Polymer-Drug Conjugates Codeliver a Temozolomide Intermediate and Nitric Oxide for Enhanced Chemotherapy against Glioblastoma Multiforme.

Polymer-drug conjugates (PDCs) provide possibilities for the development of multiresponsive drug delivery and release platforms utilized in cancer therapy. The delivery of Temozolomide (TMZ, a DNA methylation agent) by PDCs has been developed to improve TMZ stability under physiological conditions for the treatment of glioblastoma multiforme (GBM); however, with inefficient chemotherapeutic efficacy. In this work, we synthesized an amphiphilic triblock copolymer (P1-SNO) with four pendant functionalities, including (1) a TMZ intermediate (named MTIC) as a prodrug moiety, (2) a disulfide bond as a redox-responsive trigger to cage MTIC, (3) S-nitrosothiol as a light/heat-responsive donor of nitric oxide (NO), and (4) a poly(ethylene glycol) chain to enable self-assembly in aqueous media. P1-SNO was demonstrated to liberate MTIC in the presence of reduced glutathione and release gaseous NO upon exposure to light or heat. The in vitro results revealed a synergistic effect of released MTIC and NO on both TMZ-sensitive and TMZ-resistant GBM cells. The environment-responsive PDC system for codelivery of MTIC and NO is promising to overcome the efficacy issue in TMZ-based cancer therapy.

Reactive Reductive Species Participating Photodynamic Therapy for Cancer Treatment.

Although the development of oxidative photodynamic therapy (O-PDT) based on reactive oxygen species (ROS) has led to great progress in cancer treatment, tumor hypoxia, cellular adaptation and intrinsic antioxidant defenses are still obstacles at this stage. Fortunately, with the discovery and development of reactive reductive species (RRS) in the PDT process, reductive PDT (R-PDT) is receiving increasing research interest. R-PDT with oxygen-independence is an effective reduction therapy that promises excellent therapeutic efficacy in extremely hypoxic or even anaerobic environments. In the concept, we introduce representative strategies to boost the type-I photosensitizing pathway, and then focus on the most recent R-PDT involving hydrogen radical (H⋅) and the single electron transfer (SET) process.

Synthesis and DNA Cleavage Activity of Cationic Methylene-Blue-Backboned Polymers.

Methods of DNA cleavage have broad bioapplications in gene editing, disease treatment, and biosensor design. The traditional method for DNA cleavage is mainly through oxidation or hydrolysis mediated by small molecules or transition metal complexes. However, DNA cleavage by artificial nucleases using organic polymers has been rarely reported. Methylene blue has been extensively studied in the fields of biomedicine and biosensing due to its excellent singlet oxygen yield, redox properties, and good DNA affinity. Methylene blue mainly relies on light and oxygen for DNA cleavage, and the cutting rate is slow. Here, we synthesize cationic methylene-blue-backboned polymers (MBPs) that can bind DNA efficiently and induce DNA cleavage through free radical mechanisms in the absence of light and exogenous reagents, showing high-efficiency nuclease activity. In addition, MBPs with different structures showed selectivity for DNA cleavage, and the cleavage efficiency of the flexible structure was significantly higher than that of the rigid structure. Studies on the DNA cleavage mechanism have shown that the cleavage mechanism of MBPs is not through the common ROS-mediated oxidative cleavage pathway, but through the radical of MBP• inducing DNA cleavage. Meanwhile, MBPs can simulate topoisomerase I (Topo I)-mediated topological rearrangement of superhelical DNA. This work paved a way for the application of MBPs in the field of artificial nucleases.

Photoluminescent Properties and Mechanism of Novel Cyanine-Borondifluoride Curcuminoid Hybrids as Red-to-Near Infrared and Endoplasmic Reticulum-Targeting Dyes.

Synthesis-oriented design led us to the discovery of a series of novel cyanine-borondifluoride curcuminoid hybrids called Nanchang Red (NCR) dyes that overcome the intrinsic low synthetic yields of symmetrical cyanine-difluoroboronate (BF2 )-hybridized NIR dyes. The hybridization endows NCR dyes with high molar extinction coefficients, efficient red-to-NIR emission, and enlarged Stokes shifts. Quantum chemical calculations revealed that the asymmetrical layout of the three key electron-withdrawing and electron-donating fragments results in a special pattern of partial charge separation and inconsistent degrees of charge delocalization on their π-conjugated backbones. While the nature of the hemicyanine fragment exerts significant influence on the excitation modes of NCR dyes, the borondifluoride hemicurcuminoid fragment is the major contributor to the enlarged Stokes shifts. Cell imaging experiments illustrated that a subtle change in the N-heterocycle of the hemicyanine fragment has a remarkable effect on the subcellular localization of NCR dyes. Unlike other previously reported cyanine-BF2 hybridized dyes, which mainly target mitochondria, the benzothiazole and indole-based NCR dyes accumulate in both the endoplasmic reticulum (ER) and lipid droplets of HeLa cells, whereas the benzoxazole and quinoline-based NCR dyes stain the ER specifically.

Photocatalytic Generation of Hydrogen Radical (H⋠) with GSH for Photodynamic Therapy.

As a reactive hydrogen species, the hydrogen radical (H⋅) scarcely sees applications in tumor biological therapy due to the very limited bio-friendly sources of H⋅. In this work, we report that TAF can act as an organic photosensitizer as well as an efficient photocatalytic H⋅ generator with reduced glutathione (GSH) as a fuel. The photoactivation of TAF leads to cell death in two ways including triple amplification of oxidative stress via ferroptosis-apoptosis under normoxia and apoptosis through biological reductions under hypoxia. TAF presents excellent biosafety with ultrahigh photocytotoxicity index at an order of magnitude of 102 -103 on both normoxic and hypoxic cells. The in vitro data suggest that H⋅ therapy is promising to overcome the challenge of tumor hypoxia at low doses of both photocatalyst and light. In addition, the capability of near-infrared two-photon excitation would benefit broad biological applications.

Dithiol-Activated Bioorthogonal Chemistry for Endoplasmic Reticulum-Targeted Synergistic Chemophototherapy.

The controlled intracellular release of nitrite is still an unmet challenge due to the lack of bio-friendly donors, and the antitumor effect of nitrite is limited by its physiologically inert activity. Herein, we designed benzothiadiazole-based organic nitrite donors that are stable against bio-relevant species but selectively respond to dithiol species through SN Ar/intramolecular cyclization tandem reactions in the aqueous media. The bioorthogonal system was established to target the endoplasmic reticulum (ER) of liver cancer HepG2 cells. The nitrite and nonivamide were coupled to induce elevation of intracellular levels of calcium ions as well as reactive oxygen/nitrogen species, which resulted in ER stress and mitochondrial dysfunction. We demonstrated that a combination of photoactivation and "click to release" strategy could enhance antitumor effect in cellular level and show good potential for cancer precision therapy.

Intrinsic Mitochondrial Reactive Oxygen Species (ROS) Activate the In†Situ Synthesis of Trimethine Cyanines in Cancer Cells.

Environment-responsive in situ synthesis of molecular fluorescent dyes is challenging. Herein, we develop a photoextension strategy to make trimethine cyanines with decent conversion efficiency (up to 81 %) using 1-butyl 2,3,3-trimethyl 3H-indole derivatives as the sole precursors, and demonstrate a free radical mechanism. In the inducer-extension stage, free radicals and reactive oxygen species (ROS) were able to mediate similar reactions with no assistance of light. We explored a Mito-extension strategy to in situ synthesize trimethine cyanines in the living cells. The cellular ROS-dependence provided a foundation for preferential cyanine expression in cancer cells. Finally, we applied an iodized precursor as an intrinsic ROS-activated theranostic agent that integrated mitochondria-targeted cyanine synthesis, cell imaging and phototherapy.

Noncovalent Engineering of Apoferritin with a PEGylated [FeFe] Hydrogenase Mimic for In Situ Polymerization.

Apoferritin can act as a scaffold for functionalization in the inner and outer surfaces. However, traditional covalent modification methods have a risk of disrupting the structure and physicochemical properties of apoferritin. Herein, we report a method for designing versatile apoferritin-based nanosystems through noncovalent interaction between a PEGylated [FeFe]-hydrogenase mimic (FeFe-PEG-N3) and apoferritin. FeFe-PEG-N3 can be anchored into the threefold channels of apoferritin via program injection, at a number of ∼8 per protein. We also engineered apoferritin with an FeFe-PEG-N3/ATRP initiator conjugate for in situ and noninvasive atom transfer radical polymerization (ATRP) at the apoferritin surface. This "grafting-from" method for noncovalent apoferritin engineering has the advantages of simple preparation, good controllability, and high efficiency and affords opportunities for the construction of multifunctional apoferritin-based nanosystems for broad applications such as drug delivery and catalysis.

Visible Light and Glutathione Dually Responsive Delivery of a Polymer-Conjugated Temozolomide Intermediate for Glioblastoma Chemotherapy.

Temozolomide (TMZ) is a prodrug of 5-(3-methyltriazene-1-yl)imidazole-4-carboxamide (MTIC, short-lived) and used as a first-line therapy drug for glioblastoma multiforme (GBM). However, little progress has been made in regulating the kinetics of TMZ to MTIC degradation to improve the therapeutic effect, particularly in the case of TMZ-resistant GBM. In this work, we introduced a strategy to cage MTIC by N-acylation of the triazene moiety to boost the MTIC stability, designed a diblock copolymer-based MTIC prodrug installed with a disulfide linkage, and achieved self-assembled polymer micelles without the concern of MTIC leakage under physiological conditions. Polymer micelles could be induced to disassemble by stimuli factors such as glutathione (GSH) and visible light irradiation through thiol/sulfide exchange and homolytic sulfide scission mechanisms, which contributed to MTIC release in GSH-dependent and GSH-independent pathways. The in vitro results demonstrated that microenvironment-responsive polymeric micelles benefited the suppression of both TMZ-sensitive and TMZ-resistant GBM cells. The chemistry of polymer-MTIC prodrug provided a new option for TMZ-based glioma treatment.

One pot synthesis and self-assembly of methylene blue-backboned polymers.

Studies of methylene blue-backboned polymers (MBPs) are hindered by the limited availability of polymerization methods. Herein, we developed an oxidative polymerization method to produce MBPs. The polymerization is performed in aqueous medium, and is organic solvent-free, heavy metal-free, time-efficient (on a timescale of minutes), and does not need pre-formed methylene blue chromophores. The effects of the alkyl chains of the MBPs on the photophysical properties and self-assembly behavior (e.g., vesicles and nanorings) are significant, which highlights the possibility of controlling the MBP properties via rationally tailoring the functionality of the MBP monomers prior to polymerization. Importantly, the self-assembly structures can be predicted using the dissipative particle dynamics (DPD) simulation method.

Photoactivated In Situ Generation of Near Infrared Cyanines for Spatiotemporally Controlled Fluorescence Imaging in Living Cells.

Photoactivated trimerization of 2,3,3-trimethyl-3H-indole derivatives created near infrared fluorophore Cy5. The synthetic method is air-tolerant, photosensitizer free, metal free, and condensation agent free. Living cells make Cy5 on a time scale of minutes under white light irradiation at a low power intensity, with the monomer as the only exogenous agent. The new method is promising to find applications in cell studies for in situ spatiotemporally controlled fluorescence imaging in living cells.

Cascade Reactions by Nitric Oxide and Hydrogen Radical for Anti-Hypoxia Photodynamic Therapy Using an Activatable Photosensitizer.

Organelle-targeted activatable photosensitizers are attractive to improve the specificity and controllability of photodynamic therapy (PDT), however, they suffer from a big problem in the photoactivity under both normoxia and hypoxia due to the limited diversity of phototoxic species (mainly reactive oxygen species). Herein, by effectively photocaging a π-conjugated donor-acceptor (D-A) structure with an N-nitrosamine substituent, we established a unimolecular glutathione and light coactivatable photosensitizer, which achieved its high performance PDT effect by targeting mitochondria through both type I and type II (dual type) reactions as well as secondary radicals-participating reactions. Of peculiar interest, hydrogen radical (H•) was detected by electron spin resonance technique. The generation pathway of H• via reduction of proton and its role in type I reaction were discussed. We demonstrated that the synergistic effect of multiple reactive species originated from tandem cascade reactions comprising reduction of O2 by H• to form O2•-/HO2• and downstream reaction of O2•- with •NO to yield ONOO-. With a relatively large two-photon absorption cross section for photoexcitation in the near-infrared region (166 ± 22 GM at 800 nm) and fluorogenic property, the new photosensitizing system is very promising for broad biomedical applications, particularly low-light dose PDT, in both normoxic and hypoxic environments.

Biomimetic Polymer-Templated Copper Nanoparticles Stabilize a Temozolomide Intermediate for Chemotherapy against Glioblastoma Multiforme.

The short half-life of temozolomide (TMZ) limits its therapeutic effect on highly aggressive glioblastoma (GBM). Few approaches attempting to intervene the metabolic kinetics of TMZ are successful. Herein, we designed anionic copolymers via radical polymerization to prepare polymer-coated small copper nanoclusters, taking advantage of the role of pendent thymine groups as a template. The active and key intermediate of TMZ, typically called 3-methyl-(triazen-1-yl)imidazole-4-carboxamide (MTIC), was stabilized by copper under physiological (slightly alkaline) conditions, alleviating concerns associated with spontaneous drug degradation and nonspecific drug activation. Importantly, the complexes formed by MTIC and copper nanoclusters could catalyze the Fenton reaction to generate hydroxyl radicals and also respond to pH and glutathione to release therapeutic MTIC, which allows combined chemotherapy and chemodynamic therapy against GBM cells and paves a way for circumventing the complication of TMZ resistance.

Precise engineering of apoferritin through site-specific host-guest binding.

The surface engineering of the apoferritin shell by means of traditional chemical modifications usually suffers from site inaccuracy and insufficient conjugation. This report describes a non-covalent method for precise modulation of the apoferritin surface without alteration of amino acid residues. A bifunctional macromolecule, structured as azide-poly(ethylene glycol)-porphyrin (termed TPA), was synthesized. TPA was observed to be able to recognize and bind apoferritin in a 12 : 1 stoichiometry with a higher binding affinity than arachidonate, thanks to the specific host-guest interaction between the pocket of each two-fold channel and the porphyrin moiety. This method allows for site-specific engineering of the apoferritin surface with on demand functionalities and optimization of drug encapsulation.

Aggregation-Enhanced Two-Photon Absorption of Anionic Conjugated Polyelectrolytes.

The two-photon absorption properties of anionic poly(phenylene ethynylene)-type conjugated oligo- and polyelectrolytes are studied in molecularly dissolved and aggregated forms in aqueous solution. Several different polyvalent cations are used to induce aggregation. It is found that both materials in the aggregated form exhibit enhanced two-photon excited fluorescence (2PEF) and two-photon cross section (σ2) compared with the molecularly dissolved structures. The 2PEF and σ2 are unaffected by the nature of the polyvalent cation that is used to induce aggregation. The two-photon absorption cross section enhancement arises because of the increase in the difference dipole moment (Δµ) in the aggregates of the conjugated materials, an effect that is attributed to the introduction of charge transfer character into the aggregate excited state.

Temozolomide-Doxorubicin Conjugate as a Double Intercalating Agent and Delivery by Apoferritin for Glioblastoma Chemotherapy.

We designed a conjugated compound by coupling temozolomide (TMZ) with doxorubicin (DOX) via an acylhydrazone linkage as a potential prodrug used for glioblastoma multiforme (GBM) treatment. Viscosity and spectroscopic studies revealed that the drug conjugate could act as a nonclassical double intercalating agent. Although free TMZ is an inefficient DNA binder in comparison to DOX, the TMZ moiety interacted with DNA as an induced intercalator, arising from the synergistic effect of DOX moiety that mediated conformational changes of the DNA helix. Two binding modes were proposed to interpret the double intercalating effect of the drug conjugate on intra- and inter-DNA interactions that could cause DNA cross-linking and fibril aggregates. We also developed a delivery nanoplatform with a loading efficiency of 83% using copper-bound apoferritin as a nanocarrier. In sharp contrast to the short half-life of free TMZ, the nanocomposite was stable under physiological conditions without detectable drug decomposition after a 2 week storage, and drug release was activatable in the presence of glutathione at millimolar levels. The antitumor effect of the drug conjugate and nanocomposite against GBM cells was reported to demonstrate the potential therapeutic applications of double intercalating materials.

Folate-Modified Photoelectric Responsive Polymer Microarray as Bionic Artificial Retina to Restore Visual Function.

A high-optical-resolution artificial retina system that accurately communicates with the optic nerve is the main challenge in the modern biological science and bionic field. Here, we developed a bionic artificial retina possessing phototransduction "cells" with measurements even smaller than that of the neural cells. Using the technique of micrometer processing, we constructed a pyramid-shape periodic microarray of a photoreceptor. Each "sensing cell" took advantage of polythiophene derivative/fullerene derivative (PCBM) as a photoelectric converter. Because folic acid played an essential role in eye growth, we particularly modified the polythiophene derivatives with folic acid tags. Therefore, the artificial retina could enlarge the contact area and even recognize the nerve cells to improve the consequence of nerve stimulation. We implanted the artificial retina into blinded rats' eyes. Electrophysiological analysis revealed its recovery of photosensitive function 3 months after surgery. Our work provides an innovative idea for fabricating a high-resolution bionic artificial retina system. It shows great potential in artificial intelligence and biomedicine.

GSH and H2 O2 Co-Activatable Mitochondria-Targeted Photodynamic Therapy under Normoxia and Hypoxia.

Currently, photosensitizers (PSs) that are microenvironment responsive and hypoxia active are scarcely available and urgently desired for antitumor photodynamic therapy (PDT). Presented herein is the design of a redox stimuli activatable metal-free photosensitizer (aPS), also functioning as a pre-photosensitizer as it is converted to a PS by the mutual presence of glutathione (GSH) and hydrogen peroxide (H2 O2 ) with high specificity on a basis of domino reactions on the benzothiadiazole ring. Superior to traditional PSs, the activated aPS contributed to efficient generation of reactive oxygen species including singlet oxygen and superoxide ion through both type 1 and type 2 pathways, alleviating the aerobic requirement for PDT. Equipped with a triphenylphosphine ligand for mitochondria targeting, mito aPS showed excellent phototoxicity to tumor cells with low light fluence under both normoxic and hypoxic conditions, after activation by intracellular GSH and H2 O2 . The mito aPS was also compatible to near infrared PDT with two photon excitation (800 nm) for extensive bioapplications.

Apoferritin as a Carrier of Cu(II) Diethyldithiocarbamate and Biomedical Application for Glutathione-Responsive Combination Chemotherapy.

The antialcoholic drug disulfiram (DSF) has attractive biomedical interests for its anticancer effects, particularly in combination use with copper. CuET, the complex of copper and diethyldithiocarbamate (DTC) that is derived from disulfiram, exhibits high redox stability against glutathione, cysteine, and hydrogen peroxide, but appears reactive to ascorbic acid and superoxide ions. By virtue of the copper binding property, apoferritin is developed as a carrier of CuET to alleviate concerns of poor water solubility and nonspecific toxicity and shows superior stability and protection over human serum albumin in nanocomposite manipulation. CuET and doxorubicin are codelivered by apoferritin with excellent efficiency (>97%). The glutathione-responsive system demonstrates enhanced antitumor effect and potentials for combination tumor therapy.

Self-Assembly of Albumin and [FeFe]-Hydrogenase Mimics for Photocatalytic Hydrogen Evolution.

To imitate the protein microenvironment surrounding the Fe2S2 active site, we established a convenient method to prepare self-assembled ovalbumin (OVA) nanogels that noncovalently incorporated hydrophobic FeFe-H2ase mimic (FeFe-1) in situ with an excellent loading efficiency up to 53.9%. The OVA@FeFe-1 composite nanogels (CG) were applied to photocatalytic hydrogen evolution in aqueous solution and exhibited improved catalytic efficiency with a turnover number amplified over 15 times relative to free FeFe-1 catalysts. The enhanced activity was attributed to the enrichment of the FeFe-1 catalyst and changes of protein secondary structures and microenvironment.