Biodegradable Dextran Nanogels for RNA Interference: Focusing on Endosomal Escape and Intracellular siRNA Delivery

The successful therapeutic application of small interfering RNA (siRNA) largely relies on the development of safe and effective delivery systems that are able to guide the siRNA therapeutics to the cytoplasm of the target cell. In this report, biodegradable cationic dextran nanogels are engineered by inverse emulsion photopolymerization and their potential as siRNA carriers is evaluated. The nanogels are able to entrap siRNA with a high loading capacity, based on electrostatic interaction. Confocal microscopy and flow cytometry analysis reveal that large amounts of siRNA‐loaded nanogels can be internalized by HuH‐7 human hepatoma cells without significant cytotoxicity. Following their cellular uptake, it is found that the nanogels are mainly trafficked towards the endolysosomes. The influence of two different strategies to enhance endosomal escape on the extent of gene silencing is investigated. It is found that both the application of photochemical internalization (PCI) and the use of an influenza‐derived fusogenic peptide (diINF‐7) can significantly improve the silencing efficiency of siRNA‐loaded nanogels. Furthermore, it is shown that an efficient gene silencing requires the degradation of the nanogels. As the degradation kinetics of the nanogels can easily be tailored, these particles show potential for intracellular controlled release of short interfering RNA.     

Multifunctional Glyco‐Nanofibers: siRNA Induced Supermolecular Assembly for Codelivery In Vivo

Targeted codelivery and controlled release of drug/siRNA (small interfering RNA) in a safe and effective vehicle hold great promises for overcoming drug resistance and optimal efficacy in cancer treatment; however, rational design and preparation of such vehicles remain a critical challenge. Thus, glyco‐nanofibers (GNFs) are fabricated via supermolecular assembly of polyanionic siRNA and cationic vesicles to simultaneously deliver siRNA and doxorubicin hydrochloride (DOX) in vitro and in vivo. The vesicles are created through self‐assembly of a positively charged amphiphilic lactose derivative featuring a lactose moiety and a ferrocenium unit on either end of the molecule. The GNFs display excellent biocompatibility, enhanced cell‐penetrating ability, and hepatoma targetability. The high transport efficiency of siRNA, effective gene silencing ability, and enhanced cytotoxicity to HepG2 cells of GNFs loaded with DOX are observed in vitro. Furthermore, in vivo experiments show reduced systemic toxicity and enhanced therapeutic efficacy of DOX to both HepG2 and HepG2/ADR subcutaneous tumor‐bearing nude mice. This work proves the electrostatic self‐assembly between cationic carbohydrates and polyanionic siRNA to be a convenient and effective strategy to fabricate a single vehicle for safe and effective codelivery of drug/siRNA, which can be used to combine chemo‐ and gene‐therapy against cancers and other diseases.     

A Fabricated siRNA Nanoparticle for Ultralong Gene Silencing In Vivo

Persistent gene silencing is crucially required for the successful therapeutics of short interfering RNA (siRNA). Here, a nanoparticle‐based delivery system is presented which assembles by layering siRNAs between protease degradable polypeptides to extend the therapeutic window. These tightly packed nanoparticles are efficiently taken up by cells by endocytosis, and the fabricated siRNAs are gradually released following intracellular degradation of the polypeptide layers. During cell division, the particles are distributed to the daughter cells. Due to the slow degradation through the multiple layers, the particles continuously release siRNA in all cells. Using this controlled release construct, the in vivo gene silencing effect of siRNA is consistent for an ultralong period of time (>3 weeks) with only a single treatment.     

Controlling The Mobility Of Oligonucleotides In The Nanochannels Of Mesoporous Silica

Oligonucleotides used in gene therapy and silencing are fragile compounds that degrade easily in biological environments. Porous biocompatible carrier particles may provide a useful strategy to deliver these therapeutics to their target sites. Development of appropriate delivery vehicles, however, requires a better understanding of the oligonucleotide‐host interactions and the oligonucleotide dynamics inside carrier particles. We investigated template‐free SBA‐15 type mesoporous silica particles and report their loading characteristics with siRNA depending on the surface functionalization of their porous network. We show that the siRNA uptake capability of the particles can be controlled by the composition of the functional groups. Fluorescence recovery after photobleaching measurements revealed size‐dependent mobility of siRNA and double‐stranded DNA oligonucleotides within the functionalized silica particles and provided evidence for the stability of the oligonucleotides inside the pores. Hence, our study demonstrates the potential of mesoporous silica particles as a means for alternative gene delivery in nanomedicine.     

Fabrication of Gelatin Microgels by a “Cast” Strategy for Controlled Drug Release

In this paper, we propose a “casting” strategy to prepare intrinsically fluorescent, uniform and porous gelatin microgels with multi‐responsiveness. Gelatin microgels with tunable size were obtained by copying the structure of a porous CaCO₃ template. The diameter of the gelatin microgels was sensitive to salt concentration and pH. Doxorubicin and Rhodamine B as model drugs were loaded into the microgels via electrostatic interaction and release of the payload was triggered by changing the salt concentration and pH, respectively. Cell experiments demonstrated that the gelatin microgels had an excellent biocompatibility and biodegradability. The merits of gelatin microgels such as tunable size, biocompatibility, and stimulus responsive upload and release of positively charged small molecules will permit the microgels as excellent carriers for drug delivery. The whole manufacturing process is furthermore environmental‐friendly involving no organic solvents and surfactants.     

Lipoplex‐Loaded Microbubbles for Gene Delivery: A Trojan Horse Controlled by Ultrasound

Cationic poly(ethylene glycol)ylated (PEGylated) liposomes are one of the most important gene transfer reagents in non‐viral gene therapy. However, the low transfection efficiencies of highly PEGylated lipoplexes currently hamper their clinical use. Recently, ultrasound has been used in combination with microbubbles to enhance the uptake of genes in different cell types. However, the gene transfer efficiency still remains low in these experiments. To overcome the limitations of both techniques, we present the attachment of PEGylated lipoplexes to microbubbles via biotin–avidin–biotin linkages. Exposure of these lipoplex‐loaded microbubbles to ultrasound results in the release of unaltered lipoplexes. Furthermore, these lipoplex‐loaded microbubbles exhibit much higher transfection efficiencies than “free” PEGylated lipoplexes or naked plasmid DNA (pDNA) when combined with microbubbles and ultrasound. Interestingly, the lipoplex‐loaded microbubbles only transfect cells when exposed to ultrasound, which is promising for space‐ and time‐controlled gene transfer. Finally, this novel Trojan‐horse‐like concept can also be exploited to achieve the ultrasound‐triggered release of nanoparticles containing other therapeutic agents such as anticancer drugs.     

Reductively Dissociable siRNA‐Polymer Hybrid Nanogels for Efficient Targeted Gene Silencing

A highly efficient approach for target‐specific gene silencing based on a reductively dissociable nanogel incorporating small interfering RNA (siRNA) crosslinked with linear polyethylenimine (LPEI) via disulfide bonds is presented. Thiol‐terminated siRNA at both 3′‐ends is electrostatically complexed with thiol‐grafted LPEI. The prepared siRNA/LPEI complex contains inter‐ and intramolecular linkages, generating a mutually crosslinked siRNA/LPEI nanogel (MCN) that exhibits excellent structural stability against the addition of heparin but is readily disintegrated to biologically active, monomeric siRNA upon exposure to reductive conditions. Accordingly, the highly condensed, stable MCN shows greatly enhanced cellular uptake and gene silencing efficiency compared to the siRNA/LPEI complexes without crosslinks or with only LPEI‐mediated crosslinks.     

Supertough Hybrid Hydrogels Consisting of a Polymer Double‐Network and Mesoporous Silica Microrods for Mechanically Stimulated On‐Demand Drug Delivery

Despite their potential in various fields of bioapplications, such as drug/cell delivery, tissue engineering, and regenerative medicine, hydrogels have often suffered from their weak mechanical properties, which are attributed to their single network of polymers. Here, supertough composite hydrogels are proposed consisting of alginate/polyacrylamide double‐network hydrogels embedded with mesoporous silica particles (SBA‐15). The supertoughness is derived from efficient energy dissipation through the multiple bondings, such as ionic crosslinking of alginate, covalent crosslinking of polyacrylamide, and van der Waals interactions and hydrogen bondings between SBA‐15 and the polymers. The superior mechanical properties of these hybrid hydrogels make it possible to maintain the hydrogel structure for a long period of time in a physiological solution. Based on their high mechanical stability, these hybrid hydrogels are demonstrated to exhibit on‐demand drug release, which is controlled by an external mechanical stimulation (both in vitro and in vivo). Moreover, different types of drugs can be separately loaded into the hydrogel network and mesopores of SBA‐15 and can be released with different speeds, suggesting that these hydrogels can also be used for multiple drug release.     

Size/Charge Changeable Acidity‐Responsive Micelleplex for Photodynamic‐Improved PD‐L1 Immunotherapy with Enhanced Tumor Penetration

The checkpoint blockade‐based immunotherapy has recently emerged as a promising approach for tumor treatment, but its clinical implementation has been impeded by poor tumor penetration of the nanocarriers and activation of antitumor immune response. To overcome the obstacles, a tumor acidity‐responsive micellar nanocomplex co‐loaded with programmed death‐ligand 1 (PD‐L1)‐blockade siRNA and mitochondrion‐targeting photosensitizer for the synergistic integration of photodynamic therapy and immunotherapy is reported in the present study. The nanosystem is coated with long‐circulating polyethylene glycol (PEG) shells, which can be shed in response to the weakly acidic tumor microenvironment and lead to significant size reduction and increasing positive charge. These transitions facilitate penetration and uptake of nanocarriers against tumors. Subsequently, under the mild acidic endo/lysosome condition, the micellar nanocomplexes are rapidly protonated and disintegrated to release the PD‐L1‐blockade siRNA and photosensitizer through sponge effect. Results from in vitro and in vivo experiments collectively reveal that the nanosystem efficiently activates a photodynamic therapy‐induced immune response and silences immune resistance mediated by the checkpoint gene PD‐L1. In consequence, melanoma growth is inhibited and the recurrence rate is reduced via triggering systemic antitumor immune responses. This study offers an alternative strategy for the development of efficient antitumor immune therapy.     

IL4‐Receptor‐Targeted Dual Antitumoral Apoptotic Peptide—siRNA Conjugate Lipoplexes

Targeted delivery remains the major limitation in the development of small interfering RNA (siRNA) therapeutics. The successful siRNA multistep delivery requires precise carriers of substantial complexity. To achieve this, a monodisperse carrier is presented, synthesized by solid‐phase supported chemistry. The sequence‐defined assembly contains two oleic acids attached to a cationizable oligoaminoamide backbone in T‐shape configuration, and a terminal azide functionality for coupling to the atherosclerotic plaque‐specific peptide‐1 (AP‐1) as the cell targeting ligand for interleukin‐4 receptor (IL‐4R) which is overexpressed in a variety of solid cancers. For combined cytosolic delivery with siRNA, different apoptotic peptides (KLK, BAK, and BAD) are covalently conjugated via bioreversible disulfide linkage to the 5′‐end of the siRNA sense strand. siRNA‐KLK conjugates provide the highest antitumoral potency. The optimized targeted carrier is complexed with dual antitumoral siEG5‐KLK conjugates. The functionality of each subdomain is individually confirmed. The lipo‐oligomer confers stable assembly of siRNA conjugates into spherical 150–250 nm sized nanoparticles. Click‐shielding with dibenzocyclootyne‐PEG‐AP‐1 (DBCO‐PEG‐AP‐1) mediates an IL‐4R‐specific cell targeting and gene silencing in tumor cells. Most importantly, formulation of the siEG5‐KLK conjugate displays enhanced apoptotic tumor cell killing due to the combined effect of mitotic arrest by EG5 gene silencing and mitochondrial membrane disruption by KLK.     

A Near‐Infrared Light‐Triggered Nanocarrier with Reversible DNA Valves for Intracellular Controlled Release

A near‐infrared (NIR) light‐triggered nanocarrier is developed for intracellular controlled release with good stability, high nuclease resistance, and good biocompatibility. The nanocarrier consists of a gold nanorod core and mesoporous silica shell, capped with reversible single‐stranded DNA valves, which are manipulated by switching between the laser on/off states. Upon laser irradiation, the valves of the nanocarrier open and the cargo molecules can be released from the mesopores. When the NIR laser is turned off, the valves close and the nanocarrier stops releasing the cargo molecules. The release amount of the cargo molecules can be controlled precisely by adjusting the irradiation time and the laser on‐off cycles. Confocal fluorescence imaging shows that the nanocarrier can be triggered by the laser irradiation and the controlled release can be accomplished in living cells. Moreover, the therapeutic effect toward cancer cells can also be regulated when the chemotherapeutic drug doxorubicin is loaded into the nanocarrier. This novel approach provides an ideal platform for drug delivery by a NIR light‐activated mechanism with precise control of area, time, and especially dosage.     

Spatially Controlled Delivery of siRNAs to Stem Cells in Implants Generated by Multi‐Component Additive Manufacturing

Additive manufacturing is a promising technique in tissue engineering, as it enables truly individualized implants to be made to fit a particular defect. As previously shown, a feasible strategy to produce complex multicellular tissues is to deposit different small interfering RNA (siRNA) in porous implants that are subsequently sutured together. In this study, an additive manufacturing strategy to deposit carbohydrate hydrogels containing different siRNAs is applied into an implant, in a spatially controlled manner. When the obtained structures are seeded with mesenchymal stem (stromal) cells, the selected siRNAs are delivered to the cells and induces specific and localized gene silencing. Here, it is demonstrated how to replicate part of a patient's spinal cord from a computed tomography scan, using an additive manufacturing technique to produce an implant with compartmentalized siRNAs in the locations corresponding to distinct tissue. Hydrogel solutions loaded with different siRNA can be co‐printed together with polycaprolactone that acts as rigid mechanical support to the hydrogel. This study demonstrates a new route for the production of 3D functionalized, individualized implants which may provide great clinical benefit.     

Intracellular Microenvironment‐Responsive Dendrimer‐Like Mesoporous Nanohybrids for Traceable,Effective, and Safe Gene Delivery

In order to create advanced functional nanocarriers for efficient gene therapy, novel intracellular microenvironment‐sensitive fluorescence label‐free nanostructured dendrimer‐like silica hybrid nanocarriers are developed for traceable, effective, and safe gene delivery. Dendrimer‐like mesoporous silica nanoparticles (DMSNs) with center‐radial large pores are covalently modified with short polyethyleneimine (PEI) for efficient gene loading and binding. Autofluorescent and biodegradable PEI (AC‐PEI) responsive to the intracellular microenvironment are then coated on the gene‐loaded nanoparticles for inhibiting gene leakage from the carriers. Moreover, AC‐PEI coating not only endows intracellular microenvironment‐responsive gene release property, but also allows monitoring the gene delivery process in the absence of external labelling, owing to the pH‐ and GSH‐responsive autofluorescence and biodegradability of AC‐PEI. The resultant nanocarriers show high gene loading capacity, low cytotoxicity, stimuli‐responsive gene release, label‐free, and simultaneous fluorescence tracking, and high gene silencing capability. Thus, these developed nanocarriers hold substantial and promising potential as effective and safe gene‐delivery carriers for future scientific investigation and practical implications in gene therapy.     

Soft Nanostructured Films for Actuated Surface‐Based siRNA Delivery

Substrate‐mediated gene delivery is an emerging technology that enables spatial control of gene expression and localized delivery. This is of particular interest for siRNA where surface‐based release can greatly impact the fields of stem‐cell reprograming, wound healing, and medical device coatings in general. However, reports on the use of siRNA for substrate‐mediated delivery are scarce and have suffered from low efficiency. Here, an alternative strategy is reported by designing self‐assembled substrates that experience stimuli‐responsive topological transformations. Specifically, a methodology is established to promote the molecular organization of lipid films having 3D‐bicontinuous cubic, 2D‐inverted hexagonal, or 1D‐lamellar nanostructures encapsulating siRNA. In response to a compositional, temperature, or humidity stimulus, the nanostructures evolve from 1D‐lamellar or 2D‐hexagonal to 3D‐cubic resulting in efficient siRNA release to human cell cultures. Grazing incidence X‐ray diffraction reveals that film nanostructures are highly ordered and preferentially aligned. The results indicate that film structure substantially affects siRNA delivery, with the supported 3D‐bicontinuous cubic phase yielding the most effective reduction of gene expression. Subsequent studies suggest this enhanced performance arises due to the ability of this phase to cross cell membranes, particularly those of endocytic compartments. This work underpins that nanostructure tuning is decisive to the performance of therapeutic films.     

Controlled Release and Absorption Resonance of Fluorescent Silica‐Coated Platinum Nanoparticles

Aggregation‐driven seeded growth of uniform platinum nanoparticles and exclusive silica‐coating of the as‐grown platinum nanoparticles have been achieved successfully. Fluorescent Pt@SiO₂ nanoparticles have also been reproducibly prepared via effective co‐condensation of silanized dye molecules with tetraethylorthosilicate. The dye‐loaded Pt@SiO₂ nanoparticles have been exploited as model carriers for thermal‐responsive controlled release of guest molecules via slow hydrolysis/dissolution of silica shells; importantly, the encapsulated platinum cores have also been used as nanoprobes to simultaneously investigate their controlled release process. Meanwhile, unique size‐sensitive absorbance resonance in the dye‐loaded Pt@SiO₂ nanoparticles has been demonstrated for the first time, and it is expected to find novel application.     

Near‐Infrared Light‐Encoded Orthogonally Triggered and Logical Intracellular Release Using Gold Nanocage@Smart Polymer Shell

Gold nanocages (AuNCs) with hollow interiors, porous walls, and tunable localized surface plasmon resonance (LSPR) peaks in the NIR region represent a promising platform for therapeutic applications, and can be used for orthogonally triggered release by choosing the right laser according to the AuNC's LSPR. AuNCs are prepared with different LSPRs and covered with a smart polymer shell. Laser irradiation in resonance with the LSPR can trigger the release of a pre‐loaded effector. As a proof of concept, enzyme and substrate (prodrug) are selectively released from two different AuNCs. Enzymatic reactions only occur after successful opening of both types of AuNC capsules. The system acts as an “AND” logic gate. Furthermore, if the AuNC is loaded with isoenzyme or enzyme inhibitor, an “OR” or “INHIBIT” logic gate operation is achieved. To the best of our knowledge, no reports have combined NIR light‐encoded orthogonally triggered release with “prodrug” activation processes to realize defined different logic operations for regulating the dosage of active drug in a specific region. The design is simple and spatial/temporal to control, and provides new insights into developing NIR light‐encoded, logically controlled, intracellular release systems.     

pH‐Responsive,Light‐Triggered on‐Demand Antibiotic Release from Functional Metal–Organic Framework for Bacterial Infection Combination Therapy

To satisfy the ever‐growing demand in bacterial infection therapy and other fields of science, great effort is being devoted to the development of methods to precisely control drug release and achieve targeted use of an active substance at the right time and place. Here, a new strategy for bacterial infection combination therapy based on the light‐responsive zeolitic imidazolate framework (ZIF) is reported. A pH‐jump reagent is modified into the porous structure of ZIF nanoparticles as a gatekeeper, allowing the UV‐light (365 nm) responsive in situ production of acid, which subsequently induces pH‐dependent degradation of ZIF and promotes the release of the antibiotic loaded in the mesopores. The combination of the UV‐light, the pH‐triggered precise antibiotic release, and the zinc ions enables the light‐activated nanocomposite to significantly inhibit bacteria‐induced wound infection and accelerate wound healing, indicating a switchable and synergistic antibacterial effect. The light irradiated accumulation of acid ensures the controlled release of antibiotic and controlled degradation of ZIF, suggesting the therapeutic potential of the metal–organic frameworks‐based smart platform for controlling bacterial infection.     

MMP-2 Responsive Unidirectional Hydrogel-Electrospun Patch Loading TGF-β1 siRNA Polyplexes for Peritendinous Anti-Adhesion

Bio-derived hydrogel patch systems exhibit promising potential in localized drug delivery for the prevention and treatment of various diseases. However, the uncontrolled release from the hydrogel patch both in time and space, is not an optimal strategy for peritendinous anti-adhesion, leading to transient effect and unnecessary diffusion of therapeutics. Here, an innovative composite anti-adhesion patch is designed for on-demand and unidirectional polyplexes delivery to inhibit fibroblasts proliferation and collagen deposition by silencing fibrosis gene transforming growth factor-β1 (TGF-β1). Firstly, a metalloproteinase-2 (MMP-2) degradable hydrogel is prepared by crosslinking allyl glycidyl ether (AGE) modified carboxymethyl chitosan (CMCS-AGE) with MMP-2 substrate peptide CPLGLAGC (MMP-2 sp). Then, the hydrogel loading TGF-β1 siRNA polyplexes are attached onto polycaprolactone (PCL) electrospun fibers to form a composite bilayer patch. The hydrogel–electrospun fibers (H–E) patch shows MMP-2-responsive and unidirectional release behaviors of encapsulated TGF-β1 siRNA polyplexes and associated gene silencing effect on TGF-β1, leading to the inhibition of fibroblasts proliferation. Moreover, after implanting the H–E patch by wrapping the repaired tendon, the formation of adhesion tissue is responsively attenuated in MMP-2 overexpression microenvironment. This study presents a promising approach employing a composite bilayer patch with on-demand and unidirectional delivery strategy for peritendinous anti-adhesion.     

Dual Targeted Immunotherapy via In Vivo Delivery of Biohybrid RNAi‐Peptide Nanoparticles to Tumor‐Associated Macrophages and Cancer Cells

Lung cancer is associated with very poor prognosis and considered one of the leading causes of death worldwide. Here, highly potent and selective biohybrid RNA interference (RNAi)‐peptide nanoparticles (NPs) are presented that can induce specific and long‐lasting gene therapy in inflammatory tumor associated macrophages (TAMs), via an immune modulation of the tumor milieu combined with tumor suppressor effects. The data here prove that passive gene silencing can be achieved in cancer cells using regular RNAi NPs. When combined with M2 peptide–based targeted immunotherapy that immuno‐modulates TAMs cell population, a synergistic effect and long‐lived tumor eradication can be observed along with increased mice survival. Treatment with low doses of siRNA (ED₅₀ 0.0025–0.01 mg kg^?1) in a multi and long‐term dosing system substantially reduces the recruitment of inflammatory TAMs in lung tumor tissue, reduces tumor size (≈95%), and increases animal survival (≈75%) in mice. The results here suggest that it is likely that the combination of silencing important genes in tumor cells and in their supporting immune cells in the tumor microenvironment, such as TAMs, will greatly improve cancer clinical outcomes.     

Lung‐Endothelium‐Targeted Nanoparticles Based on a pH‐Sensitive Lipid and the GALA Peptide Enable Robust Gene Silencing and the Regression of Metastatic Lung Cancer

The development of efficient gene delivery systems targeting the lung endothelium remains a serious challenge. This study reports on the design and optimization of a multifunctional envelope‐type nanodevice (MEND) for an efficient siRNA delivery to the lung endothelium based on GALA‐peptide targeting ability. The incorporation of a pH‐sensitive lipid (YSK05) results in a dramatic improvement in silencing efficiency by enhancing endosomal escape, but this also causes a reduction in the lung selectivity. Contrary to the assumption that active targeting is largely dependent on the presence of a targeting ligand, the findings of the present study indicate that nanocarrier composition is critical for achieving the organ selectivity. Interestingly, helper lipids substantially mask the liver delivery resulting in optimum lung targeting. The optimized YSK05‐MEND is 40‐fold more efficient than a previously developed MEND, with a robust lung endothelium gene knockdown at small doses. The YSK05‐MEND strongly inhibits a metastatic lung cancer model and exerts superior control over lung metastasis compared to chemotherapy or the previously developed MEND. The YSK05‐MEND is well‐tolerated in mice after acute or chronic administration. As far as it is known, YSK05‐MEND achieves the most efficient lung endothelium gene silencing reported thus far with a median effective dose of 0.01 mg siRNA kg^?1 while minimally affecting the endothelium of other organs.