α‐Mannosyl‐Functionalized Cationic Nanohydrogel Particles for Targeted Gene Knockdown in Immunosuppressive Macrophages

Immunosuppressive M2 macrophages govern the immunophathogenic micromilieu in many severe diseases including cancer or fibrosis, thus, their re‐polarization through RNA interference is a promising concept to support combinatorial therapies. For targeted siRNA delivery, however, safe and stable carriers are required that manage cell specific transport to M2 macrophages. Here, siRNA‐loaded cationic nanogels are reported with α‐mannosyl decorated surfaces that target and modify M2 macrophages selectively. Via amphiphilic precursor block copolymers bearing one single α‐mannosyl moiety at their chain end mannosylated cationic nanohydrogel particles (ManNP) were obtained of 20 nm diameter determined by dynamic light scattering and cryogenic electron transmission microscopy. α‐Mannosyl surface modification is confirmed by agglutination with concanavalin A. SiRNA‐loaded ManNP preferentially targets the overexpressed mannose receptor CD206 on M2 macrophages, as shown by in vitro cell uptake studies in M2 polarized primary macrophages. This specificity is confirmed, since ManNP uptake could be reduced by blocking of CD206 with mannan. Effective ManNP‐guided siRNA delivery is confirmed by sequence‐specific gene knockdown of CSF‐1R in M2‐type macrophages exclusively, while the expression levels in M1‐polarized macrophages is not affected. In conclusion, α‐mannosyl‐functionalized ManNPs are promising universal siRNA carriers for targeted immunomodulatory treatment of immunosuppressive macrophages.     

Design of Potent Mannose 6‐Phosphate Analogues for the Functionalization of Lysosomal Enzymes To Improve the Treatment of Pompe Disease

Improving therapeutics delivery in enzyme replacement therapy (ERT) for lysosomal storage disorders is a challenge. Herein, we present the synthesis of novel analogues of mannose 6‐phosphate (M6P), known as AMFAs and functionalized at the anomeric position for enzyme grafting. AMFAs are non‐phosphate serum‐resistant derivatives that efficiently bind the cation‐independent mannose 6‐phosphate receptor (CI‐M6PR), which is the main pathway to address enzymes to lysosomes. One of the AMFAs was used to improve the treatment of the lysosomal myopathy Pompe disease, in which acid α‐glucosidase (GAA) is defective. AMFA grafting on a M6P‐free recombinant GAA led to a higher uptake of the GAA in adult Pompe fibroblasts in culture as compared to Myozyme, the M6P recombinant GAA. Moreover, the treatment of Pompe adult mice with the AMFA‐grafted recombinant enzyme led to a remarkable improvement, even at low doses, in muscle functionality and regeneration, whereas Myozyme had limited efficacy.     

GRP78‐Targeted HPMA Copolymer‐Photosensitizer Conjugate for Hyperthermia‐Induced Enhanced Uptake and Cytotoxicity in MCF‐7 Breast Cancer Cells

Photodynamic therapy (PDT) is a promising cancer treatment approach. However, the photosensitizers (PS) used for PDT are often limited by their poor solubility and selectivity for tumors. The goal of this study is to improve water solubility and delivery of the photosensitizer 2‐[1‐hexyloxyethyl]‐2‐divinyl pyropheophorbide‐a (HPPH) to breast cancer cells. An N‐(2‐hydroxypropyl)methacrylamide (HPMA) copolymer–HPPH photosensitizer conjugate is synthesized with heat shock receptor glucose‐regulated protein 78 (GRP78), targeting to GRP78 receptors of MCF‐7 cells, which are upregulated under mild hyperthermia. It is found that the uptake of the GRP78 targeted pep‐HPMA‐HPPH copolymer conjugate in MCF‐7 cells is improved through heat induction. Under mild hyperthermia the targeted copolymers are more effective compared to free HPPH. These results show potential for the utility of mild hyperthermia and copolymer delivery vehicles to enhance the efficacy of photodynamic therapy.     

Tumour‐Targeted Drug Delivery with Mannose‐Functionalized Nanoparticles Self‐Assembled from Amphiphilic β‐Cyclodextrins

Multivalent mannose‐functionalized nanoparticles self‐assembled from amphiphilic β‐cyclodextrins (β‐CDs) facilitate the targeted delivery of anticancer drugs to specific cancer cells. Doxorubicin (DOX)‐loaded nanoparticles equipped with multivalent mannose target units were efficiently taken up via receptor‐mediated endocytosis by MDA‐MB‐231 breast cancer cells that overexpress the mannose receptor. Upon entering the cell, the intracellular pH causes the release of DOX, which triggers apoptosis. Targeting by multivalent mannose significantly improved the capability of DOX‐loaded nanoparticles to inhibit the growth of MDA‐MB‐231 cancer cells with minimal side effects in vivo. This targeted and controlled drug delivery system holds promise as a nanotherapeutic for cancer treatment.     

A Fluorescent Probe for Rapid,High‐Contrast Visualization of Folate‐Receptor‐Expressing Tumors In Vivo

Folate receptors (FRs) are membrane proteins involved in folic acid uptake, and the alpha isoform (FR‐α) is overexpressed in ovarian and endometrial cancer cells. For fluorescence imaging of FRs in vivo, the near‐infrared (NIR) region (650–900 nm), in which tissue penetration is high and autofluorescence is low, is optimal, but existing NIR fluorescent probes targeting FR‐α show high non‐specific tissue adsorption, and require prolonged washout to visualize tumors. We have designed and synthesized a new NIR fluorescent probe, FolateSiR‐1 , utilizing a Si‐rhodamine fluorophore having a carboxy group at the benzene moiety, coupled to a folate ligand moiety through a negatively charged tripeptide linker. This probe exhibits very low background fluorescence and afforded a tumor‐to‐background ratio (TBR) of up to 83 in FR‐expressing tumor‐bearing mice within 30 min. Thus, FolateSiR‐1 has the potential to contribute to the research in the field of biology and the clinical medicine.     

Glutathione and pH‐responsive fluorescent nanogels for cell imaging and targeted methotrexate delivery

Cancer is one of the health problems that lead to death in the world, and nanotechnology was shown to have a unique potential to improve the therapeutic efficacy of anticancer agents. The nanosized drug delivery systems (DDSs) have been offered for targeting tumor tissue because of enhanced drug bioavailability and long circulation time. In this context, we reported a facial approach to prepare a novel pH and glutathione‐responsive nanogel. After that, the nanocarriers coupled with highly fluorescent quantum dots were developed. Then methotrexate (MTX) was loaded into and on the surface of nanogels by ionic interaction so that the triggered MTX release ability of the synthesized nanocarriers was verified through the assessment of in vitro drug release at simulated tumor tissue condition. The improved efficiency of the developed nanogels and their targeted performance via conjugation of MTX (as target ligand of folate receptors) were investigated through the various cell cytotoxicity studies such as 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay, 4′6‐diamidino‐2‐phenylindole (DAPI) staining, and flow cytometry. The results of various cell cytotoxicity studies concluded that the developed smart nanogels have many promising abilities for the targeted MTX delivery to cancer tissues.     

Unraveling the Uptake Mechanisms of Mannan Nanogel in Bone‐Marrow‐Derived Macrophages

The mechanisms associated with the cellular internalization of nanomedicines must be carefully considered when designing drug‐ and vaccine‐delivery systems. The cellular fate and effects of nanomedicines depend to a large extent on the cell uptake routes. A self‐assembled mannan nanogel is developed as a vaccination platform for antigen and adjuvant delivery. The mannan nanogel uptake by murine bone‐marrow‐derived macrophages is found to be time‐, concentration‐, and energy‐dependent, involving mannose‐receptor‐mediated phagocytosis and clathrin‐mediated endocytosis. The nanogel is also visualized in the cytosol suggesting endolysosomal escape. These results indicate that mannan nanogel is a promising versatile carrier for intracellular delivery of vaccines or therapeutic agents.

     

Mannose‐6‐Phosphate Receptor: A Target for Theranostics of Prostate Cancer

The development of personalized and non‐invasive cancer therapies based on new targets combined with nanodevices is a major challenge in nanomedicine. In this work, the over‐expression of a membrane lectin, the cation‐independent mannose 6‐phosphate receptor (M6PR), was specifically demonstrated in prostate cancer cell lines and tissues. To efficiently target this lectin a mannose‐6‐phosphate analogue was synthesized in six steps and grafted onto the surface of functionalized mesoporous silica nanoparticles (MSNs). These MSNs were used for in vitro and ex vivo photodynamic therapy to treat prostate cancer cell lines and primary cell cultures prepared from patient biopsies. The results demonstrated the efficiency of M6PR targeting for prostate cancer theranostic.     

Biodegradable Micelles Capable of Mannose‐Mediated Targeted Drug Delivery to Cancer Cells

A targeted micellar drug delivery system is developed from a biocompatible and biodegradable amphiphilic polyester, poly(Lac‐OCA)‐b‐(poly(Tyr(alkynyl)‐OCA)‐g‐mannose) (PLA‐b‐(PTA‐g‐mannose), that is synthesized via controlled ring‐opening polymerization of O‐carboxyanhydride (OCA) and highly efficient “Click” chemistry. Doxorubicin (DOX), a model lipophilic anticancer drug, can be effectively encapsulated into the micelles, and the mannose moiety allows active targeting of the micelles to cancer cells that specifically express mannose receptors, which thereafter enhances the anticancer efficiency of the drug. Comprised entirely of biodegradable and biocompatible polyesters, this micellar system demonstrates promising potentials for targeted drug delivery and cancer therapy.

     

A Redox‐Activatable Fluorescent Sensor for the High‐Throughput Quantification of Cytosolic Delivery of Macromolecules

Efficient delivery of biomacromolecules (e.g., proteins, nucleic acids) into cell cytosol remains a critical challenge for the development of macromolecular therapeutics or diagnostics. To date, most common approaches to assess cytosolic delivery rely on fluorescent labeling of macromolecules with an “always on” reporter and subcellular imaging of endolysosomal escape by confocal microscopy. This strategy is limited by poor signal‐to‐noise ratio and only offers low throughput, qualitative information. Herein we describe a quantitative redox‐activatable sensor (qRAS) for the real‐time monitoring of cytosolic delivery of macromolecules. qRAS‐labeled macromolecules are silent (off) inside the intact endocytic organelles, but can be turned on by redox activation after endolysosomal disruption and delivery into the cytosol, thereby greatly improving the detection accuracy. In addition to confocal microscopy, this quantitative sensing technology allowed for a high‐throughput screening of a panel of polymer carriers toward efficient cytosolic delivery of model proteins on a plate reader. The simple and versatile qRAS design offers a useful tool for the investigation of new strategies for endolysosomal escape of biomacromolecules to facilitate the development of macromolecular therapeutics for a variety of disease indications.     

Spatially Controllable DNA Condensation by a Water‐Soluble Supramolecular Hybrid of Single‐Walled Carbon Nanotubes and β‐Cyclodextrin‐Tethered Ruthenium Complexes

A supramolecular hybrid is prepared by the supramolecular surface modification of single‐walled carbon nanotube (SWCNT) with cationic β‐cyclodextrin‐tethered ruthenium complexes through a spacer molecule that contains both an adamantane and a pyrene moiety. By employing the supramolecular hybrid, spatially controllable DNA condensation along the SWCNT skeleton is achieved by anchoring cationic ruthenium complexes on the surface. Furthermore, because of the unique physiological properties of SWCNTs, the cationic supramolecular hybrid can be used as a nonviral gene delivery system with the ruthenium complexes as a fluorescent probe to monitor uptake of DNA by cells.     

Mannose‐Modified Serum Exosomes for the Elevated Uptake to Murine Dendritic Cells and Lymphatic Accumulation

The surface of bovine serum‐derived exosomes (EXOs) are modified with α‐d ‐mannose for facile interaction with mannose receptors on dendritic cells (DCs) and for efficient delivery of immune stimulators to the DCs. The surface of the EXOs is modified with polyethylene glycol (PEG) without particle aggregation (≈50 nm) via the incorporation of 1,2‐distearoyl‐sn‐glycero‐3‐phosphoethanolamine (DSPE) into the lipid layer of the EXO, compared to chemical conjugation by N‐hydroxysuccinimide activated PEG (NHS‐PEG). PEG modification onto the exosomal surface significantly decreases the non‐specific cellular uptake of the EXOs into the DCs. However, the EXOs with mannose‐conjugated PEG‐DSPE (EXO‐PEG‐man) exhibit excellent intracellular uptake into the DCs and boost the immune response by the incorporation of adjuvant, monophosphoryl lipid A (MPLA) within the EXO. After an intradermal injection, a higher retention of EXO‐PEG‐man is observed in the lymph nodes, which could be used for the efficient delivery of immune stimulators and antigens to the lymph nodes in vivo.     

Bright,Highly Water‐Soluble Triazacyclononane Europium Complexes To Detect Ligand Binding with Time‐Resolved FRET Microscopy

Luminescent europium complexes are used in a broad range of applications as a result of their particular emissive properties. The synthesis and application of bright, highly water‐soluble, and negatively charged sulfonic‐ or carboxylic acid derivatives of para‐substituted aryl–alkynyl triazacyclononane complexes are described. Introduction of the charged solubilizing moieties suppresses cellular uptake or adsorption to living cells making them applicable for labeling and performing assays on membrane receptors. These europium complexes are applied to monitor fluorescent ligand binding on cell‐surface proteins with time‐resolved Förster resonance energy transfer (TR‐FRET) assays in plate‐based format and using TR‐FRET microscopy.     

Bright,Highly Water‐Soluble Triazacyclononane Europium Complexes To Detect Ligand Binding with Time‐Resolved FRET Microscopy

Luminescent europium complexes are used in a broad range of applications as a result of their particular emissive properties. The synthesis and application of bright, highly water‐soluble, and negatively charged sulfonic‐ or carboxylic acid derivatives of para‐substituted aryl–alkynyl triazacyclononane complexes are described. Introduction of the charged solubilizing moieties suppresses cellular uptake or adsorption to living cells making them applicable for labeling and performing assays on membrane receptors. These europium complexes are applied to monitor fluorescent ligand binding on cell‐surface proteins with time‐resolved Förster resonance energy transfer (TR‐FRET) assays in plate‐based format and using TR‐FRET microscopy.     

Detection of LacZ‐Positive Cells in Living Tissue with Single‐Cell Resolution

The LacZ gene, which encodes Escherichia coli β‐galactosidase, is widely used as a marker for cells with targeted gene expression or disruption. However, it has been difficult to detect lacZ‐positive cells in living organisms or tissues at single‐cell resolution, limiting the utility of existing lacZ reporters. Herein we present a newly developed fluorogenic β‐galactosidase substrate suitable for labeling live cells in culture, as well as in living tissues. This precisely functionalized fluorescent probe exhibited dramatic activation of fluorescence upon reaction with the enzyme, remained inside cells by anchoring itself to intracellular proteins, and provided single‐cell resolution. Neurons labeled with this probe preserved spontaneous firing, which was enhanced by application of ligands of receptors expressed in the cells, suggesting that this probe would be applicable to investigate functions of targeted cells in living tissues and organisms.     

A D‐Peptide Ligand of Nicotine Acetylcholine Receptors for Brain‐Targeted Drug Delivery

Lysosomes of brain capillary endothelial cells are implicated in nicotine acetylcholine receptor (nAChR)‐mediated transcytosis and act as an enzymatic barrier for the transport of peptide ligands to the brain. A D ‐peptide ligand of nAChRs (termed ^DCDX), which binds to nAChRs with an IC₅₀ value of 84.5 nM , was developed by retro–inverso isomerization. ^DCDX displayed exceptional stability in lysosomal homogenate and serum, and demonstrated significantly higher transcytosis efficiency in an in vitro blood–brain barrier monolayer compared with the parent L ‐peptide. When modified on liposomal surface, ^DCDX facilitated significant brain‐targeted delivery of liposomes. As a result, brain‐targeted delivery of ^DCDX modified liposomes enhanced therapeutic efficiency of encapsulated doxorubicin for glioblastoma. This study illustrates the importance of ligand stability in nAChRs‐mediated transcytosis, and paves the way for developing stable brain‐targeted entities.     

Integrated Hollow Mesoporous Silica Nanoparticles for Target Drug/siRNA Co‐Delivery

A hollow mesoporous silica nanoparticle (HMSNP) based drug/siRNA co‐delivery system was designed and fabricated, aiming at overcoming multidrug resistance (MDR) in cancer cells for targeted cancer therapy. The as‐prepared HMSNPs have perpendicular nanochannels connecting to the internal hollow cores, thereby facilitating drug loading and release. The extra volume of the hollow core enhances the drug loading capacity by two folds as compared with conventional mesoporous silica nanoparticles (MSNPs). Folic acid conjugated polyethyleneimine (PEI‐FA) was coated on the HMSNP surfaces under neutral conditions through electrostatic interactions between the partially charged amino groups of PEI‐FA and the phosphate groups on the HMSNP surfaces, blocking the mesopores and preventing the loaded drugs from leakage. Folic acid acts as the targeting ligand that enables the co‐delivery system to selectively bind with and enter into the target cancer cells. PEI‐FA‐coated HMSNPs show enhanced siRNA binding capability on account of electrostatic interactions between the amino groups of PEI‐FA and siRNA, as compared with that of MSNPs. The electrostatic interactions provide the feasibility of pH‐controlled release. In vitro pH‐responsive drug/siRNA co‐delivery experiments were conducted on HeLa cell lines with high folic acid receptor expression and MCF‐7 cell lines with low folic acid receptor expression for comparison, showing effective target delivery to the HeLa cells through folic acid receptor meditated cellular endocytosis. The pH‐responsive intracellular drug/siRNA release greatly minimizes the prerelease and possible side effects of the delivery system. By simultaneously delivering both doxorubicin (Dox) and siRNA against the Bcl‐2 protein into the HeLa cells, the expression of the anti‐apoptotic protein Bcl‐2 was successfully suppressed, leading to an enhanced therapeutic efficacy. Thus, the present multifunctional nanoparticles show promising potentials for controlled and targeted drug and gene co‐delivery in cancer treatment.     

Hollow‐Core Magnetic Colloidal Nanocrystal Clusters with Ligand‐Exchanged Surface Modification as Delivery Vehicles for Targeted and Stimuli‐Responsive Drug Release

The fabrication of hierarchical magnetic nanomaterials with well‐defined structure, high magnetic response, excellent colloidal stability, and biocompatibility is highly sought after for drug‐delivery systems. Herein, a new kind of hollow‐core magnetic colloidal nanocrystal cluster (HMCNC) with porous shell and tunable hollow chamber is synthesized by a one‐pot solvothermal process. Its novelty lies in the “tunability” of the hollow chamber and of the pore structure within the shell through controlled feeding of sodium citrate and water, respectively. Furthermore, by using the ligand‐exchange method, folate‐modified poly(acrylic acid) was immobilized on the surface of HMCNCs to create folate‐targeted HMCNCs (folate‐HMCNCs), which endowed them with excellent colloidal stability, pH sensitivity, and, more importantly, folate receptor‐targeting ability. These assemblages exhibited excellent colloidal stability in plasma solution. Doxorubicin (DOX), as a model anticancer agent, was loaded within the hollow core of these folate‐HMCNCs (folate‐HMCNCs‐DOX), and drug‐release experiments proved that the folate‐HMCNCs‐DOX demonstrated pH‐dependent release behavior. The folate‐HMCNCs‐DOX assemblages also exhibited higher potent cytotoxicity to HeLa cells than free doxorubicin. Moreover, folate‐HMCNCs‐DOX showed rapid cell uptake apart from the enhanced cytotoxicity to HeLa cells. Experimental results confirmed that the synthesized folate‐HMCNCs are smart nanovehicles as a result of their improved folate receptor‐targeting abilities and also because of their combined pH‐ and magnetic‐stimuli response for applications in drug delivery.     

A Multifunctional Nanomicelle for Real‐Time Targeted Imaging and Precise Near‐Infrared Cancer Therapy

Simultaneous targeted cancer imaging, therapy and real‐time therapeutic monitoring can prevent over‐ or undertreatment. This work describes the design of a multifunctional nanomicelle for recognition and precise near‐infrared (NIR) cancer therapy. The nanomicelle encapsulates a new pH‐activatable fluorescent probe and a robust NIR photosensitizer, R16FP, and is functionalized with a newly screened cancer‐specific aptamer for targeting viable cancer cells. The fluorescent probe can light up the lysosomes for real‐time imaging. Upon NIR irradiation, R16FP‐mediated generation of reactive oxygen species causes lysosomal destruction and subsequently trigger lysosomal cell death. Meanwhile the fluorescent probe can reflect the cellular status and in situ visualize the treatment process. This protocol can provide molecular information for precise therapy and therapeutic monitoring.     

A Lysosome‐Compatible Near‐Infrared Fluorescent Probe for Targeted Monitoring of Nitric Oxide

The requirement for nitric oxide (NO) of lysosomes has motivated the development of a sophisticated fluorescent probe to monitor the distribution of this important biomolecule at the subcellular level in living cells. A near‐infrared (NIR) fluorescent Si‐rhodamine (SiRB)‐NO probe was designed based on the NO‐induced ring‐opening process of Si‐rhodamine. The probe exhibits fast chromogenic and fluorogenic responses, and high sensitivity and selectivity toward trace amounts of NO. Significantly, the spirolactam in Si‐rhodamine exhibits very good tolerance to H⁺, which in turn brings extremely low background fluorescence not only in the physiological environment but also under acidic conditions. The stability of the highly fluorescent product in acidic solution provides persistent fluorescence emission for long‐term imaging experiments. To achieve targeted imaging with improved spatial resolution and sensitivity, an efficient lysosome‐targeting moiety was conjugated to a SiRB‐NO probe, affording a tailored lysosome‐targeting NIR fluorescent Lyso‐SiRB‐NO probe. Inheriting the key advantages of its parent SiRB‐NO probe, Lyso‐SiRB‐NO is a functional probe that is suited for monitoring lysosomal NO with excellent lysosome compatibility. Imaging experiments demonstrated the monitoring of both exogenous and endogenous NO in real time by using the Lyso‐SiRB‐NO probe.