Cationized orthogonal triad as a photosensitizer with enhanced synergistic antimicrobial activity.

Single-molecule-based synergistic phototherapy holds great potential for antimicrobial treatment. Herein, we report an orthogonal molecular cationization strategy to improve the reactive oxygen species (ROS) and hyperthermia generation of heptamethine cyanine (Cy7) for photodynamic and photothermal treatments of bacterial infections. Cationic pyridine (Py) is introduced at the meso­position of the asymmetric Cy7 with intramolecular charge transfer (ICT) to construct an atypical electron-transfer triad, which reduces ΔES1-S0, circumvents rapid charge recombination, and simultaneously enhances intersystem crossing (ISC) based on spin-orbit charge-transfer ISC (SOCT-ISC) mechanism. This unique molecular construction produces anti-Stokes luminescence (ASL) because the rotatable CN bond enriched in high vibrational-rotational energy levels improves hot-band absorption (HBA) efficiency. The obtained triad exhibits higher singlet oxygen quantum yield and photothermal conversion efficiency compared to indocyanine green (ICG) under irradiation above 800 nm. Cationization with Py enables the triad to target bacteria via intense electrostatic attractions, as well as biocidal property against a broad spectrum of bacteria in the dark. Moreover, the triad under irradiation can enhance biofilm eradication performance in vitro and statistically improve healing efficacy of MRSA-infected wound in mice. Thus, this work provides a simple but effective strategy to design small-molecule photosensitizers for synergistic phototherapy of bacterial infections. STATEMENT OF SIGNIFICANCE: We developed an orthogonal molecular cationization strategy to enhance the reactive oxygen species and thermal effects of heptamethine cyanine (Cy7) for photodynamic and photothermal treatments of bacterial infections. Specifically, cationic pyridine (Py) was introduced at the meso­position of the asymmetric Cy7 to construct an atypical electron-transfer triad, which reduced ΔES1-S0, circumvented rapid charge recombination, and simultaneously enhanced intersystem crossing (ISC). This triad, with a rotatable CN bond, produced anti-Stokes luminescence due to hot-band absorption. The triad enhanced antimicrobial performance and statistically improved the healing efficacy of MRSA-infected wounds in mice. This site-specific cationization strategy may provide insights into the design of small molecule-based photosensitizers for synergistic phototherapy of bacterial infections.

Supramolecular prodrug-like nanotheranostics with dynamic and activatable nature for synergistic photothermal immunotherapy of metastatic cancer.

Synergistic photothermal immunotherapy has attracted widespread attention due to the mutually reinforcing therapeutic effects on primary and metastatic tumors. However, the lack of clinical approval nanomedicines for spatial, temporal, and dosage control of drug co-administration underscores the challenges facing this field. Here, a photothermal agent (Cy7-TCF) and an immune checkpoint blocker (NLG919) are conjugated via disulfide bond to construct a tumor-specific small molecule prodrug (Cy7-TCF-SS-NLG), which self-assembles into prodrug-like nano-assemblies (PNAs) that are self-delivering and self-formulating. In tumor cells, over-produced GSH cleaves disulfide bonds to release Cy7-TCF-OH, which re-assembles into nanoparticles to enhance photothermal conversion while generate reactive oxygen species (ROSs) upon laser irradiation, and then binds to endogenous albumin to activate near-infrared fluorescence, enabling multimodal imaging-guided phototherapy for primary tumor ablation and subsequent release of tumor-associated antigens (TAAs). These TAAs, in combination with the co-released NLG919, effectively activated effector T cells and suppressed Tregs, thereby boosting antitumor immunity to prevent tumor metastasis. This work provides a simple yet effective strategy that integrates the supramolecular dynamics and reversibility with stimuli-responsive covalent bonding to design a simple small molecule with synergistic multimodal imaging-guided phototherapy and immunotherapy cascades for cancer treatment with high clinical value.

Ligand engineering of Au44 nanoclusters for NIR-II luminescent and photoacoustic imaging-guided cancer photothermal therapy.

Developing a high-performance noninvasive probe for precise cancer theranostics is very challenging but urgently required. Herein, a novel Au nanoclusters (NCs)-based probe was designed for cancer theranostics via ligand engineering by conjugating photoluminescent (PL) Au44 NCs in the second near-infrared window (NIR-II, 1000-1700 nm) with aromatic photoacoustic (PA)/photothermal molecules through click chemistry. This design bypasses the incompatibility dilemma between photoluminescence (PL) attributes and PA/photothermal properties because the rigidity of the PA/photothermal molecules can lead to aggregation-induced emission (AIE) of the Au(i)-ligand shell of the Au NCs by constraining their nonradiative relaxation. Benefiting from strong NIR-II PL with emissions at 1080 and 1240 nm, high photothermal conversion efficiency (65.12%), low cytotoxicity, appropriate renal clearance, and enhanced permeability and retention (EPR) effect, the as-designed Au NC-based theranostic probe achieves ultradeep NIR-II PL/PA imaging-guided cancer photothermal therapy (PTT). Remarkably, 16 days after photothermal treatment guided by NIR-II PL/PA imaging, mice were all healed without tumor recurrence, while the average life span of the mice in the control groups was only 17-21 days. This study is interesting because it provides a paradigm for designing a metal NC-based theranostics probe, and it may add fundamentally and methodologically to noninvasive imaging-guided disease therapy.

Zwitterionic rhodamine-CPT prodrug nanoparticles with GSH/H2O2 responsiveness for cancer theranostics.

Rationale: Fluorescently traceable prodrugs, which can monitor their biodistribution in vivo and track the kinetics of drug delivery in living cells, are promising for constructing theranostic medicines. However, due to their charge and hydrophobicity, most of the fluorescently traceable prodrugs exhibit high protein binding and non-specific tissue retention affecting in vivo distribution and toxicity, with high background signals. Methods: Herein, the zwitterionic rhodamine (RhB) and camptothecin (CPT) were bridged with a disulfide bond to construct a tumorous heterogeneity-activatable prodrug (RhB-SS-CPT). The interaction of zwitterionic RhB-SS-CPT with proteins was detected by UV and fluorescence spectroscopy, and further demonstrated by molecular docking studies. Then, intracellular tracking and cytotoxicity of RhB-SS-CPT were determined in tumor and normal cells. Finally, the in vivo biodistribution, pharmacokinetics, and anticancer efficacy of RhB-SS-CPT were evaluated in a mouse animal model. Results: The tumorous heterogeneity-activatable RhB-SS-CPT prodrug can self-assemble into stable nanoparticles in water based on its amphiphilic structure. Particularly, the zwitterionic prodrug nanoparticles reduce the non-specific binding to generate a low background signal for better identification of cancerous lesions, achieve rapid internalization into cancer cells, selectively release bioactive CPT as a cytotoxic agent in response to high levels of GSH and H2O2, and exhibit high fluorescence that contributes to the visual chemotherapy modality. In addition, the RhB-SS-CPT prodrug nanoparticles show longer circulation time and better antitumor activity than free CPT in vivo. Interestingly, the zwitterionic nature allows RhB-SS-CPT to be excreted through the renal route, with fewer side effects. Conclusions: Zwitterionic features and responsive linkers are important considerations for constructing potent prodrugs, which provide some useful insights to design the next-generation of theranostic prodrugs for cancer.

Programmable multistage small-molecule nano-photosensitizer for multimodal imaging-guided photothermal therapy.

Photothermal therapy has become a promising approach as precision medicine to allow spatial control of therapeutic effect only in the site of interest. However, the full potential of PTT has not been realized due to the lack of simple photosensitizers (PSs) that can overcome multistage biological barriers and improve theranostic efficiency. Here, we develop a small molecule-based PS to enhance tumor-specific PTT by programming multistage transport and activation properties in molecular architecture. This PS can self-assemble into stable nanoparticles that accumulate passively in tumor, and then actively internalize through ligand-mediated endocytosis. Subsequently, the programmable degradable linkers are selectively cleaved, enabling size shrinkage for better tumor penetration, binding albumin to enhance the near-infrared fluorescence for low-background imaging, and activating photothermal conversion for tumor suppression. The self-delivery process can be programmed, representing the first multistage small-molecule nano-photosensitizer that overcomes multiple biological barriers and improves the PTT index of tumor. STATEMENT OF SIGNIFICANCE: Photothermal therapy has become a promising approach as precision medicine, but has not been realized due to the lack of simple photosensitizers that can overcome multistage biological barriers and improve theranostic efficiency. In this contribution, we solve this dilemma by developing a small molecule-based photosensitizer by programming multistage transport and activation properties in molecular architecture, which could self-assemble into stable nanoparticles that accumulate passively in tumor, and actively internalized through ligand-mediated endocytosis. Subsequently, the programmable activation by ROS triggered size reduction for tumor penetration and minimized the phototoxicity to normal tissue. The activatable fluorescence and photothermal properties made the photosensitizer intrinsically suitable for multimodal imaging-guided PTT, providing a promising supramolecular nanomedicine towards tumor precise diagnosis and therapy.

Lighting up Self-Quenching Nanoaggregates with Protein Corona for Simultaneous Intraoperative Imaging and Photothermal Theranostics of Metastatic Cancer.

Near-infrared (NIR) photothermal transduction agents (PTAs) with large rigid π-extended and planar structures are prone to aggregate in a physiological environment where their emission is often quenched due to the strong intermolecular dipole-dipole or π-π interactions. This aggregation-caused quenching effect greatly impedes their applications in image-guided photothermal theranostics. Herein, we made an interesting finding that engineering a bioinspired protein corona (PC), once thermodynamically stabilized in preferred orientations on PTA nanoaggregates, can produce brilliant NIR fluorescence with a high quantum yield (∼6.2%) without compromising their photothermal properties. Both experimental data and computational modeling suggest that the mechanism of fluorescence enhancement is due to the high-affinity binding of nano-sized PTA to albumin, which regulates the molecular conformation and aggregation state of PTA. High spatial and temporal resolution imaging of albumin PC-coated PTA aggregates enables image-guided photothermal therapy for cancer cells in sentinel lymph nodes to remarkably inhibit pulmonary metastasis. Such a treatment combined with the surgical removal of the primary tumor can prolong animal survival, which is a promising candidate for clinical applications in the treatment of advanced metastatic cancers.

Reducing thermal damage to adjacent normal tissue with dual thermo-responsive polymer via thermo-induced phase transition for precise photothermal theranosis.

Photothermal therapy has been extensively studied to improve the light-to-heat efficiency for tumor ablation, but could cause severe damage to adjacent healthy tissue due to the thermal transfer, the random distribution of photothermal agents (PTAs), or combination hereof. Herein, we solve this dilemma with a material design strategy to develop a P(AAm-co-AN)-b-P(NIPAM-co-DMAa)-b-P(AAm-co-AN) ABA triblock copolymer by RAFT polymerization, which exhibits both UCST and LCST dual thermo-responsive behaviors in aqueous solution. The P(AAm-co-AN) block with appropriate AN content allows to finely tune its UCST to ∼ 43°C, which can effectively co-assemble with camptothecin (CPT) and Cy7-TCF, a near-infrared (NIR) PTA, realizing the photo-activated "on-demand" release of CPT and Cy7-TCF. The LCST of P(NIPAM-co-DMAa) segment is adjusted to ∼ 53°C by varying DMAa content, enabling an irreversible sol-to-gel transition. The heat transfer in hydrogel and heat dissipation at the interface of hydrogel-adjacent tissue are limited, resulting in selectively cell killing in tumor, with little hyperthermia in adjacent tissues. Moreover, the hydrogel continues to release CPT to enhance the synergistic efficacy of PTT with chemotherapy. These results suggest that dual thermo-responsive polymer can contribute PTT with high selectivity and negligible side effects for precise medicine. STATEMENT OF SIGNIFICANCE: Photothermal therapy exploits the susceptibility of tumor cells toward external light-induced hyperthermia, but can cause severe damage to adjacent healthy tissue due to thermal transfer, random distribution of photothermal agents (PTAs), or combination hereof. Here, we solve this dilemma by developing a P(AAm-co-AN)-b-P(NIPAM-co-DMAa)-b-P(AAm-co-AN) triblock copolymer with UCST and LCST dual thermo-responsive behaviors, realizing the sequential micelle-unimer-hydrogel phase transitions. The polymer can effectively encapsulate PTA/drug, achieve long systemic circulation, accumulate in tumor through EPR effect, regulate drug release by controlling tumor temperature above UCST via irradiation, and finally exhibit a sol-gel transition, eradicating the heat transfer to adjacent tissue. This represents a practicable strategy to guide the design of next-generation polymeric vector that can contribute PTT with negligible side effects.

Tumor Customized 2D Supramolecular Nanodiscs for Ultralong Tumor Retention and Precise Photothermal Therapy of Highly Heterogeneous Cancers.

Target therapy for highly heterogeneous cancers represents a major clinical challenge due to the lack of recurrent therapeutic targets identified in these tumors. Herein, the authors report a tumor-customized targeting photothermal therapy (PTT) strategy for highly heterogeneous cancers, by which 2D supramolecular self-assembled nanodiscs are modified with tumor-specific binding peptides identified by phage display techniques. Taking osteosarcoma (OS) as a model heterogeneous cancer, an OS targeting peptide (OTP) is first selected after biopanning and is demonstrated to successfully bind to this heterogeneous cancer cells/tissues. Successful conjugation of OTP to heptamethine cyanine (Cy7)-based 2D nanodiscs Cy7-TCF (2-dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran,TCF) enables the 2D nanodiscs to specifically target the heterogeneous tumor. Notably, a single dose injection of this targeted nanodisc (T-ND) not only effectively induces enhanced photothermal tumor ablation under near-infrared light, but also exhibits sevenfold increase of tumor retention time (more than 24 days) compared to generic nanomedicine. Thus, the authors' findings suggest that the combination of phage display-based affinity peptides selection and 2D supramolecular nanodiscs leads to the development of a platform technology for highly heterogeneous cancers precise therapy, offering specific tumor targeting, ultralong tumor retention, and precise PTT.

A Simple Small Molecule with Synergistic Passive and Active Dual-Targeting Effects for Imaging-Guided Photothermal Cancer Therapy.

Photothermal therapy allows spatiotemporal control of the treatment effect only at the site of the disease and provides promising opportunities for imaging-guided precision therapy. However, the development of photothermal transduction agents (PTAs) for tumor-specific accumulation and precision imaging, avoiding toxicity to the surrounding healthy tissue, is still challenging. Herein, a cyclooxygenase-2-specific small-organic-molecule-based PTA (Cy7-TCF-IMC) is developed, which can self-assemble into nanosaucers having unique photothermal and photoacoustic properties. Specifically, the self-assembling nature of Cy7-TCF-IMC affords preferential accumulation in tumors arising from synergistic passive enhanced permeability and retention effects and active targeting for precision theranostics. Antitumor therapy results show that these Cy7-TCF-IMC nanosaucers are highly photoacoustic imaging-guided PTAs for tumor ablation. These findings suggest the self-assembled Cy7-TCF-IMC nanosaucer represents a new paradigm as a single-component supramolecular medicine that can synergistically optimize passive and active targeting, thereby improving the therapeutic index of cancer and future clinical outcomes.

Esterase-Responsive Polypeptide Vesicles as Fast-Response and Sustained-Release Nanocompartments for Fibroblast-Exempt Drug Delivery.

Enzyme-responsive polypeptide vesicles have attracted considerable attention for precision theranostics because of their biocompatibility, biodegradability, and unique secondary conformation transition triggered by the catalytic actions of enzymes. These promising potentials of polypeptide vesicles could be limited in a drug delivery system by the very slow enzyme diffusion rate into vesicles that could reduce the efficacy of the drug. On the other hand, stimuli-responsive polymeric vesicles that respond to stimuli can undergo microstructure destruction for the burst release of drugs, which would penetrate through the membrane of dead cells and the tumor extracellular matrix, inducing acute toxicity to neighboring cells. Here, we designed amphiphilic PEG-polypeptide copolymers containing esterase-labile carbamate-caged primary amines. It was found that the diblock can self-assemble into vesicular structures. Esterase-triggered self-immolative decaging reactions could quickly release the primary amine moiety of monomers that can undergo an amidation reaction for transition of the bilayer of vesicles from hydrophobic to partially hydrophilic. This esterase-responsive process retains the nanostructure of vesicles but permeabilizes the vesicle membrane, which can afford the sustained release of encapsulating drugs. These esterase-responsive polypeptide vesicles mediate selective cytotoxicity in cancer cells with high esterase expression over normal fibroblasts with low esterase, enabling the potent anticancer chemotherapy with minimized side effects.

Intriguing H-Aggregates of Heptamethine Cyanine for Imaging-Guided Photothermal Cancer Therapy.

Organic small-molecule-based photothermal agents such as cyanine dyes have received increasing attention in developing novel cancer therapies with potential clinical utility but suffer from poor stability, low photothermal efficiency, and limited accumulation at tumor sites in molecular forms. Self-assembly of small-molecule dyes into supramolecular assemblies may address these concerns by controlling the molecular organization of dye monomers to form structures of a higher order. Among them, H-aggregates of dyes favor face-to-face contacts with strongly overlapping areas, which always have a negative connotation to exhibit low or no fluorescence in most cases but may emanate energy in nonradiative forms such as heat for photothermal cancer therapy applications. Here, the synergistic self-assembly of cyanine dyes into H-aggregates is developed as a new supramolecular strategy to fabricate small-molecule-based photothermal nanomaterials. Compared to the free cyanine dyes, the H-aggregates assembled from pyrene or tetraphenylethene (TPE) conjugating cyanine exhibit the expected absorption spectral blue shift and fluorescence self-quenching but unique photothermal properties. Remarkably, the obtained H-aggregates are saucer-shaped nanoparticles that exhibit passive tumor-targeting properties to induce imaging-guided photothermal tumor ablation under irradiation. This supramolecular strategy presented herein may open up new opportunities for constructing next-generation small-molecule-based self-assembly nanomaterials for PTT cancer therapy in clinics.

Engineering a "PEG-g-PEI/DNA nanoparticle-in- PLGA microsphere" hybrid controlled release system to enhance immunogenicity of DNA vaccine.

Controlled release strategies of DNA vaccine hold promise for the design of in vivo vaccination platforms, yet the formulation and sustained delivery still pose a substantial challenge. In this study, we developed a novel hybrid dual-particulate delivery system, nanoparticle-in-microsphere (NIM), to integrate the advantages of nano-sized polymer/DNA polyplex with the sustained-release microsphere for DNA vaccine delivery. The nano-sized cores, consisting of polyethylene glycol-graft-polyethylenimine (PEG-g-PEI)/DNA polyplexes, were formulated into PLGA microspheres using a solid-in-oil-in-water (S/O/W) emulsion. The PEG block was used as stabilizing excipient to make DNA soluble and stable in organic solvent to prevent the inactivation of DNA at aqueous-organic interface during encapsulation. The fashion of DNA in dry solid state greatly increased the encapsulation efficiency of DNA in NIMs. This new formulation exhibited a burst release less than 15% and then sustain release close to zero-order kinetics in physiological environment. In addition, the microspheres showed pH-sensitivity and degraded faster in lysosomal compartments, which contributed to the accelerated intracellular release kinetics of DNA. Finally, intramuscular injection of NIMs encoding HIV proteins elicited distinct humoral and cellular immune response in mice at low dose. These results thus may aid NIM-based vaccination towards more extensive clinical evaluations.

Supramolecular Nanodiscs Self-Assembled from Non-Ionic Heptamethine Cyanine for Imaging-Guided Cancer Photothermal Therapy.

Supramolecular nanomedicines, which use supramolecular design to improve the precision and effectiveness of pharmaceutical practice and optimize pharmacokinetic profiles, have gathered momentum to battle cancer and other incurable diseases, for which traditional small-molecular and macromolecular drugs are less effective. However, the lack of clinical approval of supramolecular assembly-based medicine underscores the challenges facing this field. A 2D nanodisc-based supramolecular structure is formed by a non-ionic heptamethine cyanine (Cy7) dye, which generates fluorescence self-quenching but unique photothermal and photoacoustic properties. These Cy7-based supramolecular nanodiscs exhibit passive tumor-targeting properties to not only visualize the tumor by near-infrared fluorescence imaging and photoacoustic tomography but also induce photothermal tumor ablation under irradiation. Due to the nature of organic small molecule, they induce undetectable acute toxicity in mice and can be eliminated by the liver without extrahepatic metabolism. These findings suggest that the self-assembling cyanine discs represent a new paradigm in drug delivery as single-component supramolecular nanomedicines that are self-delivering and self-formulating, and provide a platform technology for synergistic clinical cancer imaging and therapy.

A water-soluble two-dimensional supramolecular organic framework with aggregation-induced emission for DNA affinity and live-cell imaging.

A water-soluble two-dimensional supramolecular organic framework (2D SOF) was prepared via self-assembly of cucurbit[8]uril (CB[8]) and a three-arm flat linker molecule, which contains a benzene ring as the core and three Brooker's merocyanine (BM) analogs as arms. The strong host-guest interactions between BM and CB[8] and the directional head-to-tail stacking modes between the BM arms synergistically led to the formation of a 2D SOF. The structure of the 2D SOF was verified by 1H NMR, 2D 1H NMR NOESY, and DLS characterizations, while the monolayer structure was characterized by Cryo-TEM and AFM measurements. The 2D SOF exhibited an obvious AIE enhancement effect in H2O. In addition, DNA induced photoluminescence enhancement was observed for the monomer. As a result, this AIEgen-based 2D SOF could feature not only as a cell visualizer but also as a tracker for the nucleus in biological imaging due to the dynamic assembly process.