Release Of siRNA Research Articles

Solid Lipid-Polymer Hybrid Nanoplatform for Topical Delivery of siRNA: In Vitro Biological Activity and Permeation Studies.

Small interfering RNA (siRNA) molecules have limited transfection efficiency and stability, necessitating the use of delivery systems to be effective in gene knockdown therapies. In this regard, lipid-polymeric nanocarriers have emerged as a promising class of nanoparticles for siRNA delivery, particularly for topical applications. We proposed the use of solid lipid-polymer hybrid nanoparticles (SLPHNs) as topical delivery systems for siRNA. This approach was evaluated by assessing the ability of SLPHNs-siRNA complexes to internalize siRNA molecules and both to penetrate skin layers in vitro and induce gene knocking down in a skin cell line. The SLPHNs were formed by a specific composition of solid lipids, a surfactant polymer as a dispersive agent, and a cationic polymer as a complexing agent for siRNA. The optimized nanocarriers exhibited a spherical shape with a smooth surface. The average diameter of the nanoparticles was found to be 200 nm, and the zeta potential was measured to be +20 mV. Furthermore, these nanocarriers demonstrated excellent stability when stored at 4 °C over a period of 90 days. In vitro and in vivo permeation studies showed that SLPHNs increased the cutaneous penetration of fluorescent-labeled siRNA, which reached deeper skin layers. Efficacy studies were conducted on keratinocytes and fibroblasts, showing that SLPHNs maintained cell viability and high cellular uptake. Furthermore, SLPHNs complexed with siRNA against Firefly luciferase (siLuc) reduced luciferase expression, proving the efficacy of this nanocarrier in providing adequate intracellular release of siRNA for silencing specific genes. Based on these results, the developed carriers are promising siRNA delivery systems for skin disease therapy.

β-Glucan-Mediated Oral Codelivery of 5FU and Bcl2 siRNA Attenuates Stomach Cancer.

Based on cancer-related deaths, stomach cancer is ranked fifth, and first among Hispanics. Lack of technologies for early diagnosis and unavailability of target-specific therapeutics are largely the causes of the poor therapeutic outcomes from existing chemotherapeutics. Currently available therapeutic modalities are invasive and require systemic delivery, although the cancer is localized in the stomach at its early stage. Therefore, we hypothesize that an oral local delivery approach can extend the retention duration of the therapeutics modalities within the stomach and thereby enhance therapeutic efficacy. To accomplish this, we have developed a ß-glucan (BG)-based oral delivery vehicle that can adhere to the mucus lining of the stomach for an extended period while controlling the release of Bcl2 siRNA and 5-fluorouracil (5FU) payload for over 6 h. We found that Bcl2 siRNA selectively knocked down the Bcl2 gene in a C57BL/6 stomach cancer mouse model followed by upregulation of apoptosis and remission of cancer. BG was found to be very effective in maintaining the stability of siRNA for at least 6 h, when submerged in simulated gastric juice tested in vitro. To investigate the potential therapeutic effects in vivo, we used a stomach cancer mouse model, where C57BL/6 mice were treated with 5FU, BG/5FU, siRNA, BG/siRNA, and BG/5FU/siRNA. Higher inhibition of Bcl2 and therapeutic efficacy were observed in mice treated with BG/5FU/siRNA confirmed with Western blotting and a TUNEL assay. Significant reduction in the tumor region was observed with histology (H&E) and immunohistochemistry (Ki67, TUNEL, and Bcl2) analyses. Overall, the oral formulation shows improved efficacy with nonsignificant side effects compared to the conventional treatment tested in the gastric cancer mouse model.

Macrophage-Cancer Hybrid Membrane-Camouflaged Nanoplatforms for HIF-1α Gene Silencing-Enhanced Sonodynamic Therapy of Glioblastoma.

Fabrication of ingenious nanomedicines to penetrate the blood-brain barrier (BBB) and blood-brain-tumor barrier (BBTB) for efficient glioblastoma (GBM) therapy remains a big challenge. In this work, macrophage-cancer hybrid membrane-camouflaged nanoplatforms were fabricated for target gene silencing-enhanced sonodynamic therapy (SDT) of GBM. The J774.A.1 macrophage cell membrane and the U87 glioblastoma cell membrane were fused to create a hybrid biomembrane (JUM) with good BBB penetration and glioblastoma targeting capability for camouflaging. The ZIF-8 nanoparticles were synthesized for indocyanine green (ICG) and HIF-1α siRNA encapsulation (ICG-siRNA@ZIF-8, ISZ) with a high loading efficiency. After accumulation in the tumor sites, the pH sensitivity of the nanoplatform enabled release of ICG and HIF-1α siRNA in the tumor cells. Then, the expression of HIF-1α could be efficiently inhibited by the released HIF-1α siRNA to increase the SDT efficiency under hypoxic conditions. In vitro and in vivo experiments revealed that ISZ@JUM showed good BBB penetration and brain tumor-targeting capability and could achieve effective gene silencing-enhanced SDT, demonstrating great promise for clinical applications.

Smart Telomerase-Gated DNA cage for precise siRNA release in cancer cells

Oligonucleotide nanoparticles (ONPs) have been utilized as a small interfering RNA (siRNA) delivery system because of their multiple advantages. However, the release of siRNA by ONPs inner cells mainly relies on acidic environments and high concentrations of ATP. These conditions do not allow the specific release of siRNA in tumor cells, which would prevent the side effects that result from the release of siRNA in normal cells. In this study, a prism oligonucleotide nanoparticle (pONP)-based delivery system was designed, which targeted with telomeric primers (TS) that can be extended by cancer-specific telomerase for specifically releasing siRNA inside telomerase-positive cancer cells. In vitro tests confirmed that this cage facilitated the release of siRNA specifically into telomerase-positive HeLa and MDA-MB-231 tumor cells. The release was so precise that almost no siRNA was released into telomerase-negative cells. In vivo tests also confirmed that this cage could release sibcl-2 specifically in tumor cells in an MDA-MB-231 orthotopic breast cancer mouse model. Moreover, the cage has a long half-life (e.g., 2.83 h in FBS) in the circulatory system and protects the cargo from nuclease degradation; thus, the released sibcl-2 effectively silences the translation of target cancer-related mRNA (bcl-2), similar to the sibcl-2 delivered by Lipo6000 in the telomerase-positive environment of cancer cells. pONP/TS/siRNA is readily prepared using a simple sequence design together with one-step self-assembly. This nanoparticle cage is a promising delivery method for precise siRNA release into targeted telomerase-positive cancer cells.

SiRNA delivery mediated by pH and redox responsive p(DEAEMA-co-HEMA-g-PEGMA) nanogels

Small interfering RNAs (siRNA) have enabled novel, specific, and efficient treatment of diseases, including cancer. To obtain sufficient and well-controlled intracellular delivery, a drug delivery system is essential. Herein a delivery system for siRNA, composed of cationic biodegradable nanogels, is presented. p(DEAEMA-co-HEMA-g-PEGMA) nanogels with varying ratios of 2-diethyl aminoethyl methacrylate (DEAEMA) and 2-Hydroxyethyl methacrylate (HEMA), crosslinked with tetraethylene glycol dimethacrylate or disulfide-crosslinker bis(2-methacryloyloxy ethyl), are synthesized. The pH-depended nanogel swelling facilitates siRNA loading and endosomal disruption. The disulfide-crosslinker ensures siRNA release by GSH-mediated particle disassociating. The nanogels are within the sub-100nm range in their collapsed state, have a PDI ≥0.4, and a ζ-potential ranging from approximately 5-10 in the swollen state and 22–30V in the collapsed state and have a swelling ratio up to 1.6 in diameter. Furthermore, the nanogels show good serum-stability, and are capable of delivering siRNA with acceptable cytotoxicity levels. The nanogels induced >90% eGFP-silencing in lung cancer cells, and gene-silencing of the cancer target POLR2A led to siRNA-induced cell death in squamous carcinoma cells. The findings suggest that the nanogel-system may function as a carrier vehicle for cytoplasmic delivery of siRNA.

Retinol-binding protein-hijacking nanopolyplex delivering siRNA to cytoplasm of hepatic stellate cell for liver fibrosis alleviation

Activated hepatic stellate cell (aHSC) is mainly responsible for deposition of extracellular collagen matrix that causes liver fibrosis. Although several siRNAs adequately inhibited HSC activation in vitro, they were demonstrated poor RNAi efficiency in vivo. Developing HSC-targeting and cytoplasmic delivery nanocarrier is highly essential to acquire a desirable siRNA therapeutic index for anti-liver fibrosis. Here, we developed a unique crosslinking nanopolyplex (called T-C-siRNA) modified by vitamin A (VA) with the well-designed natures, including the negative charge, retinol-binding protein (RBP) hijacking, and cytoplasmic siRNA release in response to ROS and cis diol molecules. The nanopolyplex was given a yolk-shell-like shape, camouflage ability in blood, and HSC-targeting capability by hijacking the endogenous ligand RBP via surface VA. PDGFR-β siRNA (siPDGFR-β) supplied via T-C-siPDGFR-β nanopolyplex dramatically reduced HSC activation and its production of pro-fibrogenic proteins in vitro and in vivo. Furthermore, T-C-siPDGFR-β nanopolyplex effectively alleviated CCl4-induced liver injury, decreased hepatic collagen sediment, and recovered liver function in mice. This study provides a sophisticated method for HSC-targeting cytoplasmic RNA delivery using endogenous ligand hijacking and dual sensitivity of ROS and cis diol compounds.

Cation-free siRNA-cored nanocapsules for tumor-targeted RNAi therapy.

Cation-associated cytotoxicity limits the systemic administration of RNA delivery in vivo, demanding the development of non-cationic nanosystems. In this study, cation-free polymer-siRNA nanocapsules with disulfide-crosslinked interlayer, namely T-SS(-), were prepared via the following steps: 1) complexation of siRNA with a cationic block polymer cRGD-poly(ethylene glycol)-b-poly[(2-aminoethanethiol)aspartamide]-b-poly{N'-[N-(2-aminoethyl)-2-ethylimino-1-aminomethyl]aspartamide}, abbreviated as cRGD-PEG-PAsp(MEA)-PAsp(C=N-DETA), 2) interlayer crosslinking via disulfide bond in pH 7.4 solution, and 3) removal of cationic DETA pendant at pH 5.0 via breakage of imide bond. The cationic-free nanocapsules with siRNA cores not only showed great performance (such as efficient siRNA encapsulation, high stability in serum, cancer cell targeting via cRGD modification, and GSH-triggered siRNA release), but also achieved tumor-targeted gene silencing in vivo. Moreover, the nanocapsules loaded with siRNA against polo-like kinase 1 (siRNA-PLK1) significantly inhibited tumor growth without showing cation-associated toxicity side effects and remarkably improved the survival rate of PC-3 tumor-bearing mice. The cation-free nanocapsules could potentially serve as a safe and effective platform for siRNA delivery. STATEMENT OF SIGNIFICANCE: Cation-associated toxicity limits the clinical translation of cationic carriers for siRNA delivery. Recently, several non-cationic carriers, such as siRNA micelles, DNA-based nanogels, and bottlebrush-architectured poly(ethylene glycol), have been developed to deliver siRNA. However, in these designs, siRNA as a hydrophilic macromolecule was attached to the nanoparticle surface instead of being encapsulated. Thus, it was easily degraded by serum nuclease and often induced immunogenicity. Herein, we demonstrate a new type of cation-free siRNA-cored polymeric nanocapsules. The developed nanocapsules not only showed capacities including efficient siRNA encapsulation, high stability in serum, and cancer cell targeting via cRGD modification, but also achieved an efficient tumor-targeted gene silencing in vivo. Importantly, unlike cationic carriers, the nanocapsules exhibited no cation-associated side effects.

A cathepsin B/GSH dual-responsive fluorinated peptide for effective siRNA delivery to cancer cells

Small interfering RNA (siRNA) can be exploited to silence specific genes associated with cancer development, and successful siRNA therapy is highly dependent on the efficiency of the siRNA delivery vector. Herein, a well-designed novel redox- and enzyme-responsive fluorinated polyarginine (PFC-PR) was developed to be used as an anti-cancer siRNA carrier. The multiple guanidine groups could provide positive charges and bind with siRNA efficiently, and further fluorination modification enhanced the interaction with siRNA, resulting in a more stable PFC-PR/siRNA nanocomplex, improving serum tolerance, and promoting cellular uptake and endosome escape. Meanwhile, the PFC-PR was responsive to overexpressed cathepsin B and high levels of glutathione in cancer cells, conferring its ability to enhance siRNA release within cancer cells and making it cancer-targeting. Consequently, PFC-PR showed good biocompatibility and high gene silencing efficiency, which could inhibit cancer cell growth when delivered the siRNA targeting vascular endothelial growth factor, suggesting that it can be potentially used for anti-cancer gene therapy applications.

Single-cell quantification and dose-response of cytosolic siRNA delivery

Endosomal escape and subsequent cytosolic delivery of small interfering RNA (siRNA) therapeutics is believed to be highly inefficient. Since it has not been possible to quantify cytosolic amounts of delivered siRNA at therapeutic doses, determining delivery bottlenecks and total efficiency has been difficult. Here, we present a confocal microscopy-based method to quantify cytosolic delivery of fluorescently labeled siRNA during lipid-mediated delivery. This method enables detection and quantification of sub-nanomolar cytosolic siRNA release amounts from individual release events with measures of quantitation confidence for each event. Single-cell kinetics of siRNA-mediated knockdown in cells expressing destabilized eGFP unveiled a dose-response relationship with respect to knockdown induction, depth and duration in the range from several hundred to thousands of cytosolic siRNA molecules. Accurate quantification of cytosolic siRNA, and the establishment of the intracellular dose-response relationships, will aid the development and characterization of novel delivery strategies for nucleic acid therapeutics.

A Multifunctional Hybrid Nanocarrier for Non-Invasive siRNA Delivery to the Retina.

Drug therapy for retinal diseases (e.g., age-related macular degeneration, the leading cause of blindness) is generally performed by invasive intravitreal injection because of poor drug delivery caused by the blood-retinal barrier (BRB). This study aimed to develop a nanocarrier for the non-invasive delivery of small interfering RNA (siRNA) to the posterior segment of the eye (i.e., the retina) by eyedrops. To this end, we prepared a hybrid nanocarrier based on a multifunctional peptide and liposomes, and the composition was optimized. A cytoplasm-responsive stearylated peptide (STR-CH2R4H2C) was used as the multifunctional peptide because of its superior ability to enhance the complexation, cell permeation, and intracellular dynamics of siRNA. By adding STR-CH2R4H2C to the surface of liposomes, intracellular uptake increased regardless of the liposome surface charge. The STR-CH2R4H2C-modified cationic nanocarrier demonstrated significant siRNA transfection efficiency with no cytotoxicity, enhanced siRNA release from endosomes, and effectively suppressed vascular endothelial growth factor expression in rat retinal pigment epithelium cells. The 2.0 mol% STR-CH2R4H2C-modified cationic nanocarrier enhanced intraocular migration into the retina after instillation into rat eyes.

Upconversion nanoparticle-based optogenetic nanosystem for photodynamic therapy and cascade gene therapy.

Most photosensitizer molecules used for the photodynamic therapy (PDT) are chemically-synthesized organic photosensitizer dyes which show several limitations such as unsatisfactory cell uptake, weak selectivity and off-target phototoxicity. Recently, genetically-encoded photosensitizers have attracted increasing attentions which provide the targeted cell elimination with single-cell precision. However, their applications are mainly limited by the shallow tissue penetration depth of the excitation light and the low cell apoptosis ratio. Herein, we developed a feasible upconversion nanoparticle (UCNP)-based optogenetic nanosystem with three-in-one functional integration: bio-imaging, NIR-triggered PDT and cascade gene therapy. Firstly, the mitochondria-targeted genetically-encoded photosensitizer was constructed and transfected into cancer cells. Then, the functional upconversion nanoprobe was constructed with the mitochondria targetability and then the siRNA was loaded on the surface of UCNPs via the reactive oxygen species (ROSs) sensitive chemical bond. After the transfection and incubation, both of the upconversion nanoprobe and the genetically-encoded photosensitizer were accumulated in the mitochondria of cancer cells. Under the NIR irradiation, the emission of UCNPs could excite the expressed protein photosensitizer to generate ROSs which then stimulated the release of siRNAs in a controllable manner, achieving PDT and cascade gene therapy. Since the generation of ROSs and the release of siRNA occurred in the mitochondria in-situ, the mitochondria-mediated cell apoptosis signal pathway would be activated to induce cell apoptosis and subsequently inhibit tumor growth. To the best of our knowledge, this is the first report about NIR laser-activated, organelle-localized genetically-encoded photosensitizers developed for cascade therapy, which will widen the application of optogenetic tools in the tumor therapy. STATEMENT OF SIGNIFICANCE: The application of genetically-encoded photosensitizers in photodynamic therapy (PDT) is mainly limited by the shallow tissue penetration depth of the excitation light and unsatisfactory therapeutic performance. In this experiment, we developed an upconversion nanoparticles-based optogenetic nanosystem to enhance the PDT and cascade gene therapy for malignant tumors. The expressed genetically-encoded photosensitizers were accumulated in the mitochondria, which were activated in situ by the upconversion nanoprobe. Besides, the photogenerated reactive oxygen species (ROSs) stimulated the release of siRNAs in a controllable manner. To the best of our knowledge, this is the first report about NIR laser-activated, genetically-encoded photosensitizers developed for organelle-localized controllable cascade therapy. We hope this work can accelerate the application of genetically-encoded photosensitizers in the tumor therapy.

High-Capacity Mesoporous Silica Nanocarriers of siRNA for Applications in Retinal Delivery.

The main cause of subretinal neovascularisation in wet age-related macular degeneration (AMD) is an abnormal expression in the retinal pigment epithelium (RPE) of the vascular endothelial growth factor (VEGF). Current approaches for the treatment of AMD present considerable issues that could be overcome by encapsulating anti-VEGF drugs in suitable nanocarriers, thus providing better penetration, higher retention times, and sustained release. In this work, the ability of large pore mesoporous silica nanoparticles (LP-MSNs) to transport and protect nucleic acid molecules is exploited to develop an innovative LP-MSN-based nanosystem for the topical administration of anti-VEGF siRNA molecules to RPE cells. siRNA is loaded into LP-MSN mesopores, while the external surface of the nanodevices is functionalised with polyethylenimine (PEI) chains that allow the controlled release of siRNA and promote endosomal escape to facilitate cytosolic delivery of the cargo. The successful results obtained for VEGF silencing in ARPE-19 RPE cells demonstrate that the designed nanodevice is suitable as an siRNA transporter.

Oral Delivery of siRNA Using Fluorinated, Small-Sized Nanocapsules toward Anti-Inflammation Treatment.

Oral delivery of small interfering RNA (siRNA) provides a promising paradigm for treating diseases that require regular injections. However, the multiple gastrointestinal (GI) and systemic barriers often lead to inefficient oral absorption and low bioavailability of siRNA. Technologies that can overcome these barriers are still lacking, which hinders the clinical potential of orally delivered siRNA. Herein, small-sized, fluorinated nanocapsules (F-NCs) are developed to mediate efficient oral delivery of tumor necrosis factor α (TNF-α) siRNA for anti-inflammation treatment. The NCs possess a disulfide-cross-linked shell structure, thus featuring robust stability in the GI tract. Because of their small size (≈30nm) and fluorocarbon-assisted repelling of mucin adsorption, the best-performing F3 -NCs show excellent mucus penetration and intestinal transport capabilities without impairing the intestinal tight junction, conferring the oral bioavailability of 20.4% in relative to intravenous injection. The disulfide cross-linker can be cleaved inside target cells, causing NCs dissociation and siRNA release to potentiate the TNF-α silencing efficiency. In murine models of acute and chronic inflammation, orally delivered F3 -NCs provoke efficient TNF-α silencing and pronounced anti-inflammatory efficacies. This study therefore provides a transformative strategy for oral siRNA delivery, and will render promising utilities for anti-inflammation treatment.

Development of hyaluronic acid-anchored polycaprolactone nanoparticles for efficient delivery of PLK1 siRNA to breast cancer.

PlK1 has a significant role in the development of breast cancer. Thus, silencing of PlK1 gene may arrest the growth of breast cancer. However, the in vivo stability of PlK1 siRNA after injection remains a challenge to target the specific site. The delivery of siPlK1 RNA via viral vector and amine group-terminated dendrimer is associated with immune reaction and cellular cytotoxicity. Thus, in the present study, hyaluronic acid-functionalized and -thiolated polycaprolactone nanoparticles (SH-HPP NPs) were developed for enhancing the targeting capabilities of siRNA towards human breast cancer cells. NPs displayed size in the range of 180-217nm, and with sustain and pH-dependent release of siRNA up to 120h. The in vitro treatments with siRNA-containing NPs showed the high number of necrotic cells and the cell cycle arrest at the G2/M phase. The gene expression analysis depicts the decrease of endogenous PLK1 siRNA expression on MCF-7 cells upon PLK1 NPs treatment. In vitro cytotoxicity experiments demonstrated effective anticancer properties against MCF-7. Finally, in vivo results showed that substantial tumor inhibition was achieved with PLK1 siRNA-containing SH-HPP NPs in comparison of the control group. Hence, HPP NPs have enormous potential for the selective delivery of siRNA, i.e., breast cancer cells.

Design, strategies, and therapeutics in nanoparticle-based siRNA delivery systems for breast cancer.

Utilizing small interfering RNA (siRNA) as a treatment for cancer, a disease largely driven by genetic aberrations, shows great promise. However, implementing siRNA therapy in clinical practice is challenging due to its limited bioavailability following systemic administration. An attractive approach to address this issue is the use of a nanoparticle (NP) delivery platform, which protects siRNA and delivers it to the cytoplasm of target cells. We provide an overview of design considerations for using lipid-based NPs, polymer-based NPs, and inorganic NPs to improve the efficacy and safety of siRNA delivery. We focus on the chemical structure modification of carriers and NP formulation optimization, NP surface modifications to target breast cancer cells, and the linking strategy and intracellular release of siRNA. As a practical example, recent advances in the development of siRNA therapeutics for treating breast cancer are discussed, with a focus on inhibiting cancer growth, overcoming drug resistance, inhibiting metastasis, and enhancing immunotherapy.

Engineering a Novel Self-Assembled Multi-siRNA Nanocaged Architecture with Controlled Enzyme-Mediated siRNA Release.

The RNA interference (RNAi) chemical and structural design space has evolved since its original definitions. Although this has led to the development of RNAi molecules that are starting to address the issues of silencing efficiency and delivery to target organs and cells, there is an on-going interest to improve upon their properties to attain wider therapeutic applicability. Taking advantage of the flexibility given by DNA and RNA structural and chemical properties, we here investigated unconventional RNAi encoding structures, designated by caged-siRNA structures (CsiRNAs), to explore novel features that could translate into advantageous properties for cellular delivery and intracellular activity. Using the principles of controlled nucleic acid self-assembly, branched DNA-RNA hybrid intermediates were formed, ultimately leading to the assembly of a "closed" structure encompassing multiple RNAi units. The RNAi active regions are further triggered by an encoded RNAse H-mediated release mechanism, while the overall structure possesses easily addressable anchors for hybridization-based functionalization with active biological moieties. We confirmed the production of correct structures and demonstrated that the encoded RNAi sequences maintain gene silencing activity even within this novel unconventional nanoarchitecture, aided by the intracellularly triggered RNAse H release mechanism. With this design, functionalization is easily achieved with no negative effects on the silencing activity, warranting further development of these novel molecular structures as a multi-RNAi platform for therapeutic delivery.

Monitoring the in vivo siRNA release from lipid nanoparticles based on the fluorescence resonance energy transfer principle

The siRNA-loaded lipid nanoparticles have attracted much attention due to its significant gene silencing effect and successful marketization. However, the in vivo distribution and release of siRNA still cannot be effectively monitored. In this study, based on the fluorescence resonance energy transfer (FRET) principle, a fluorescence dye Cy5-modified survivin siRNA was conjugated to nanogolds (Au-DR-siRNA), which were then wrapped with lipid nanoparticles (LNPs) for monitoring the release behaviour of siRNA in vivo. The results showed that once Au-DR-siRNA was released from the LNPs and cleaved by the Dicer enzyme to produce free siRNA in cells, the fluorescence of Cy5 would change from quenched state to activated state, showing the location and time of siRNA release. Besides, the LNPs showed a significant antitumor effect by silencing the survivin gene and a CT imaging function superior to iohexol by nanogolds. Therefore, this work provided not only an effective method for monitoring the pharmacokinetic behaviour of LNP-based siRNA, but also a siRNA delivery system for treating and diagnosing tumors.

CD70-Targeted Micelles Enhance HIF2α siRNA Delivery and Inhibit Oncogenic Functions in Patient-Derived Clear Cell Renal Carcinoma Cells.

The majority of clear cell renal cell carcinomas (ccRCCs) are characterized by mutations in the Von Hippel−Lindau (VHL) tumor suppressor gene, which leads to the stabilization and accumulation of the HIF2α transcription factor that upregulates key oncogenic pathways that promote glucose metabolism, cell cycle progression, angiogenesis, and cell migration. Although FDA-approved HIF2α inhibitors for treating VHL disease-related ccRCC are available, these therapies are associated with significant toxicities such as anemia and hypoxia. To improve ccRCC-specific drug delivery, peptide amphiphile micelles (PAMs) were synthesized incorporating peptides targeted to the CD70 marker expressed by ccRCs and anti-HIF2α siRNA, and the ability of HIF2α-CD27 PAMs to modulate HIF2α and its downstream targets was evaluated in human ccRCC patient-derived cells. Cell cultures were derived from eight human ccRCC tumors and the baseline mRNA expression of HIF2A and CD70, as well as the HIF2α target genes SLC2A1, CCND1, VEGFA, CXCR4, and CXCL12 were first determined. As expected, each gene was overexpressed by at least 63% of all samples compared to normal kidney proximal tubule cells. Upon incubation with HIF2α-CD27 PAMs, a 50% increase in ccRCC-binding was observed upon incorporation of a CD70-targeting peptide into the PAMs, and gel shift assays demonstrated the rapid release of siRNA (>80% in 1 h) under intracellular glutathione concentrations, which contributed to ~70% gene knockdown of HIF2α and its downstream genes. Further studies demonstrated that knockdown of the HIF2α target genes SLC2A1, CCND1, VEGFA, CXCR4, and CXCL12 led to inhibition of their oncogenic functions of glucose transport, cell proliferation, angiogenic factor release, and cell migration by 50−80%. Herein, the development of a nanotherapeutic strategy for ccRCC-specific siRNA delivery and its potential to interfere with key oncogenic pathways is presented.

Transformable Nanotheranostics Capable of Sensoring Nucleic Acid Drugs’ Spatiotemporal Behaviors

AbstractNanotheranostics facilitates personalized treatment by providing imaging capabilities for nucleic acid drug vectors. However, available nanotheranostics are not yet capable of indicating the release behavior of nucleic acid drugs. Herein, four guidelines for the design of nanotheranostics are proposed. Under the guidance, transformable nanotheranostics are prepared that can sense the spatiotemporal behaviors of siRNA accumulation and release to facilitate personalized oncology treatment. The nanotheranostics sense the spatiotemporal accumulation behavior of siRNA in tumors through computerized tomography. Besides, the tumor cytoplasmic microenvironment mediates both siRNA release and nanotherapeutic agent transformation simultaneously. The transformation‐activated magnetic resonance imaging (MRI) signals sense the spatiotemporal release behavior of siRNA. Moreover, the therapeutic effect of survivin‐targeted siRNA can be diagnosed and confirmed by contrast‐enhanced CT/MRI multi‐modal imaging in real‐time. The transformable nanotheranostics further promote personalized oncotherapy by nucleic acid drugs. These four guidelines are expected to direct the research and development of nucleic acid drug‐based nanotheranostics. This study also provides a new perspective on real‐time sensoring the spatiotemporal behaviors of nucleic acid drugs in vivo.

Brain‐Targeting Metastatic Tumor Cell Membrane Cloaked Biomimetic Nanomedicines Mediate Potent Chemodynamic and RNAi Combinational Therapy of Glioblastoma

AbstractThe complex genetic heterogeneity of glioblastoma (GBM) together with the existence of blood brain barrier (BBB) renders GBM essentially incurable. In this paper, a brain‐targeting metastatic tumor cell membrane cloaked biomimetic nanomedicine that potently induces cascade chemodynamic and RNA interference (RNAi) is developed for combinational GBM therapy. The designed nanomedicines are constructed from metastatic melanoma cell membrane, charge‐conversional middle layer, and siRNA complexed polyethyleneimine xanthate (PEX). PEX chelates the abundant copper ions in tumor cells and then reacts with intracellular glutathione (GSH), resulting in GSH depletion and the reduction of Cu2+ to Cu+, followed by reacting with H2O2 and producing •OH via Fenton‐like reaction. Interestingly, the acidic conditions in endo/lysosomes accelerate siRNA release, inducing potent gene silencing of the anti‐apoptotic B‐cell lymphoma 2 (Bcl‐2), lead to multiple molecular cascades that readily induces GBM cell apoptosis. The results also demonstrate that this nanomedicine exhibits efficient BBB penetration via decreasing the tightness and interacting with the adhesion molecules in endothelial cells. More importantly, improved therapeutic efficacy with extended survival rates are achieved in both local melanoma and orthotopic GBM tumor models, suggesting that the chemodynamic and gene combinatorial therapy mediated by the biomimetic nanomedicines shows promising potential in treating GBM and brain metastases.