Targeted Delivery of siRNA‐Generating DNA Nanocassettes Using Multifunctional Nanoparticles

Molecular therapy using a small interfering RNA (siRNA) has shown promise in the development of novel therapeutics. Various formulations have been used for in vivo delivery of siRNAs. However, the stability of short double‐stranded RNA molecules in the blood and efficiency of siRNA delivery into target organs or tissues following systemic administration have been the major issues that limit applications of siRNA in human patients. In this study, multifunctional siRNA delivery nanoparticles are developed that combine imaging capability of nanoparticles with urokinase plasminogen activator receptor‐targeted delivery of siRNA expressing DNA nanocassettes. This theranostic nanoparticle platform consists of a nanoparticle conjugated with targeting ligands and double‐stranded DNA nanocassettes containing a U6 promoter and a shRNA gene for in vivo siRNA expression. Targeted delivery and gene silencing efficiency of firefly luciferase siRNA nanogenerators are demonstrated in tumor cells and in animal tumor models. Delivery of survivin siRNA expressing nanocassettes into tumor cells induces apoptotic cell death and sensitizes cells to chemotherapy drugs. The ability of expression of siRNAs from multiple nanocassettes conjugated to a single nanoparticle following receptor‐mediated internalization should enhance the therapeutic effect of the siRNA‐mediated cancer therapy.     

Pulmonary Codelivery of Doxorubicin and siRNA by pH‐Sensitive Nanoparticles for Therapy of Metastatic Lung Cancer

A pulmonary codelivery system that can simultaneously deliver doxorubicin (DOX) and Bcl2 siRNA to the lungs provides a promising local treatment strategy for lung cancers. In this study, DOX is conjugated onto polyethylenimine (PEI) by using cis‐aconitic anhydride (CA, a pH‐sensitive linker) to obtain PEI‐CA‐DOX conjugates. The PEI‐CA‐DOX/siRNA complex nanoparticles are formed spontaneously via electrostatic interaction between cationic PEI‐CA‐DOX and anionic siRNA. The drug release experiment shows that DOX releases faster at acidic pH than at pH 7.4. Moreover, PEI‐CA‐DOX/Bcl2 siRNA complex nanoparticles show higher cytotoxicity and apoptosis induction in B16F10 cells than those treated with either DOX or Bcl2 siRNA alone. When the codelivery systems are directly sprayed into the lungs of B16F10 melanoma‐bearing mice, the PEI‐CA‐DOX/Bcl2 siRNA complex nanoparticles exhibit enhanced antitumor efficacy compared with the single delivery of DOX or Bcl2 siRNA. Compared with systemic delivery, most drug and siRNA show a long‐term retention in the lungs via pulmonary delivery, and a considerable number of the drug and siRNA accumulate in tumor tissues of lungs, but rarely in normal lung tissues. The PEI‐CA‐DOX/Bcl2 siRNA complex nanoparticles are promising for the treatment of metastatic lung cancer by pulmonary delivery with low side effects on the normal tissues.     

Lipid‐PEG‐Folate Encapsulated Nanoparticles with Aggregation Induced Emission Characteristics: Cellular Uptake Mechanism and Two‐Photon Fluorescence Imaging

Folate functionalized nanoparticles (NPs) that contain fluorogens with aggregation‐induced emission (AIE) characteristics are fabricated to show bright far‐red/near‐infrared fluorescence, a large two‐photon absorption cross section and low cytotoxicity, which are internalized into MCF‐7 cancer cells mainly through caveolae‐mediated endocytosis. One‐photon excited in vivo fluorescence imaging illustrates that these AIE NPs can accumulate in a tumor and two‐photon excited ex vivo tumor tissue imaging reveals that they can be easily detected in the tumor mass at a depth of 400 μm. These studies indicate that AIE NPs are promising alternatives to conventional TPA probes for biological imaging.     

Cellular Internalization of Rod‐Like Nanoparticles with Various Surface Patterns: Novel Entry Pathway and Controllable Uptake Capacity

The cellular internalization of rod‐like nanoparticles (NPs) is investigated in a combined experimental and simulation study. These rod‐like nanoparticles with smooth, abacus‐like (i.e., beads‐on‐wires), and helical surface patterns are prepared by the cooperative self‐assembly of poly(γ‐benzyl‐l ‐glutamate)‐block‐poly(ethylene glycol) (PBLG‐b‐PEG) block copolymers and PBLG homopolymers. All three types of NPs can be internalized via endocytosis. Helical NPs exhibit the best endocytic efficacy, followed by smooth NPs and abacus‐like NPs. Coarse‐grained molecular dynamics simulations are used to examine the endocytic efficiency of these NPs. The NPs with helical and abacus‐like surfaces can be endocytosed via novel “standing up” (tip entry) and “gyroscope‐like” (precession) pathways, respectively, which are distinct from the pathway of traditional NPs with smooth surfaces. This finding indicates that the cellular internalization capacity and pathways can be regulated by introducing stripe patterns (helical and abacus‐like) onto the surface of rod‐like NPs. The results of this study may lead to novel applications of biomaterials, such as advanced drug delivery systems.     

One‐Step,Room‐Temperature Synthesis of Glutathione‐Capped Iron‐Oxide Nanoparticles and their Application in In Vivo T1‐Weighted Magnetic Resonance Imaging

The room‐temperature, aqueous‐phase synthesis of iron‐oxide nanoparticles (IO NPs) with glutathione (GSH) is reported. The simple, one‐step reduction involves GSH as a capping agent and tetrakis(hydroxymethyl)phosphonium chloride (THPC) as the reducing agent; GSH is an anti‐oxidant that is abundant in the human body while THPC is commonly used in the synthesis of noble‐metal clusters. Due to their low magnetization and good water‐dispersibility, the resulting GSH‐IO NPs, which are 3.72 ± 0.12 nm in diameter, exhibit a low r₂ relaxivity (8.28 mm ^?1s^?1) and r₂/r₁ ratio (2.28)—both of which are critical for T₁ contrast agents. This, together with the excellent biocompatibility, makes these NPs an ideal candidate to be a T₁ contrast agent. Its capability in cellular imaging is illustrated by the high signal intensity in the T₁‐weighted magnetic resonance imaging (MRI) of treated HeLa cells. Surprisingly, the GSH‐IO NPs escape ingestion by the hepatic reticuloendothelial system, enabling strong vascular enhancement at the internal carotid artery and superior sagittal sinus, where detection of the thrombus is critical for diagnosing a stroke. Moreover, serial T₁‐ and T₂‐weighted time‐dependent MR images are resolved for a rat's kidneys, unveiling detailed cortical‐medullary anatomy and renal physiological functions. The newly developed GSH‐IO NPs thus open a new dimension in efforts towards high‐performance, long‐circulating MRI contrast agents that have biotargeting potential.     

A Layer‐by‐Layer ZnO Nanoparticle–PbS Quantum Dot Self‐Assembly Platform for Ultrafast Interfacial Electron Injection

Absorbent layers of semiconductor quantum dots (QDs) are now used as material platforms for low‐cost, high‐performance solar cells. The semiconductor metal oxide nanoparticles as an acceptor layer have become an integral part of the next generation solar cell. To achieve sufficient electron transfer and subsequently high conversion efficiency in these solar cells, however, energy‐level alignment and interfacial contact between the donor and the acceptor units are needed. Here, the layer‐by‐layer (LbL) technique is used to assemble ZnO nanoparticles (NPs), providing adequate PbS QD uptake to achieve greater interfacial contact compared with traditional sputtering methods. Electron injection at the PbS QD and ZnO NP interface is investigated using broadband transient absorption spectroscopy with 120 femtosecond temporal resolution. The results indicate that electron injection from photoexcited PbS QDs to ZnO NPs occurs on a time scale of a few hundred femtoseconds. This observation is supported by the interfacial electronic‐energy alignment between the donor and acceptor moieties. Finally, due to the combination of large interfacial contact and ultrafast electron injection, this proposed platform of assembled thin films holds promise for a variety of solar cell architectures and other settings that principally rely on interfacial contact, such as photocatalysis.     

A Poly(Propyleneimine) Dendrimer‐Based Polyplex‐System for Single‐Chain Antibody‐Mediated Targeted Delivery and Cellular Uptake of SiRNA

Therapeutics based on small interfering RNAs (siRNAs) offer a great potential to treat so far incurable diseases or metastatic cancer. However, the broad application of siRNAs using various nonviral carrier systems is hampered by unspecific toxic side effects, poor pharmacokinetics due to unwanted delivery of siRNA‐loaded nanoparticles into nontarget organs, or rapid renal excretion. In order to overcome these obstacles, several targeting strategies using chemically linked antibodies and ligands have emerged. This study reports a new modular polyplex carrier system for targeted delivery of siRNA, which is based on transfection‐disabled maltose‐modified poly(propyleneimine)‐dendrimers (mal‐PPI) bioconjugated to single chain fragment variables (scFvs). To achieve targeted delivery into tumor cells expressing the epidermal growth factor receptor variant III (EGFRvIII), monobiotinylated anti‐EGFRvIII scFv fused to a Propionibacterium shermanii transcarboxylase‐derived biotinylation acceptor (P‐BAP) is bioconjugated to mal‐PPI through a novel coupling strategy solely based on biotin–neutravidin bridging. In contrast to polyplexes containing an unspecific control scFv‐P‐BAP, the generated EGFRvIII‐specific polyplexes are able to exclusively deliver siRNA to tumor cells and tumors by receptor‐mediated endocytosis. These results suggest that receptor‐mediated uptake of otherwise noninternalized mal‐PPI‐based polyplexes is a promising avenue to improve siRNA therapy of cancer, and introduce a novel strategy for modular bioconjugation of protein ligands to nanoparticles.     

Synthesis and Assembly of Click‐Nucleic‐Acid‐Containing PEG–PLGA Nanoparticles for DNA Delivery

Co‐delivery of both chemotherapy drugs and siRNA from a single delivery vehicle can have a significant impact on cancer therapy due to the potential for overcoming issues such as drug resistance. However, the inherent chemical differences between charged nucleic acids and hydrophobic drugs have hindered entrapment of both components within a single carrier. While poly(ethylene glycol)‐block‐poly(lactic‐co‐glycolic acid) (PEG–PLGA) copolymers have been used successfully for targeted delivery of chemotherapy drugs, loading of DNA or RNA has been poor. It is demonstrated that significant amounts of DNA can be encapsulated within PLGA‐containing nanoparticles through the use of a new synthetic DNA analog, click nucleic acids (CNAs). First, triblock copolymers of PEG‐CNA‐PLGA are synthesized and then formulated into polymer nanoparticles from oil‐in‐water emulsions. The CNA‐containing particles show high encapsulation of DNA complementary to the CNA sequence, whereas PEG‐PLGA alone shows minimal DNA loading, and non‐complementary DNA strands do not get encapsulated within the PEG‐CNA‐PLGA nanoparticles. Furthermore, the dye pyrene can be successfully co‐loaded with DNA and lastly, a complex, larger DNA sequence that contains an overhang complementary to the CNA can also be encapsulated, demonstrating the potential utility of the CNA‐containing particles as carriers for chemotherapy agents and gene silencers.     

Bright Quantum‐Dot‐Sized Single‐Chain Conjugated Polyelectrolyte Nanoparticles: Synthesis,Characterization and Application for Specific Extracellular Labeling and Imaging

We report a simple method to fabricate quantum‐dot‐sized nanoparticles (NPs) from poly[9,9‐bis((6‐N,N,N‐trimethylammonium)hexyl)fluorene‐alt‐co‐2,1,3‐benzo­xadiazole dibromide] (PFBD). The transmission electron microscope results reveal that the obtained NPs have a mean diameter of ≈4 nm, which is composed of a single PFBD chain. The NPs show bright fluorescence with an emission maximum at ≈636 nm and a quantum yield of ≈26% in water. The fluorescence properties of the NPs are characterized by steady fluorescence microscopy, fluorescence dynamic study and single nanoparticle microscopy, which show superior brightness over commercial quantum dots QD655. The NPs are further conjugated with streptavidin to yield PFBD‐SA NPs, which serve as a specific extracellular labeling and imaging probe with high specificity and good photostability.     

Memantine‐Loaded PEGylated Biodegradable Nanoparticles for the Treatment of Glaucoma

Glaucoma is a multifactorial neurodegenerative disease associated with retinal ganglion cells (RGC) loss. Increasing reports of similarities in glaucoma and other neurodegenerative conditions have led to speculation that therapies for brain neurodegenerative disorders may also have potential as glaucoma therapies. Memantine is an N‐methyl‐d ‐aspartate (NMDA) antagonist approved for Alzheimer's disease treatment. Glutamate‐induced excitotoxicity is implicated in glaucoma and NMDA receptor antagonism is advocated as a potential strategy for RGC preservation. This study describes the development of a topical formulation of memantine‐loaded PLGA‐PEG nanoparticles (MEM‐NP) and investigates the efficacy of this formulation using a well‐established glaucoma model. MEM‐NPs <200 nm in diameter and incorporating 4 mg mL^?1 of memantine were prepared with 0.35 mg mL^?1 localized to the aqueous interior. In vitro assessment indicated sustained release from MEM‐NPs and ex vivo ocular permeation studies demonstrated enhanced delivery. MEM‐NPs were additionally found to be well tolerated in vitro (human retinoblastoma cells) and in vivo (Draize test). Finally, when applied topically in a rodent model of ocular hypertension for three weeks, MEM‐NP eye drops were found to significantly (p < 0.0001) reduce RGC loss. These results suggest that topical MEM‐NP is safe, well tolerated, and, most promisingly, neuroprotective in an experimental glaucoma model.     

Safety‐by‐Design of Metal Oxide Nanoparticles Based on the Regulation of their Energy Edges

The safety of metal oxide (MOx) nanoparticles (NPs) has been highly concerned because of their wide application and potential toxicological injury. The safe‐by‐design strategy is usually developed to make safer MOx NPs based on regulation of their physicochemical properties. In the present study, manganese oxide (Mn₃O₄) NPs, as a representative of insoluble toxic MOx NPs, are doped with a series of transition metal to regulate their conduction band energy (E_c) out of biological redox potential range (BRPR) or Fermi energy (E_f) far away from valence band energy (E_v), aiming at completely eliminating the toxicity or significantly reducing the toxicity. It is found that all these M‐doping cannot move E_c of Mn₃O₄ NPs out of the BRPR but zinc (Zn)‐, copper (Cu)‐, and chromium (Cr)‐doping do move E_f far away from E_v, where Zn‐doping results in the largest |E_f ? E_v| value. Various abiotic, in vitro and in vivo assessments reveal that Zn‐, Cu‐, and Cr‐doped Mn₃O₄ NPs can generate lower amount of ?OH and trigger weaker injury than Mn₃O₄ NPs, where Zn‐doped Mn₃O₄ NPs show the lowest toxicity. Regulating E_f far away from E_v becomes a feasible safe‐by‐design approach to achieve safe MOx NPs.     

WO3 Nanofiber‐Based Biomarker Detectors Enabled by Protein‐Encapsulated Catalyst Self‐Assembled on Polystyrene Colloid Templates

A novel catalyst functionalization method, based on protein‐encapsulated metallic nanoparticles (NPs) and their self‐assembly on polystyrene (PS) colloid templates, is used to form catalyst‐loaded porous WO₃ nanofibers (NFs). The metallic NPs, composed of Au, Pd, or Pt, are encapsulated within a protein cage, i.e., apoferritin, to form unagglomerated monodispersed particles with diameters of less than 5 nm. The catalytic NPs maintain their nanoscale size, even following high‐temperature heat‐treatment during synthesis, which is attributed to the discrete self‐assembly of NPs on PS colloid templates. In addition, the PS templates generate open pores on the electrospun WO₃ NFs, facilitating gas molecule transport into the sensing layers and promoting active surface reactions. As a result, the Au and Pd NP‐loaded porous WO₃ NFs show superior sensitivity toward hydrogen sulfide, as evidenced by responses (R_air/R_gas) of 11.1 and 43.5 at 350 °C, respectively. These responses represent 1.8‐ and 7.1‐fold improvements compared to that of dense WO₃ NFs (R_air/R_gas = 6.1). Moreover, Pt NP‐loaded porous WO₃ NFs exhibit high acetone sensitivity with response of 28.9. These results demonstrate a novel catalyst loading method, in which small NPs are well‐dispersed within the pores of WO₃ NFs, that is applicable to high sensitivity breath sensors.     

Highly efficient gene silencing and bioimaging based on fluorescent carbon dots <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis>

Small interfering RNA (siRNA) is an attractive therapeutic candidate for sequencespecific gene silencing to treat incurable diseases using small molecule drugs.However,its efficient intracellular delivery has remained a challenge.Here,we have developed a highly biocompatible fluorescent carbon dot (CD),and demonstrate a functional siRNA delivery system that induces efficient gene knockdown in vitro and in vivo.We found that CD nanoparticles (NPs) enhance the cellular uptake of siRNA,via endocytosis in tumor cells,with low cytotoxicity and unexpected immune responses.Real-time study of fluorescence imaging in live cells shows that CD NPs favorably localize in cytoplasm and successfully release siRNA within 12 h.Moreover,we demonstrate that CD NP-mediated siRNA delivery significantly silences green fluorescence protein (GFP) expression and inhibits tumor growth in a breast cancer cell xenograft mouse model of tumor-specific therapy.We have developed a multi functional siRNA delivery vehicle enabling simultaneous bioimaging and efficient downregulation of gene expression,that shows excellent potential for gene therapy.     

Water‐Dispersible,Multifunctional, Magnetic,Luminescent Silica‐Encapsulated Composite Nanotubes

A multifunctional one‐dimensional nanostructure incorporating both CdSe quantum dots (QDs) and Fe₃O₄ nanoparticles (NPs) within a SiO₂‐nanotube matrix is successfully synthesized based on the self‐assembly of preformed functional NPs, allowing for control over the size and amount of NPs contained within the composite nanostructures. This specific nanostructure is distinctive because both the favorable photoluminescent and magnetic properties of QD and NP building blocks are incorporated and retained within the final silica‐based composite, thus rendering it susceptible to both magnetic guidance and optical tracking. Moreover, the resulting hydrophilic nanocomposites are found to easily enter into the interiors of HeLa cells without damage, thereby highlighting their capability not only as fluorescent probes but also as possible drug‐delivery vehicles of interest in nanobiotechnology.     

Iron‐Oxide‐Based Nanovector for Tumor Targeted siRNA Delivery in an Orthotopic Hepatocellular Carcinoma Xenograft Mouse Model

Hepatocellular carcinoma (HCC) is one of the deadliest cancers worldwide. Small interfering RNA (siRNA) holds promise as a new class of therapeutics for HCC, as it can achieve sequence‐specific gene knockdown with low cytotoxicity. However, the main challenge in the clinical application of siRNA lies in the lack of effective delivery approaches that need to be highly specific and thus incur low or no systemic toxicity. Here, a nonviral nanoparticle‐based gene carrier is presented that can specifically deliver siRNA to HCC. The nanovector (NP‐siRNA‐GPC3 Ab) is made of an iron oxide core coated with chitosan‐polyethylene glycol (PEG) grafted polyethyleneimine copolymer, which is further functionalized with siRNA and conjugated with a monoclonal antibody (Ab) against human glypican‐3 (GPC3) receptor highly expressed in HCC. A rat RH7777 HCC cell line that coexpresses human GPC3 and firefly luciferase (Luc) is established to evaluate the nanovector. The nanoparticle‐mediated delivery of siRNA against Luc effectively suppresses Luc expression in vitro without notable cytotoxicity. Significantly, NP‐siLuc‐GPC3 Ab administered intravenously in an orthotopic model of HCC is able to specifically bound to tumor and induce remarkable inhibition of Luc expression. The findings demonstrate the potential of using this nanovector for targeted delivery of therapeutic siRNA to HCC.     

Supramolecular Glyco‐nanoparticles Toward Immunological Applications

Glyco‐mimicking nanoparticles (glyco‐NPs) with Förster resonance energy transfer (FRET) donor and acceptor groups formed via dynamic covalent bond of benzoboroxole and sugar from two complementary polymers are prepared. The glyco‐NPs are proved to be quite stable under physiological conditions but sensitive to pH. So the glyco‐NPs can be internalized by dendritic cells with integrity and nontoxicity and then dissociate within the acidic organelles. This particle dissociation is directly observed and visualized in vitro, for the first time via the FRET measurements and fluorescent microscopy. This feature makes controlled release of drug or protein by glyco‐NPs possible, i.e., when model antigen Ovalbumin is loaded in the glyco‐NPs, the released Ovalbumin in dendritic cells stimulates T cells more efficiently than the free Ovalbumin itself as a result of the enhanced antigen processing and presentation. Thus, the results enlighten a bright future of the glyco‐NPs in immunotherapy.     

Amorphous Silica Nanoparticles Promote Monocyte Adhesion to Human Endothelial Cells: Size‐Dependent Effect

There is evidence that nanoparticles can induce endothelial dysfunction. Here, the effect of monodisperse amorphous silica nanoparticles (SiO₂‐NPs) of different diameters on endothelial cells function is examined. Human endothelial cell line (EA.hy926) or primary human pulmonary artery endothelial cells (hPAEC) are seeded in inserts introduced or not above triple cell co‐cultures (pneumocytes, macrophages, and mast cells). Endothelial cells are incubated with SiO₂‐NPs at non‐cytotoxic concentrations for 12 h. A significant increase (up to 2‐fold) in human monocytes adhesion to endothelial cells is observed for 18 and 54 nm particles. Exposure to SiO₂‐NPs induces protein expression of adhesion molecules (ICAM‐1 and VCAM‐1) as well as significant up‐regulation in mRNA expression of ICAM‐1 in both endothelial cell types. Experiments performed with fluorescent‐labelled monodisperse amorphous SiO₂‐NPs of similar size evidence nanoparticle uptake into the cytoplasm of endothelial cells. It is concluded that exposure of human endothelial cells to amorphous silica nanoparticles enhances their adhesive properties. This process is modified by the size of the nanoparticle and the presence of other co‐cultured cells.     

pH‐Responsive Isoniazid‐Loaded Nanoparticles Markedly Improve Tuberculosis Treatment in Mice

Tuberculosis is a major global health problem for which improved therapeutics are needed to shorten the course of treatment and combat emergence of drug resistance. Mycobacterium tuberculosis, the etiologic agent of tuberculosis, is an intracellular pathogen of mononuclear phagocytes. As such, it is an ideal pathogen for nanotherapeutics because macrophages avidly ingest nanoparticles even without specific targeting molecules. Hence, a nanoparticle drug delivery system has the potential to target and deliver high concentrations of drug directly into M. tuberculosis‐infected cells—greatly enhancing efficacy while avoiding off‐target toxicities. Stimulus‐responsive mesoporous silica nanoparticles of two different sizes, 100 and 50 nm, are developed as carriers for the major anti‐tuberculosis drug isoniazid in a prodrug configuration. The drug is captured by the aldehyde‐functionalized nanoparticle via hydrazone bond formation and coated with poly(ethylene imine)–poly(ethylene glycol) (PEI–PEG). The drug is released from the nanoparticles in response to acidic pH at levels that naturally occur within acidified endolysosomes. It is demonstrated that isoniazid‐loaded PEI–PEG‐coated nanoparticles are avidly ingested by M. tuberculosis‐infected human macrophages and kill the intracellular bacteria in a dose‐dependent manner. It is further demonstrated in a mouse model of pulmonary tuberculosis that the nanoparticles are well tolerated and much more efficacious than an equivalent amount of free drug.     

Simultaneous Imaging of Endogenous Survivin mRNA and On‐Demand Drug Release in Live Cells by Using a Mesoporous Silica Nanoquencher

The design of multifunctional drug delivery systems capable of simultaneous target detection, imaging, and therapeutics in live mammalian cells is critical for biomedical research. In this study, by using mesoporous silica nanoparticles (MSNs) chemically modified with a small‐molecule dark quencher, followed by sequential drug encapsulation, MSN capping with a dye‐labeled antisense oligonucleotide, and bioorthogonal surface modification with cell‐penetrating poly(disulfide)s, the authors have successfully developed the first mesoporous silica nanoquencher (qMSN), characterized by high drug‐loading and endocytosis‐independent cell uptake, which is able to quantitatively image endogenous survivin mRNA and release the loaded drug in a manner that depends on the survivin expression level in tumor cells. The authors further show that this novel drug delivery system may be used to minimize potential cytotoxicity encountered by many existing small‐molecule drugs in cancer therapy.