major routes for the internalization of PCL-R15/siRNA nanoplexes. The intracellular release of siRNA from nanoplexes was confirmed by fluorescence resonance energy transfer assay. It was also noticed that the internalized PCL-R15/siRNA nanoplexes were transported through digestive routes and trapped in lysosomes, which may be the bottleneck for efficient siRNA delivery of PCL-R15/siRNA nanoplexes. This study investigated the relationship between the polymer structure of PCL-R15 and the cellular interaction patterns, which may render implications on the rational design of polyarginine-based siRNA delivery systems.

Charge Conversional Biomimetic Nanocomplexes as a Multifunctional Platform for Boosting Orthotopic Glioblastoma RNAi Therapy.

Nanotechnology-based RNA interference (RNAi) has shown great promise in overcoming the limitations of traditional clinical treatments for glioblastoma (GBM). However, because of the complexity of brain physiology, simple blood-brain barrier (BBB) penetration or tumor-targeting strategies cannot entirely meet the demanding requirements of different therapeutic delivery stages. Herein, we developed a charge conversional biomimetic nanoplatform with a three-layer core-shell structure to programmatically overcome persistent obstacles in siRNA delivery to GBM. The resulting nanocomplex presents good biocompatibility, prolonged blood circulation, high BBB transcytosis, effective tumor accumulation, and specific uptake by tumor cells in the brain. Moreover, red blood cell membrane (RBCm) disruption and effective siRNA release can be further triggered elegantly by charge conversion from negative to positive in the endo/lysosome (pH 5.0-6.5) of tumor cells, leading to highly potent target-gene silencing with a strong anti-GBM effect. Our study provides an intelligent biomimetic nanoplatform tailored for systemically siRNA delivery to GBM, leveraging Angiopep-2 peptide-modified, immune-free RBCm and charge conversional components. Improved therapeutic efficacy, higher survival rates, and minimized systemic side effects were achieved in orthotopic U87MG-luc human glioblastoma tumor-bearing nude mice.

Silencing of IL-6 and STAT3 by siRNA loaded hyaluronate-N,N,N-trimethyl chitosan nanoparticles potently reduces cancer cell progression

The immunosuppressive nature of the tumor microenvironment is a critical problem that should be considered before the design of immunotherapies. Interleukin (IL)-6 and its related downstream molecules such as signal transducer and activator of transcription (STAT)3 play an important role in the cancer progression, which can be considered as potential therapeutic targets. In the present study, we generated the active-targeted hyaluronate (HA) recoated N, N, N-trimethyl chitosan (TMC) nanoparticles (NPs) to deliver IL-6- and STAT3-specific small interfering RNAs (siRNAs) to the CD44-expressing cancer cells. We utilized the interaction between HA and CD44 to increase the specificity and efficacy of cellular uptake in NPs. The results showed that the synthesized NPs had efficient physicochemical characteristics, high transfection efficiency, low toxicity, and controlled siRNA release. siRNA-loaded NPs significantly inhibited the IL-6/STAT3 expression, which was associated with blockade of proliferation, colony formation, migration, and angiogenesis in cancer cells. These findings imply the potential of HA-TMC NPs as potent vectors in gene therapy and their application for the silencing of IL-6 and STAT3, as a novel anti-cancer combination therapeutic strategy, for the first time.

Silencing KLK12 expression via RGDfC-decorated selenium nanoparticles for the treatment of colorectal cancer in vitro and in vivo.

Short interfering RNA (siRNA) has been investigated as a promising modality of cancer treatment due to its capability to target specific target genes for downregulation. However, the successful application of this strategy depends on producing a safe and effective carrier system for delivering siRNA to the tumor. Thus, investigation of siRNA delivery carriers is a fundamental step in the field of siRNA-based therapeutics. In the current research, the surface of selenium nanoparticles (SeNPs) were modified with the tumor-targeted molecular RGDfC peptide with positive charge to synthetize the biocompatible siRNA carrier RGDfC-SeNPs. Subsequently, KLK12-siRNA was loaded onto the surface of RGDfC-SeNPs to create functionalized nanoparticles (RGDfC-Se@siRNA) that we tested for in vitro and in vivo antitumor efficacy. We measured significantly greater particle uptake in HT-29 colorectal cancer cells relative to HUVECs, providing evidence for the targeted delivery of RGDfC-Se@siRNA. We found that RGDfC-Se@siRNA could enter HT-29 cells primarily via clathrin-mediated endocytosis. Further, these particles experienced faster siRNA release in an acidic microenvironment compared to pH7.4. The results from quantitative PCR and Western blot assays suggested that the target gene of KLK12 in HT-29 cells were obviously silenced by RGDfC-Se@siRNA. The further biological studies showed that treatment with RGDfC-Se@siRNA had ability to suppress the proliferation and migration/invasion of HT-29 cells, and triggered HT-29 cells apoptosis. RGDfC-Se@siRNA could induce the mitochondrial membrane potential (MMP) disruption and enhance the reactive oxygen species (ROS) generation in HT-29 cells, indicating that RGDfC-Se@siRNA induced the HT-29 cells apoptosis possibly by a ROS-mediated mitochondrial dysfunction pathway. Importantly, the in vivo antitumor study also verified that RGDfC-Se@siRNA could significantly suppress the growth of tumor in vivo. In addition, we did not observe any signs of systemic or tissue-specific toxicity after administration of RGDfC-Se@siRNA in mice. As a whole, these findings suggest that RGDfC-Se@siRNA has promising potential as a therapy for colorectal cancer.

Vapor nanobubble is the more reliable photothermal mechanism for inducing endosomal escape of siRNA without disturbing cell homeostasis

Strategies for controlled delivery of therapeutic siRNA into living cells are in high demand as endosomal escape remains the most prominent bottleneck at the intracellular level. Photothermal properties of gold nanoparticles (AuNP) can be used to overcome the endosomal membrane barrier upon laser irradiation by two mechanisms: endosomal rupture by mechanical energy from water vapor nanobubbles (VNBs), or permeabilization of the endosomal membrane by heat diffusion. Here we evaluated how both mechanisms influence cargo release, transfection efficiency, acute cytotoxicity and cell homeostasis. Using a siRNA/AuNP drug delivery system we found that the in vitro release of siRNA from the AuNP carrier occurs equally efficiently by VNB formation or heat generation. Heat-mediated endosomal escape happened more efficiently in cells that had more particles per endosome, resulting in variable siRNA-induced downregulation (20–50%). VNB-mediated endosomal escape did not dependent on the number of AuNP per endosome, yielding high downregulations (50–60%) independent of the cell type. Effects on cell homeostasis by whole transcriptome analysis, showed a quick recover after 24 h or 48 h for either of both photothermal mechanisms. We conclude that VNBs are more consistent to induce efficient endosomal escape and gene silencing independent of the cell type without long lasting effects on cell homeostasis.