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.

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.

Fabrication of Acidic pH-Cleavable Polymer for Anticancer Drug Delivery Using a Dual Functional Monomer.

The preparation of tumor acidic pH-cleavable polymers generally requires tedious postpolymerization modifications, leading to batch-to-batch variation and scale-up complexity. To develop a facile and universal strategy, we reported in this study design and successful synthesis of a dual functional monomer, a-OEGMA that bridges a methacrylate structure and oligo(ethylene glycol) (OEG) units via an acidic pH-cleavable acetal link. Therefore, a-OEGMA integrates (i) the merits of commercially available oligo(ethylene glycol) monomethyl ether methacrylate (OEGMA) monomer, i.e., hydrophilicity for extracellular stabilization of particulates and a polymerizable methacrylate for adopting controlled living radical polymerization (CLRP), and (ii) an acidic pH-cleavable acetal link for efficiently intracellular destabilization of polymeric carriers. To demonstrate the advantages of a-OEGMA ( Mn = 500 g/mol) relative to the commercially available OEGMA ( Mn = 300 g/mol) for drug delivery applications, we prepared both acidic pH-cleavable poly(ε-caprolactone)21- b-poly( a-OEGMA)11 (PCL21- b-P( a-OEGMA)11) and pH-insensitive analogues of PCL21- b-P(OEGMA)18 with an almost identical molecular weight (MW) of approximately 5.0 kDa for the hydrophilic blocks by a combination of ring-opening polymerization (ROP) of ε-CL and subsequent atom transfer radical polymerization (ATRP) of a-OEGMA or OEGMA. The pH-responsive micelles self-assembled from PCL21- b-P( a-OEGMA)11 showed sufficient salt stability, but efficient acidic pH-triggered aggregation that was confirmed by the DLS and TEM measurements as well as further characterizations of the products after degradation. In vitro drug release study revealed significantly promoted drug release at pH 5.0 relative to the release profile recorded at pH 7.4 due to the loss of colloidal stability and formation of micelle aggregates. The delivery efficacy evaluated by flow cytometry analyses and an in vitro cytotoxicity study in A549 cells further corroborated greater cellular uptake and cytotoxicity of Dox-loaded pH-sensitive micelles of PCL21- b-P( a-OEGMA)11 relative to the pH-insensitive analogues of PCL21- b-P(OEGMA)18. This study therefore presents a facile and robust means toward tumor acidic pH-responsive polymers as well as provides one solution to the trade-off between extracellular stability and intracellular high therapeutic efficacy of drug delivery systems using a novel monomer of a-OEGMA with dual functionalities.

Recent advances for enhanced photodynamic therapy: from new mechanisms to innovative strategies.

Photodynamic therapy (PDT) has been developed as a potential cancer treatment approach owing to its non-invasiveness, spatiotemporal control and limited side effects. Currently, great efforts have been made to improve the PDT effect in terms of safety and efficiency. In this review, we highlight recent advances in innovative strategies for enhanced PDT, including (1) the development of novel radicals, (2) design of activatable photosensitizers based on the TME and light, and (3) photocatalytic NADH oxidation to damage the mitochondrial electron transport chain. Additionally, the new mechanisms for PDT are also presented as an inspiration for the design of novel PSs. Finally, we discuss the current challenges and future prospects in the clinical practice of these innovative strategies. It is hoped that this review will provide a new angle for understanding the relationship between the intratumoural redox environment and PDT mechanisms, and new ideas for the future development of smart PDT systems.

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.

One-pot fabrication of dual-redox sensitive, stabilized supramolecular nanocontainers for potential programmable drug release using a multifunctional cyclodextrin unit.

Facile engineering of ß-cyclodextrin (ß-CD)-based supramolecular nanocontainers with simultaneous enhanced extracellular stability and efficient intracellular biosignals-triggered destabilization generally suffers from multistep synthesis and tedious purification process, thus remains a significant challenge for the scale-up production and clinical translation of ß-CD-based supramolecular nanomedicine. To address these issues, we reported in this study a one-pot preparation of dual-redox sensitive, stabilized supramolecular nanocontainers for potential programmable drug release by self-crosslinking of a multifunctional ß-CD unit that integrates a host cavity for oxidation-mediated reversible complexation with ferrocence (Fc) guest molecule and lipoic acids (LAs)-decorated primary and secondary faces for reversible in-situ crosslinking by the reducible disulfide links. The resulting doxorubicin (DOX)-loaded nanoparticles showed, on one hand, enhanced colloidal stability and high DOX loading capacity with a drug loading content (DLC) of approximately 11.3% due to the crosslinked structure, and on the other hand, a programmable destruction of the supramolecular micelles triggered by a simultaneous adoption of intracellular glutathione (GSH) and reactive oxygen species (ROS) toward a complete structural destruction for promoted drug release with enhanced therapeutic efficiency. Notably, an optimized DOX-loaded micelle formation, DOX@CL P1 showed greater cytotoxicity with an IC50 of 2.94 ± 0.25 µg/mL than free DOX (6.00 ± 0.56 µg/mL) in Bel-7402 cancer liver cells, but a significantly reduced side effect relative to free DOX in L02 normal liver cells. In vivo animal study in Bel-7402 tumor-bearing BALB/c mice further confirmed prolonger elimination half-life time, efficient tumor accumulation, enhanced therapeutic efficiency and compromised systemic toxicity of this micelle construct. Therefore the multifunctional CD unit developed in this study offers an extremely straightforward and robust strategy with respect to dual-redox responsive, stabilized supramolecular nanocontainers with potential programmable controlled release properties for clinical translations.

Synthesis of cyclic graft polymeric prodrugs with heterogeneous grafts of hydrophilic OEG and reducibly conjugated CPT for controlled release.

Fabrication of cyclic graft (cg) copolymer-based polymeric prodrugs by conjugation of drug molecules to cg copolymers via a dynamic covalent bond capable of responding to biorelevant signals integrates simultaneously the merits of cg copolymers and polymeric prodrugs for enhanced stability of nanocarriers and precise modulation of drug release kinetics. To completely eliminate the compromised drug conjugation efficiency due to the steric hindrance of hydrophilic grafts, it will be useful to develop cg polymeric prodrugs with heterogeneous grafts composed of hydrophilic polymers and drug species, respectively. For this purpose, we reported in this study the synthesis of cyclic graft polymeric prodrugs with heterogeneous grafts of hydrophilic oligo (ethylene glycol) (OEG) and reducibly conjugated camptothecin (CPT), cg-poly(oligo(ethylene glycol) monomethyl ether methacrylate)-b-poly((2-hydroxyethyl methacrylate)-disulfide link-camptothecin) (cg-P(OEGMA)-b-P(HEMA-SS-CPT), cg-prodrugs), via an integrated strategy of a previously reported diblock copolymer-based template and post-polymerization intermolecular click conjugation of a reducible CPT prodrug. The micelles self-assembled from cg-prodrugs on one hand had sufficient salt stability due to the branched cg structure, and on the other hand showed a reduction-triggered cleavage of the disulfide link for a promoted CPT release. Most importantly, we uncovered two interesting phenomena of the cg-based polymeric prodrugs as delivery vehicles: (i) the dimensions of both self-assemblies formed by the cg and bottlegraft (bg) polymers depend substantially on the molecular size of the cg and bg polymers likely due to the steric hindrance of the grafted structures of the cg and bg molecules and relatively low aggregation number of the self-assembled structures, and (ii) cg-prodrug-based micelles exhibited greater in vitro cytotoxicity against cancer cells despite the lower drug loading content (DLC) than the bg-based analogues, which results primarily from the faster reduction-triggered degradation and drug release as well as the greater cellular uptake efficiency of the former micelle prodrugs. Taken together, the developed cg-prodrugs provide great potential for chemotherapy, and the aforementioned interesting results will definitely inspire more upcoming studies on the future design and development of novel cg polymers for biomedical applications.

Synthesis of Thermosensitive Conjugated Triblock Copolymers by Sequential Click Couplings for Drug Delivery and Cell Imaging.

The elegant integration of an excellent light-emitting segment and a biorelevant signal-responsive moiety could generate advanced polymeric delivery systems with simultaneously favorable diagnostic and therapeutic functions with respect to cancer theranostics. Although polymeric delivery systems based on fluorescent polyfluorene (PF) or thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm) have been extensively developed, the preparation of a ternary polymer formulation composed of a PF block, a PNIPAAm sequence, and a hydrophilic moiety remains rarely explored likely because of the difficulty in integrating different synthesis strategies for polymer synthesis. To this end, herein we reported the design and controlled synthesis of a PF- and PNIPAAm-based amphiphilic triblock copolymer, PF11-b-PNIPAAm120-b-poly(oligo(ethylene glycol) monomethyl ether methacrylate)17 (PF11-b-PNIPAAm120-b-POEGMA17), with a well-defined structure by a strategy of sequential click couplings between Suzuki-coupling-generated PF and atom-transfer radical polymerization (ATRP)-produced PNIPAAm and POEGMA. The as-prepared triblock copolymers can self-assemble into micelles with a core-shell-corona (CSC) structure that is composed of an inner hydrophobic core of the PF moiety for fluorescent tracking and drug encapsulation, a thermosensitive middle shell of PNIPAAm block for thermomodulated drug loading and release, and a hydrophilic outer corona of the POEGMA segment for micelle stabilization. Interestingly, the doxorubicin (DOX)-loaded micelles prepared at 25 °C had a greater drug loading capacity than the analogues fabricated at 37 °C due to the better stability of the former formulation, leading to its higher in vitro cytotoxicity in HeLa cells. Together with the integration of a localized hyperthermia-triggered drug release profile and efficiently intracellular trafficking of the nanocarriers by monitoring the fluorescence of the PF moiety, this formulation demonstrates a great potential for cancer theranostics.

Synthesis of stimuli-responsive nanosized ring-like colloids and cyclic polymers via a dual-template approach.

Ring-like particles have received considerable attention due to their unique interior cavity and properties. However, the preparation of stimuli-responsive nanosized rings with internal size smaller than 100 nm remains unexplored likely due to the challenges encountered in their synthesis. The successful fulfillment of this target will not only significantly enrich the family of ring-like nanoparticles but also build a connection that bridges ring-like nanoparticles and cyclic polymers. For this purpose, we report in this study a controlled synthesis of stimuli-responsive ring-like colloids and cyclic polymers using both star-shaped polymers and ß-cyclodextrin (ß-CD) as the dual templates. The first template comprising star-shaped polymers generated a ring-like structure and adoption of ß-CD as the second template further restricted the ring thickness to the height of a ß-CD, leading to the generation of stimuli-responsive nanosized ring-like colloids with ring thickness less than 1 nm, which shifted the ring-like structure to cyclic polymers with reversible cross-linked disulfide bridges. The reported "dual-template" approach is thus a valuable alternative to the current synthetic strategies toward stimuli-responsive ring-like colloids and cyclic polymers.

Fabrication of supramolecular star-shaped amphiphilic copolymers for ROS-triggered drug release.

Star-shaped copolymers with branched structures can form unimolecular micelles with better stability than the micelles self-assembled from conventional linear copolymers. However, the synthesis of star-shaped copolymers with precisely controlled degree of branching (DB) suffers from complicated sequential polymerizations and multi-step purification procedures, as well as repeated optimizations of polymer compositions. The use of a supramolecular host-guest pair as the block junction would significantly simplify the preparation. Moreover, the star-shaped copolymer-based unimolecular micelle provides an elegant solution to the tradeoff between extracellular stability and intracellular high therapeutic efficacy if the association/dissociation of the supramolecular host-guest joint can be triggered by the biologically relevant stimuli. For this purpose, in this study, a panel of supramolecular star-shaped amphiphilic block copolymers with 9, 12, and 18 arms were designed and fabricated by host-guest complexations between the ring-opening polymerization (ROP)-synthesized star-shaped poly(ε-caprolactone) (PCL) with 3, 4, and 6 arms end-capped with ferrocene (Fc) (PCL-Fc) and the atom transfer radical polymerization (ATRP)-produced 3-arm poly(oligo ethylene glycol) methacrylates (POEGMA) with different degrees of polymerization (DPs) of 24, 30, 47 initiated by ß-cyclodextrin (ß-CD) (3Br-ß-CD-POEGMA). The effect of DB and polymer composition on the self-assembled properties of the five star-shaped copolymers was investigated by dynamic light scattering (DLS), transmission electron microscopy (TEM), and fluorescence spectrometery. Interestingly, the micelles self-assembled from 12-arm star-shaped copolymers exhibited greater stability than the 9- and 18-arm formulations. The potential of the resulting supramolecular star-shaped amphiphilic copolymers as drug carriers was evaluated by an in vitro drug release study, which confirmed the ROS-triggered accelerated drug release from the doxorubicin (DOX)-loaded supramolecular star-shaped micelles due to the oxidation-induced dissociation of ß-CD/Fc pair and the consequent loss of the colloidal stability of the star-shaped micelles. Studies of the delivery efficacy by an in vitro cytotoxicity study further indicated that higher DBs and longer hydrophilic arm compromised the therapeutic efficacy of the DOX-loaded supramolecular star-shaped micelles, resulting in significantly reduced cytotoxicity, as measured by increased IC50 value. Overall, our results revealed that the screening of hydrophilic block by DB and MW for an optimized star-shaped copolymer should balance the stability versus therapeutic efficacy tradeoff for a comprehensive consideration. Therefore, the 12-arm star-shaped copolymer with POEGMA30 is the best formulation tested.

Fabrication of Conjugated Amphiphilic Triblock Copolymer for Drug Delivery and Fluorescence Cell Imaging.

An elegant integration of light-emitting segments into the structure of polymeric delivery systems endows the resulting self-assembled nanovehicles with the diagnostic ability toward an enhanced therapeutic efficiency. A variety of polyfluorene (PF)-based binary delivery systems were designed and developed successfully, but PF-based ternary formulations remain rarely explored, likely due to the synthetic challenge. To develop a universal synthesis strategy toward linear conjugated amphiphilic triblock copolymer for cancer theranostics, herein we focused on the functionalization of the PF terminus for further chain extension and prepared well-defined PF-based amphiphilic triblock copolymers, PF-b-poly(ε-caprolactone)-b-poly(oligo(ethylene glycol) monomethyl ether methacrylate) (PF-b-PCL-b-POEGMA), by integrated state-of-the-art polymer chemistry techniques, including Suzuki reaction, ring-opening polymerization, atom transfer radical polymerization, and click coupling. The resulting conjugated amphiphilic triblock copolymers can self-assembe into core-shell-corona (CSC) micelles with PF block constructing the inner hydrophobic core for fluorescent tracking, PCL segment forming the hydrophobic middle shell for drug encapsulation, and POEGMA moiety building the hydrophilic outer corona for particulate stabilization. Interestingly, the CSC micelles with hydrophobic PCL middle layer show a greater drug loading capacity as well as a higher fluorescence quantum yield (Φ) relative to the core-shell micelles self-assembled from the control of PF-b-POEGMA diblock copolymers without PCL sequence due to having more hydrophobic spaces and better separation of PF sequence provided simultaneously by the PCL central block. The efficient cellular uptake of the anticancer drug doxorubicin-loaded CSC micelles together with the in vitro cytotoxicity against the HeLa cells makes the conjugated amphiphilic triblock copolymers developed herein a promising platform for simultaneous cell image and drug delivery, thus offering great potential for cancer theranostics.

Fabrication of biocleavable shell cross-linked hybrid micelles for controlled drug release using a reducible silica monomer.

We report in this communication the first preparation of triblock copolymer-based biocleavable shell cross-linked (SCL) hybrid micelles using a reducible silica monomer that integrates a polymerizable methacrylate structure and in situ cross-linkers of silica precursors via a disulfide bond. The monomer developed herein offers a highly straightforward and robust strategy toward bioreducible silica-based hybrid nanoparticles for controlled drug release.