Universal crRNA Acylation Strategy for Robust Photo-Initiated One-Pot CRISPR-Cas12a Nucleic Acid Diagnostics.

Spatiotemporal regulation of clustered regularly interspaced short palindromic repeats (CRISPR) system is attractive for precise gene editing and accurate molecular diagnosis. Although many efforts have been made, versatile and efficient strategies to control CRISPR system are still desirable. Here, we proposed a universal and accessible acylation strategy to regulate the CRISPR-Cas12a system by efficient acylation of 2'-hydroxyls (2'-OH) on crRNA strand with photolabile agents (PLGs). The introduction of PLGs confers efficient suppression of crRNA function and rapid restoration of CRISPR-Cas12a reaction upon short light exposure regardless of crRNA sequences. Based on this strategy, we constructed a universal PhotO-Initiated CRISPR-Cas12a system for Robust One-pot Testing (POIROT) platform integrated with recombinase polymerase amplification (RPA), which showed two orders of magnitude more sensitive than the conventional one-step assay and comparable to the two-step assay. For clinical sample testing, POIROT achieved high-efficiency detection performance comparable to the gold-standard quantitative PCR (qPCR) in sensitivity and specificity, but faster than the qPCR method. Overall, we believe the proposed strategy will promote the development of many other universal photo-controlled CRISPR technologies for one-pot assay, and even expand applications in the fields of controllable CRISPR-based genomic editing, disease therapy, and cell imaging.

Synthesis of Dendrimer-Like Molecules with Partial Carbon Chain via Iterative Single Unit Monomer Insertions.

Carbon-chain dendritic polymers hold unique properties and promising applications. However, synthesizing carbon-chain dendrimers, beyond conjugated ones, remains a challenge. Here, the use of the iterative single unit monomer insertion technique for synthesizing 2.5 generation partial-carbon-chain dendrimers (G2.5) is described, utilizing bismaleimide as the core, a maleimide-trithiocarbonate conjugate as the branching unit, and indene as the spacer unit, following a divergent growth strategy. The optimized conditions for synthesizing the maleimide-trithiocarbonate branching unit are a bismaleimide to trithiocarbonate ratio of 5:1 and a reaction time of 30 min. The structures are verified using 1H nuclear magnetic resonance, gel permeation chromatography, and matrix-assisted laser desorption/ionization-time of flight mass spectra. A four-arm star polymer is then synthesized using the G2.5 as the core. This synthesis of a partial-carbon-chain dendrimer establishes a foundational step toward creating all-carbon-chain ones and may open new application avenues in material science.

Cationic Single-Unit Monomer Insertion (cSUMI): From Discrete Oligomers to the α-/ω-End and In-Chain Sequence-Regulated Polymers.

Single-unit monomer insertion (SUMI) has become an important strategy for the synthesis of sequence-controlled vinyl polymers due to its strong versatility and high efficiency. However, all reported SUMI processes are based on a free-radical mechanism, resulting in a limited number of monomer types being applicable to SUMI or a limited number of sequences of structural units that SUMI can synthesize. Herein, we developed a novel SUMI based on a cationic mechanism (cSUMI), which operates through a degenerative (similar to radical SUMI) but cationic chain transfer process. By optimizing the chain transfer agent (CTA) and monomer pairs, a high-efficiency cSUMI was achieved for vinyl ether and styrene monomers. Based on this reaction, a range of discrete oligomers containing vinyl ether and styrene moieties, and even α-/ω-end and in-chain sequence-regulated polymers were synthesized, most of which cannot be achieved by radical SUMI. In addition, we explored the application of these sequence-regulated polymers in the preparation of miktoarm star polymers, delivery of photosensitizers, and solubilization of fluorescence probes. The development of SUMI with a new mechanism will certainly broaden the scope of structures and sequences in precise vinyl-based polymers.

Oscillating the local milieu of polymersome interiors via single input-regulated bilayer crosslinking and permeability tuning.

The unique permselectivity of cellular membranes is of crucial importance to maintain intracellular homeostasis while adapting to microenvironmental changes. Although liposomes and polymersomes have been widely engineered to mimic microstructures and functions of cells, it still remains a considerable challenge to synergize the stability and permeability of artificial cells and to imitate local milieu fluctuations. Herein, we report concurrent crosslinking and permeabilizing of pH-responsive polymersomes containing Schiff base moieties within bilayer membranes via enzyme-catalyzed acid production. Notably, this synergistic crosslinking and permeabilizing strategy allows tuning of the mesh sizes of the crosslinked bilayers with subnanometer precision, showing discriminative permeability toward maltooligosaccharides with molecular sizes of ~1.4-2.6 nm. The permselectivity of bilayer membranes enables intravesicular pH oscillation, fueled by a single input of glucose. This intravesicular pH oscillation can further drive the dissipative self-assembly of pH-sensitive dipeptides. Moreover, the permeabilization of polymersomes can be regulated by intracellular pH gradient as well, enabling the controlled release of encapsulated payloads.

High-Fidelity End-Functionalization of Poly(ethylene glycol) Using Stable and Potent Carbamate Linkages.

Commercial PEG-amine is of unreliable quality, and conventional PEG functionalization relies on esterification and etherification steps, suffering from incomplete conversion, harsh reaction conditions, and functional-group incompatibility. To solve these challenges, we propose an efficient strategy for PEG functionalization with carbamate linkages. By fine-tuning terminal amine basicity, stable and high-fidelity PEG-amine with carbamate linkage was obtained, as seen from the clean MALDI-TOF MS pattern. The carbamate strategy was further applied to the synthesis of high-fidelity multi-functionalized PEG with varying reactive groups. Compared to with an ester linkage, amphiphilic PEG-PS block copolymers bearing carbamate junction linkage exhibits preferential self-assembly tendency into vesicles. Moreover, nanoparticles of the latter demonstrate higher drug loading efficiency, encapsulation stability against enzymatic hydrolysis, and improved in vivo retention at the tumor region.

Ratiometric Fluorescent Mapping of pH and Glutathione Dictates Intracellular Transport Pathways of Micellar Nanoparticles.

Visualization of intracellular transport pathways is crucial to investigate the internalization mechanism and understand the intracellular behavior of nanomaterials. Herein, we rationalized the design of micellar nanoparticles (NPs) for ratiometric fluorescent mapping of intracellular pH and glutathione (GSH), two essential parameters for maintaining normal cellular functions. Specifically, pH-sensitive naphthalimide-based probe (NPI) and pH-inert rhodamine B (RhB) were covalently labeled to double hydrophilic block copolymers (DHBCs) using the thiolactone chemistry, enabling the covalent attachment of NPI and RhB to one molecule with a redox-responsive disulfide linkage. The dually labeled DHBCs exhibited blue/orange dual emissions in acidic pH, which was further converted into green/orange dual emissions in neutral pH because of the deprotonation of NPI moieties and the sole green emission in the presence of GSH at neutral pH because of the decreased Förster resonance energy transfer efficiency between an NPI donor and an RhB acceptor as a result of GSH-mediated cleavage of disulfide bonds. These remarkable ratiometric fluorescence changes allowed for not only the simultaneous mapping of the intracellular pH and GSH but also the intracellular transport pathways of internalized NPs.

Angiopep2-Conjugated Star-Shaped Polyprodrug Amphiphiles for Simultaneous Glioma-Targeting Therapy and MR Imaging.

The development of valuable theranostic agents for overcoming the blood-brain barrier (BBB) to achieve efficient imaging-guided glioma-targeting delivery of therapeutics remains a great challenge for personalized glioma therapy. We herein developed a novel functional star-shaped polyprodrug amphiphile (denoted as CPP-2) via a combination of successive reversible addition-fragmentation chain transfer (RAFT) polymerization and click functionalization. In a diluted solution, the star amphiphile existed as structurally stable unimolecular micelles, containing hydrophobic cores conjugated with reduction-responsive camptothecin prodrugs Camptothecin (CPT) prodrug monomer (CPTM) and a tertiary amine monomer (2-(diethylamine) ethyl methacrylate, DEA) and hydrophilic oligo-(ethylene glycol) monomethyl ether methacrylat (OEGMA) outer coronas covalently decorated with dual-targeting moieties Angiopep2 (ANG) and small magnetic resonance imaging (MRI) contrast agents DOTA-Gd. In vitro and in vivo data in this study demonstrated that the ANG-modified micelles were capable of efficiently penetrating the BBB and delivering loaded cargoes such as CPT and Gd3+ contrast agents to glioma cells, leading to a considerably enhanced t1 relaxivity as well as antiglioma efficacy. Simultaneously, the targeted antiglioma efficacy and noninvasive MR imaging for a visualized therapy were realized. These collective findings augured well for the star polyprodrug amphiphiles to be utilized as a novel theranostic platform for clinical application in glioma therapy.

Emerging Applications of Fluorogenic and Non-fluorogenic Bifunctional Linkers.

Homo- and hetero-bifunctional linkers play vital roles in constructing a variety of functional systems, ranging from protein bioconjugates with drugs and functional agents, to surface modification of nanoparticles and living cells, and to the cyclization/dimerization of synthetic polymers and biomolecules. Conventional approaches for assaying conjugation extents typically rely on ex situ techniques, such as mass spectrometry, gel electrophoresis, and size-exclusion chromatography. If the conjugation process involving bifunctional linkers was rendered fluorogenic, then in situ monitoring, quantification, and optical tracking/visualization of relevant processes would be achieved. In this review, conventional non-fluorogenic linkers are first discussed. Then the focus is on the evolution and emerging applications of fluorogenic bifunctional linkers, which are categorized into hetero-bifunctional single-caging fluorogenic linkers, homo-bifunctional double-caging fluorogenic linkers, and hetero-bifunctional double-caging fluorogenic linkers. In addition, stimuli-cleavable bifunctional linkers designed for both conjugation and subsequent site-specific triggered release are also summarized.

Concurrent Drug Unplugging and Permeabilization of Polyprodrug-Gated Crosslinked Vesicles for Cancer Combination Chemotherapy.

Combination chemotherapy with both hydrophobic and hydrophilic therapeutic drugs is clinically vital toward the treatment of persistent cancers. Though conventional liposomes and polymeric vesicles possessing hydrophobic bilayers and aqueous interiors can serve as codelivery nanocarriers, it remains a considerable challenge to achieve synchronized release of both types of drugs due to distinct encapsulation mechanisms; premature release of water-soluble cargos from unstable liposomes and ruptured vesicles is also a major concern. Herein, the fabrication of physiologically stable polyprodrug-gated crosslinked vesicles (GCVs) via the self-assembly of camptothecin (CPT) polyprodrug amphiphiles and in situ bilayer crosslinking through traceless sol-gel reaction is reported. Polyprodrug-GCVs possess high CPT loading (>30 wt%) and minimized leakage of encapsulated hydrophilic doxorubicin (DOX) hydrochloride due to the suppressed permeability of crosslinked membrane, exhibiting extended blood circulation (t 1/2 > 13 h) with caged cytotoxicity in physiological circulation. Upon cellular uptake by cancer cells, cytosolic reductive milieu-triggered CPT unplugging from vesicle bilayers is demonstrated to generate hydrophilic mesh channels and make the membrane highly permeable. Concurrently, it will promote DOX corelease from hydrophilic lumen (≈36-fold increase). The reduction-activated combination chemotherapeutic potency based on polyprodrug-GCVs is confirmed by both in vitro and in vivo explorations.

Photo- and Reduction-Responsive Polymersomes for Programmed Release of Small and Macromolecular Payloads.

We report on the preparation of photo- and reduction-responsive diblock copolymers through reversible addition-fragmentation chain transfer (RAFT) polymerization of a coumarin-based disulfide-containing monomer (i.e., CSSMA) using a poly(ethylene oxide) (PEO)-based macroRAFT agent. The resulting amphiphilic PEO- b-PCSSMA copolymers self-assembled into polymersomes with hydrophilic PEO shielding coronas and hydrophobic bilayer membranes. Upon irradiating the polymersomes with visible light (e.g., 430 nm), the coumarin moieties within the bilayer membranes were cleaved with the generation of primary amine groups, which spontaneously underwent inter/intrachain amidation reactions with the ester moieties, thereby tracelessly cross-linking and permeating the bilayer membranes. Notably, this process only gave rise to the release of small molecule payloads (e.g., doxorubicin hydrochloride, DOX) while large molecule encapsulants (e.g., Texas red-labeled dextran, TR-dextran) were retained within the cross-linked polymersomes due to the preservation of the integrity of the vesicular nanostructures. However, cross-linked polymersomes undergo further structural disintegration upon incubation with glutathione (GSH) due to the scission of disulfide linkages, resulting in the release of macromolecular payloads. Thus, dual-stimuli responsive polymersomes with tracelessly cross-linkable characteristics enable sequential release of payloads with spatiotemporal precision, which could be of promising applications in synergistic loading and programmed release of therapeutics.

Anti-inflammatory polymersomes of redox-responsive polyprodrug amphiphiles with inflammation-triggered indomethacin release characteristics.

Inflammation serves as a natural defense mechanism to protect living organisms from infectious diseases. Nonsteroidal anti-inflammatory drugs (NSAIDs) can help relieve inflammatory reactions and are clinically used to treat pain, fever, and inflammation, whereas long-term use of NSAIDs may lead to severe side effects including gastrointestinal damage and cardiovascular toxicity. Therefore, it is of increasing importance to configure new dosing strategies and alleviate the side effects of NSAIDs. Towards this goal, glutathione (GSH)-responsive disulfide bonds and hydrogen peroxide (H2O2)-reactive phenylboronic ester linkages were utilized as triggering moieties in this work to design redox-responsive prodrug monomers and polyprodrug amphiphiles based on indomethacin (IND) drug. Note that IND is a widely prescribed NSAID in the clinic. Starting from three types of redox-reactive IND prodrug monomers, redox-responsive polyprodrug amphiphiles were synthesized through reversible addition-fragmentation chain transfer (RAFT) polymerizations of prodrug monomers using poly(ethylene oxide) (PEO)-based macroRAFT agent. The resultant polyprodrug amphiphiles with high IND loading contents (>33 wt%) could self-assemble into polymersomes with PEO shielding coronas and redox-responsive bilayer membranes composed of IND prodrugs. Upon incubation with GSH or H2O2, controlled release of intact IND in the active form from polyprodrug polymersomes was actuated by GSH-mediated disulfide cleavage reaction and H2O2-mediated oxidation of phenylboronic ester moieties, respectively, followed by self-immolative degradation events. Furthermore, in vitro studies at the cellular level revealed that redox-responsive polymersomes could efficiently relieve inflammatory responses induced by lipopolysaccharide (LPS) in RAW264.7 macrophage cells.

Near-Infrared Light-Activated Photochemical Internalization of Reduction-Responsive Polyprodrug Vesicles for Synergistic Photodynamic Therapy and Chemotherapy.

The use of intracellular reductive microenvironment to control the release of therapeutic payloads has emerged as a popular approach to design and fabricate intelligent nanocarriers. However, these reduction-responsive nanocarriers are generally trapped within endolysosomes after internalization and are subjected to unwanted disintegration, remarkably compromising the therapeutic performance. Herein, amphiphilic polyprodrugs of poly(N,N-dimethylacrylamide-co-EoS)-b-PCPTM were synthesized via sequential reversible addition-fragmentation chain transfer (RAFT) polymerization, where EoS and CPTM are Eosin Y- and camptothecin (CPT)-based monomers, respectively. An oil-in-water (O/W) emulsion method was applied to self-assemble the amphiphilic polyprodrugs into hybrid vesicles in the presence of hydrophobic oleic acid (OA)-stabilized upconversion nanoparticles (UCNPs, NaYF4:Yb/Er), rendering it possible to activate the EoS photosensitizer under a near-infrared (NIR) laser irradiation with the generation of singlet oxygen (1O2) through the energy transfer between UCNPs and EoS moieties. Notably, the in situ generated singlet oxygen (1O2) can not only exert its photodynamic therapy (PDT) effect but also disrupt the membranes of endolysosomes and thus facilitate the endosomal escape of internalized nanocarriers (i.e., photochemical internalization (PCI)). Cell experiments revealed that the hybrid vesicles could be facilely taken up by endocytosis. Although the internalized hybrid vesicles were initially trapped within endolysosomes, a remarkable endosomal escape into the cytoplasm was observed under 980 nm laser irradiation as a result of the PCI effect of 1O2. The escaped hybrid vesicles subsequently underwent GSH-triggered CPT release in the cytosol, thereby activating the chemotherapy process. The integration of PDT module into the design of reduction-responsive nanocarriers provides a feasible approach to enhance the therapeutic performance.

Doubly Caged Linker for AND-Type Fluorogenic Construction of Protein/Antibody Bioconjugates and In Situ Quantification.

In situ quantification of the conjugation efficiency of azide-terminated synthetic polymers/imaging probes and thiol-functionalized antibodies/proteins/peptides was enabled by a doubly caged profluorescent and heterodifunctional core molecule C1 as a self-sorting bridging unit. Orthogonal dual "click" coupling of C1 with azide- and thiol-functionalized precursors led to highly fluorescent bioconjugates, whereas single-click products remained essentially nonfluorescent. Integration with FRET processes was also possible. For the construction of antibody-probe conjugates from an anti-carcinoembryonic antigen and a quinone-caged profluorescent naphthalimide derivative, the dual "click" coupling process with C1 was monitored on the basis of the emission turn-on of C1, whereas prominent changes in FRET ratios occurred for antibody-imaging-probe conjugates when specifically triggered by quinone oxidoreductase (NQO1), which is overexpressed in various types of cancer cells.

Photo- and thermo-responsive multicompartment hydrogels for synergistic delivery of gemcitabine and doxorubicin.

Hydrogels have found promising applications in drug delivery due to their biocompatibility, high drug loading capability, and tunable release profiles. However, hydrogel-based carriers are primarily employed for delivering hydrophilic payloads while hydrophobic drugs cannot be efficiently delivered due to the lack of hydrophobic domains within conventional hydrogel matrices. Herein, we report that thermo- and photo-responsive hydrogels could be constructed from amphiphilic triblock copolymers, poly(N-isopropylacrylamide)-b-poly(4-acryloylmorpholine)-b-poly(2-((((2-nitrobenzyl)oxy)carbonyl) amino)ethyl methacrylate) (PNIPAM-b-PNAM-b-PNBOC), and the resulting hydrogels could be further engineered a new carrier for both hydrophilic gemcitabine (GCT) and hydrophobic doxorubicin (DOX). PNIPAM-b-PNAM-b-PNBOC triblock copolymers were first self-assembled into micelles with hydrophobic photosensitive PNBOC cores, hydrophilic PNAM inner shells, and thermoresponsive PNIPAM coronas below the lower critical solution temperature (LCST), while hydrogels of physically cross-linked micellar nanoparticles were achieved at elevated polymer concentrations and high temperatures above the critical gelation temperature (CGT). Rheological experiments revealed that the CGT was highly dependent on polymer compositions and concentrations, that is, a longer hydrophobic PNBOC block or a higher polymer concentration led to a decreased CGT. However, the CGT prior to UV irradiation (CGT0) could be drastically elevated after UV irradiation (CGTUV) as a result of UV irradiation-induced concurrently cross-linking and hydrophobic-to-hydrophilic transition within PNBOC cores. As such, gel-to-sol transition could be accomplished by either temperature decrease or exposure to UV irradiation at a fixed temperature lower than the CGTUV. Note that both GCT and DOX could be simultaneously encapsulated into the hydrogels due to the coexistence of extramicellar aqueous phase and hydrophobic micellar cores. Intriguingly, the subsequent co-release of GCT and DOX could be regulated by taking advantage of either temperature or UV irradiation-mediated gel-to-sol transitions.

Engineering Intracellular Delivery Nanocarriers and Nanoreactors from Oxidation-Responsive Polymersomes via Synchronized Bilayer Cross-Linking and Permeabilizing Inside Live Cells.

Reactive oxygen species (ROS) and oxidative stress are implicated in various physiological and pathological processes, and this feature provides a vital biochemical basis for designing novel therapeutic and diagnostic nanomedicines. Among them, oxidation-responsive micelles and vesicles (polymersomes) of amphiphilic block copolymers have been extensively explored; however, in previous works, oxidation by ROS including H2O2 exclusively leads to microstructural destruction of polymeric assemblies. For oxidation-responsive polymersomes, fast release of encapsulated hydrophilic drugs and bioactive macromolecules will occur upon microstructural disintegration. Under certain application circumstances, this does not meet design requirements for sustained-release drug nanocarriers and long-acting in vivo nanoreactors. Also note that conventional polymersomes possess thick hydrophobic bilayers and compromised membrane permeability, rendering them as ineffective nanocarriers and nanoreactors. We herein report the fabrication of oxidation-responsive multifunctional polymersomes exhibiting intracellular milieu-triggered vesicle bilayer cross-linking, permeability switching, and enhanced imaging/drug release features. Mitochondria-targeted H2O2 reactive polymersomes were obtained through the self-assembly of amphiphilic block copolymers containing arylboronate ester-capped self-immolative side linkages in the hydrophobic block, followed by surface functionalization with targeting peptides. Upon cellular uptake, intracellular H2O2 triggers cascade decaging reactions and generates primary amine moieties; prominent amidation reaction then occurs within hydrophobic bilayer membranes, resulting in concurrent cross-linking and hydrophobic-to-hydrophilic transition of polymersome bilayers inside live cells. This process was further utilized to achieve integrated functions such as sustained drug release, (combination) chemotherapy monitored by fluorescence and magnetic resonance (MR) imaging turn-on, and to construct intracellular fluorogenic nanoreactors for cytosolic thiol-containing bioactive molecules.

Enzyme-Responsive Polymeric Vesicles for Bacterial-Strain-Selective Delivery of Antimicrobial Agents.

Antimicrobial resistance poses serious public health concerns and antibiotic misuse/abuse further complicates the situation; thus, it remains a considerable challenge to optimize/improve the usage of currently available drugs. We report a general strategy to construct a bacterial strain-selective delivery system for antibiotics based on responsive polymeric vesicles. In response to enzymes including penicillin G amidase (PGA) and ß-lactamase (Bla), which are closely associated with drug-resistant bacterial strains, antibiotic-loaded polymeric vesicles undergo self-immolative structural rearrangement and morphological transitions, leading to sustained release of antibiotics. Enhanced stability, reduced side effects, and bacterial strain-selective drug release were achieved. Considering that Bla is the main cause of bacterial resistance to ß-lactam antibiotic drugs, as a further validation, we demonstrate methicillin-resistant S. aureus (MRSA)-triggered release of antibiotics from Bla-degradable polymeric vesicles, in vitro inhibition of MRSA growth, and enhanced wound healing in an in vivo murine model.

pH-Regulated Reversible Transition Between Polyion Complexes (PIC) and Hydrogen-Bonding Complexes (HBC) with Tunable Aggregation-Induced Emission.

The mimicking of biological supramolecular interactions and their mutual transitions to fabricate intelligent artificial systems has been of increasing interest. Herein, we report the fabrication of supramolecular micellar nanoparticles consisting of quaternized poly(ethylene oxide)-b-poly(2-dimethylaminoethyl methacrylate) (PEO-b-PQDMA) and tetrakis(4-carboxylmethoxyphenyl)ethene (TPE-4COOH), which was capable of reversible transition between polyion complexes (PIC) and hydrogen bonding complexes (HBC) with tunable aggregation-induced emission (AIE) mediated by solution pH. At pH 8, TPE-4COOH chromophores can be directly dissolved in aqueous milieu without evident fluorescence emission. However, upon mixing with PEO-b-PQDMA, polyion complexes were formed by taking advantage of electrostatic interaction between carboxylate anions and quaternary ammonium cations and the most compact PIC micelles were achieved at the isoelectric point (i.e., [QDMA(+)]/[COO(-)] = 1), as confirmed by dynamic light scattering (DLS) measurement. Simultaneously, fluorescence spectroscopy revealed an evident emission turn-on and the maximum fluorescence intensity was observed near the isoelectric point due to the restriction of intramolecular rotation of TPE moieties within the PIC cores. The kinetic study supported a micelle fusion/fission mechanism on the formation of PIC micelles at varying charge ratios, exhibiting a quick time constant (τ1) relating to the formation of quasi-equilibrium micelles and a slow time constant (τ2) corresponding to the formation of final equilibrium micelles. Upon deceasing the pH of PIC micelles from 8 to 2 at the [QDMA(+)]/[COO(-)] molar ratio of 1, TPE-4COOH chromophores became gradually protonated and hydrophobic. The size of micellar nanoparticles underwent a remarkable decrease, whereas the fluorescence intensity exhibited a further increase by approximately 7.35-fold, presumably because of the formation of HBC micelles comprising cationic PQDMA coronas and PEO/TPE-4COOH hydrogen-bonded cores, an inverted micellar structures compared to initial PIC micelles. Moreover, the pH-mediated schizophrenic micellar transition from PIC to HBC with tunable AIE characteristic was reversible.

pH-Responsive Tumor-Targetable Theranostic Nanovectors Based on Core Crosslinked (CCL) Micelles with Fluorescence and Magnetic Resonance (MR) Dual Imaging Modalities and Drug Delivery Performance.

The development of novel theranostic nanovectors is of particular interest in treating formidable diseases (e.g., cancers). Herein, we report a new tumor-targetable theranostic agent based on core crosslinked (CCL) micelles, possessing tumor targetable moieties and fluorescence and magnetic resonance (MR) dual imaging modalities. An azide-terminated diblock copolymer, N3-POEGMA-b-P(DPA-co-GMA), was synthesized via consecutive atom transfer radical polymerization (ATRP), where OEGMA, DPA, and GMA are oligo(ethylene glycol)methyl ether methacrylate, 2-(diisopropylamino)ethyl methacrylate, and glycidyl methacrylate, respectively. The resulting diblock copolymer was further functionalized with DOTA(Gd) (DOTA is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakisacetic acid) or benzaldehyde moieties via copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) chemistry, resulting in the formation of DOTA(Gd)-POEGMA-b-P(DPA-co-GMA) and benzaldehyde-POEGMA-b-P(DPA-co-GMA) copolymers. The resultant block copolymers co-assembled into mixed micelles at neutral pH in the presence of tetrakis[4-(2-mercaptoethoxy)phenyl]ethylene (TPE-4SH), which underwent spontaneous crosslinking reactions with GMA residues embedded within the micellar cores, simultaneously switching on TPE fluorescence due to the restriction of intramolecular rotation. Moreover, camptothecin (CPT) was encapsulated into the crosslinked cores at neutral pH, and tumor-targeting pH low insertion peptide (pHLIP, sequence: AEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTCG) moieties were attached to the coronas through the Schiff base chemistry, yielding a theranostic nanovector with fluorescence and MR dual imaging modalities and tumor-targeting capability. The nanovectors can be efficiently taken up by A549 cells, as monitored by TPE fluorescence. After internalization, intracellular acidic pH triggered the release of loaded CPT, killing cancer cells in a selective manner. On the other hand, the nanovectors labeled with DOTA(Gd) contrast agents exhibited increased relaxivity (r1 = 16.97 mM-1·s-1) compared to alkynyl-DOTA(Gd) small molecule precursor (r1 = 3.16 mM-1·s-1). Moreover, in vivo MRI (magnetic resonance imaging) measurements revealed CCL micelles with pHLIP peptides exhibiting better tumor accumulation and MR imaging performance as well.

Reversibly Switching Bilayer Permeability and Release Modules of Photochromic Polymersomes Stabilized by Cooperative Noncovalent Interactions.

We report on the fabrication of photochromic polymersomes exhibiting photoswitchable and reversible bilayer permeability from newly designed poly(ethylene oxide)-b-PSPA (PEO-b-PSPA) diblock copolymers, where SPA is spiropyran (SP)-based monomer containing a unique carbamate linkage. Upon self-assembling into polymersomes, SP moieties within vesicle bilayers undergo reversible phototriggered isomerization between hydrophobic spiropyran (SP, λ2 > 450 nm irradiation) and zwitterionic merocyanine (MC, λ1 < 420 nm irradiation) states. For both SP and MC polymersomes, their microstructures are stabilized by multiple cooperative noncovalent interactions including hydrophobic, hydrogen bonding, π-π stacking, and paired electrostatic (zwitterionic) interactions, with the latter two types being exclusive for MC polymersomes. Control experiments using analogous block copolymers of hydrophobic SP monomer with a carbonate linkage (SPO) and conventional spiropyran methacrylate monomer (SPMA) containing a single ester functionality were then conducted, revealing that carbamate-incurred hydrogen bonding interactions in PEO-b-PSPA are crucial for polymersome stabilization in the zwitterionic MC state. Moreover, reversible phototriggered SP-to-MC polymersome transition is accompanied by membrane polarity and permeability switching from being nonimpermeable to selectively permeable toward noncharged, charged, and zwitterionic small molecule species below critical molar masses. Intriguingly, UV-actuated MC polymersomes possess two types of release modules: (1) sustained release upon short UV irradiation duration by taking advantage of the unexpectedly slow spontaneous MC-to-SP transition kinetics (t1/2 > 20 h) under dark conditions; (2) on-demand and switchable release under alternated UV-vis light irradiation. We further demonstrate photoswitchable spatiotemporal release of 4',6-diamidino-2-phenylindole (DAPI, cell nuclei-staining dye) within living HeLa cells.