Gas-generating polymersomes-based amplified photoimmunotherapy for abscopal effect and tumor metastasis inhibition

Due to the heterogeneity of tumors,single phototherapy cannot completely ablate tumors and inhibit tumor metastasis.To overcome these,we formulated targeted and multifunctional polymersomes ABC@ICGIMQ-LHRH(AIRL)that encapsulated Toll-like receptor(TLR)7/8 agonist imiquimod(IMQ)and photosensitizer indocyanine green(ICG)in the hydrophobic layer as well as bubble-generator NH_(4)HCO_(3) in the hydrophilic cavity to inhibit the growth of primary and distant tumors,and prevent tumor metastasis through synergistic photoimmunotherapy.The AIRL polymersomes exhibited uniform and stable size,and high drug encapsulation efficiency,acid/reduction/laser responsiveness,excellent photothermal conversion efficiency,effective reactive oxygen species generation,high tumor accumulation.AIRL could be effectively internalized by dendritic cells(DCs),achieve lysosome escape and enhance DCs maturation.The synergistic photoimmunotherapy via AIRL polymersomes remarkably promoted the differentiation and activation of T cells,elevated strong systemic immune response to eradicate primary tumors and inhibit the growth of distant tumors.Simultaneously,the endurable immunological memory prevented tumor metastasis,which provided a promising nanoplatform for the combination therapy of cancer.

Efficient and targeted drug/siRNA co-delivery mediated by reversibly crosslinked polymersomes toward anti-inflammatory treatment of ulcerative colitis (UC)

Co-delivery of anti-inflammatory siRNA and hydrophilic drug provides a promising approach for the treatment of ulcerative colitis (UC). However, lack of a suitable and efficient co-delivery carrier poses critical challenge against their utilization. We herein developed macrophage-targeting, reversibly crossli nked polymersomes (TKPR-RCP) based on the TKPR-modified, poly(ethyle ne glycol)-b-poly(trimethylene carbonate-codithiolane trimethylene carbonate)-b-polyethylenimine (PEG-P(TMC-DTC)-PEI) triblock copolymer, which could efficiently encapsulate TNF-α siRNA and dexamethasone sodium phosphate (DSP) in their hydrophilic core. The cationic PEI segments provided additional electrostatic interactions with cargo molecules to promote the encapsulatiion, and disulfide crosslinking of the polymersome membrane endowed the TKPR-RCP with high colloidal stability. Because the cationic PEI was embedded in the hydrophilic core, the polymersomes displayed neutral surface charge and thus possessed high serum stability. The TKPR-RCP co-encapsulating TNF-α siRNA and DSP could be efficiently internalized by macrophages (~98%) and undergo redox-responsive membrane de-crosslinking to accelerate cargo release in the cytoplasm, thus inducing efficient gene silencing and anti-inflammatory effect .Intravenous injectio n of the co-delivery TKPR-RCP mediated pote nt and cooperative anti-inflammatory effect in inflamed colons of UC mice, and significantly prevented animals from colonic injury. This study therefore provides a promising approach for the co-delivery of hydrophilic drug/siRNA toward the treatment of inflammatory bowel diseases.

Fabrication of Polymersomes with Controllable Morphologies through Dewetting w/o/w Double Emulsion Droplets

In this work, monodisperse giant polymersomes are fabricated by dewetting of water-in-oil-in-water double emulsion droplets which are assembled by amphiphilic block copolymer molecules in a microfluidic device. The dewetting process can be tuned by solvation between solvent and amphiphilic block copolymer to get polymersomes with controllable morphology. Good solvent （chloroform and toluene） hinders dewetting process of double emulsion droplets and gets acornlike polymersomes or patched polymersomes. On the other hand, poor solvent （hexane） accelerates the dewetting process and achieves complete separation of inner water phase from oil phase to form complete bilayer polymersomes. In addition, twin polymersomes with bilayer membrane structure are formed by this facile method. The formation mechanism for different polymersomes is discussed in detail.

Polymersomes:From Macromolecular Self-Assembly to Particle Assembly

What is the most favorite and original chemistry developed in your research group?Ring-opening polymerization-induced self-assembly of N-carboxyanhydrides(NCA-PISA),and fusion-induced particle assembly(FIPA).How do you get into this specific field?Could you please share some experiences with our readers?NCA-PISA was developed to solve the biodegradability problem of nanoparticles by traditional PISA,while FIPA was inspired by nature.

Non-spherical polymersomes driven by directional aromatic interactions

Polymersomes,nanoscopic polymer vesicles self-assembled from synthetic block copolymers,are ideal candidates for drug delivery and synthetic biology,due to their characteristic of high stability,chemical

Folate-targeted co-delivery polymersomes for efficient photo-chemo-antiangiogenic therapy against breast cancer and in vivo evaluation via OCTA/NIRF dual-modal imaging

Intelligent nanoplatform that combines multimodal imaging and therapeutic effects holds great promise for precise and efficient cancer therapy.Herein,folate-targeted polymersomes with stimuli-responsiveness were fabricated and evaluated by near-infrared fluorescence(NIRF)and optical coherence tomography angiography(OCTA)dual-imaging for photo-chemo-antiangiogenic therapy against cancer.The folate-targeted polymersomes(FA-MIT-SIPS)not only integrated ammonium bicarbonate(ABC)and mitoxantrone(MIT)into their hydrophilic cavity but also encapsulated indocyanine green(ICG)and sorafenib(SOR)within their hydrophobic layer.NIRF imaging demonstrated that FA-MIT-SIPS effectively accumulated and retained in the tumors.Upon 808 nm laser irradiation,the ICG produced hyperthermia and reactive oxygen species(ROS)for efficient photothermal and photodynamic therapy.In addition,the decomposition of ABC in responsive to acidic tumor environment and ICG-induced hyperthermia accelerated drug release.The released MIT accumulated in nucleus to inhibit DNA synthesis,while the released SOR destructed tumor vascularization.Notably,OCTA imaging was applied to observe the tumor blood flow upon the combination therapy,demonstrating that FA-MIT-SIPS obviously decreased the vessels area density.Moreover,the synergistic photo-chemo-antiangiogenic therapy of FA-MIT-SIPS achieved excellent antitumor effect with 40%of the 4T1 tumor-bearing mice being completely cured without recurrence.The multifunctional polymersomes provide a promising dual-modal imaging-evaluated synergistic strategy for tumor therapy.

Synthesis and self-assembly of poly(ethylene glycol)-block-poly(N-3-(methylthio)propyl glycine)and their oxidation-sensitive polymersomes

Amphiphilic block copolymers poly(ethylene glycol)-block-poly(N-3-(methylthio)propyl glycine)(PEGb-PMeSPG)were synthesized via ring-opening polymerization of N-3-(methylthio)propyl glycine Nthiocarboxyanhydride(MeSPG-NTA)initiated by amino-terminated PEG.The self-assemblies of three PEG-b-PMeSPG copolymers with different PMeSPG block lengths were first prepared by nanoprecipitation method using THF and DMF,respectively,as the organic solvent,and their morphologies were studied by Cryo-EM and DLS.To prepare polymersomes loaded with glucose oxidase(GOx),double emulsion method followed by extrusion treatment was employed.The oxidation-responsive disruption of polymersomes was achieved upon the introduction of glucose because of the oxidants generated insitu by GOx/glucose.

Polymersomes scalably fabricated via flash nano- precipitation are non-toxic in non-human primates and associate with leukocytes in the spleen and kidney following intravenous administration

Vesicular nanocarrier formulations confer the ability to deliver hydrophobic and hydrophilic cargos simultaneously to cells of interest in vivo. While liposomal formulations reached the clinic long ago, younger technologies such as polymeric vesicles （polymersomes） have yet to make the transition to clinical approval and use, in part due to difficulties in ensuring their safe and scalable production. In this work, we demonstrate the scalable production of poly（ethylene glycol）-block-poly（propylene sulfide） （PEG-bl-PPS） polymersomes via flash nanoprecipitation, and further show the safe administration of these nanocarriers to mice and non-human primates. In mice, PEG-bl-PPS polymersomes were found to be well tolerated at up to 200 mg/（kg.week）. Following the administration of a more relevant 20 mg/（kg.week） dosage in non-human primates, polymersomes were found to associate with numerous phagocytic immune cell populations, including a remarkable 68% of plasmacytoid dendritic cells and 〉 95% of macrophages in the spleen, while showing no toxicity or abnormalities in the liver, kidney, spleen, or blood. Despite the presence of a dense PEG corona, neither anti-PEG antibodies nor complement activation were detected. This work provides evidence of the translatability of PEG-bI-PPS polymersomes into the clinic for therapeutic applications in humans.

Controlling Membrane Phase Separation of Polymersomes for Programmed Drug Release

Programmed release of small molecular drugs from polymersomes is of great importance in drug delivery.A significant challenge is to adjust the membrane permeability in a well-controlled manner.Herein,we propose a strategy for controlling membrane phase separation by photo-cross-linking of the membrane-forming blocks with different molecular architectures.We synthesized three amphiphilic block copolymers with different membrane-forming blocks,which are poly(ethylene oxide)_(43)-b-poly((ε-caprolactone)_(45)-stat-((α-(cinnamoyloxymethyl)-1,2,3-triazol)caprolactone)_(25))(PEO_(43)-b-P(CL_(45)-stat-CTCL_(25))),PEO_(43)-b-P(CL_(108)-stat-CTCL_(16)),and PEO_(43)-b-PCTCL_(4)-b-PCL_(79).These polymers were self-assembled into polymersomes using either a solvent-switch or powder rehydration method,and the obtained polymersomes were characterized by dynamic light scattering and transmission electron microscopy.Then the phase separation patterns within the polymersome membranes were investigated by mesoscopic dynamics(MesoDyn)simulations.To further confirm the change of the membrane permeability that resulted from the phase separation within the membrane,doxorubicin,as a small molecular drug,was loaded and released from the polymersomes.Due to the incompatibility between membrane-forming moieties(PCTCL and PCL),phase separation occurs and the release rate can be tuned by controlling the membrane phase pattern or by photo-cross-linking.Moreover,besides the compacting effect by formation of chemical bonds in the membrane,the cross-linking process can act as a driving force to facilitate the rearrangement and re-orientation of the phase pattern,which also influences the drug release behavior by modulating the cross-membrane distribution of the amorphous PCTCL moieties.In this way,the strategy of focusing on the membrane phase separation for the preparation of the polymersomes with finely tunable drug release rate can be envisioned and designed accordingly,which is of great significance in the field of delivery vehicles for programmed drug release.

Hybrid lipopolymer vesicle drug delivery and release systems

Hybrid lipopolymer vesicles are membrane vesicles that can be self-assembled on both the micro-and nano-scale.On the nanoscale,they are potential novel smart materials for drug delivery systems that could combine the relative strengths of liposome and polymersome drug delivery systems without their respective weaknesses.However,little is known about their properties and how they could be tailored.Currently,most methods of investigation are limited to the microscale.Here we provide a brief review on hybrid vesicle systems with a specific focus on recent developments demonstrating that nanoscale hybrid vesicles have different properties from their macroscale counterparts.

Bioinspired menstrual blood-derived stem cells-conditioned medium/polymersome nanoparticles for the treatment of carbon tetrachloride-induced acute liver failure in mice

Acute liver failure is a life-threatening syndrome,for which liver transplantation is presently the most effective treatment.Unfortunately,such treatment is extremely limited by a shortage of donor organs.Stem cell therapy offers a promising treatment strategy for acute liver failure.Yet,therapeutic efficacy and potential are hampered by administration route and safety concerns.In this work,we fabricated menstrual blood-derived stem cells-conditioned medium/polymersome hybrid nanoparticles that were self-assembled from amphiphilic block copolymers via the direct hydra-tion method and encapsulated therapeutic bioactive factors within the aqueous core of vesicles.The merit of vesicular architecture enabled the loading capacity of dis-tinct proteins and the maintenance of biological activity.These hybrid nanoparticles can be steadily taken up into cytoplasm and promote hepatocyte proliferation in vitro.Prolonged in vivo circulation time brought higher accumulation in livers.The therapeutic nanoparticles alleviated hepatic injury and promoted liver recovery in mice with carbon tetrachloride-induced liver failure.Considering the feasibility and benefit of the hybrid nanoparticle therapy,it provided a potential strategy to treat acute liver failure.

Polymersome-mediated cytosolic delivery of cyclic dinucleotide STING agonist enhances tumor immunotherapy

Cyclic dinucleotides(CDNs)as stimulator of interferon genes(STING)agonists capable of inducing strong antitumor innate immune response are highly promising for tumor immunotherapy.The efficacy of these CDNs is,however,reduced greatly by their fast clearance,poor cell uptake and inefficient cytosolic transportation.Here,we report that reduction-responsive biodegradable chimaeric polymersomes(CPs)markedly enhance tumor retention and cytosolic delivery of a synthetic CDN,ADU-S100,and bolster STING pathway activation in the tumor microenvironment and tumor draining lymph nodes,giving significantly better tumor repression and survival of B16F10 melanoma-bearing mice compared with free CDN control.The superiority of CPs-mediated CDN delivery is further verified in combination therapy with low-dose fractionated radiation,which brings about clearly stronger and longer-term immunotherapeutic effects and protection against tumor re-challenge.The development of nano-STING agonists that are able to overcome the delivery barriers of CDNs represents an effective strategy to potentiate cancer immunotherapy.

Effect of Water Content on the Size and Membrane Thickness of Polystyrene-block-Poly(ethylene oxide) Vesicles

The asymmetric amphiphilic block copolymer polystyrene962-block-poly（ethylene oxide）227 （PS962-b-PEO227） eanforms mieelles with N, N-dimethylforrnamide （DMF） as co-solvent and water as selected solvent, and when the water content of the mixed solvent is higher than 4.5 wt%, the vesicle will be dominated. This work finds that once vesicles are formed in the DMF-water mixed solvent, the vesicle size and membrane thickness can be tuned by further increasing water content. As the water fraction elevated from 4.8 wt% to 13.0 wt%, the vesicle size dercreases from 246 nm to 150 nm, while the membrane thickness increases from 28 nm to 42 nm. In addition, the block copolymer packing and the free energy are analyzed as the vesicle size becomes small and the membrane becomes thick.

Recent Progress in Fluorescent Vesicles with Aggregation-induced Emission

Fluorescent vesicles have recently attracted increasing attention because of their potential applications in bioimaging,diagnostics, and theranostics, for example, in vivo study of the delivery and the distribution of active substances. However, fluorescent vesicles containing conventional organic dyes often suffer from the problem of aggregation-caused quenching(ACQ) of fluorescence.Fluorescent vesicles working with aggregation-induced emission(AIE) offer an extraordinary tool to tackle the ACQ issue, showing advantages such as high emission efficiency, superior photophysical stability, low background interference, and high sensitivity. AIE fluorescent vesicles represent a new type of fluorescent and functional nanomaterials. In this review, we summarize the recent advances in the development of AIE fluorescent vesicles. The review is organized according to the chemical structures and architectures of the amphiphilic molecules that constitute the AIE vesicles, i.e., small-molecule amphiphiles, amphiphilic polymers, and amphiphilic supramolecules and supramacromolecules. The studies on the applications of these AIE vesicles as stimuli-responsive vesicles,fluorescence-guided drug release carriers, cell imaging tools, and fluorescent materials based on fluorescence resonance energy transfer(FRET) are also discussed.

Highly uniform ultrasound-sensitive nanospheres produced by a pH-induced micelle-to-vesicle transition for tumor-targeted drug delivery

Although gas-filled microbubbles with high echogenicity are widely applied in clinical ultrasonography, the micron scale particle size impedes their use in the treatment of solid tumors, which are accessible to objects less than several hundred nanometers. We herein propose an unusual approach involving a pH-induced core-shell micelle-to-vesicle transition to prepare ultrasound-sensitive polymeric nanospheres （polymersomes in structure） possessing multiple features, including nanosize, monodispersity, and incorporation of a phase- transitional imaging agent into the aqueous lumen. These features are not achievable via the conventional double-emulsion method for polymersome preparation. The nanospheres were constructed based on a novel triblock copolymer with dual pH sensitivity. The liquid-to-gas phase transition of the imaging agent induced by external low-frequency ultrasound may destroy the nanospheres for a rapid drug release, with simultaneous tissue-penetrating drug delivery inside a tumor. These effects may provide new opportunities for the development of an effective cancer therapy with few adverse effects.

pH-responsive Polymersome Based on PMCP-b-PDPA as a Drug Delivery System to Enhance Cellular Internalization and Intracellular Drug Release

Choline phosphate （CP） as a novel zwitterion possesses specific and excellent properties compared with phosphorylcholine （PC）, as well as its polymer, such as poly（2-（methacryloyloxy）ethyl choline phosphate） （PMCP）, has been studied extensively due to its unique characteristics of rapid cellular internalization via the special quadrupole interactions with the cell membrane. Recently, we reported a novel PMCP-based drug delivery system to enhance the cellular internalization where the drug was conjugated to the polymer via reversible acylhydrazone bond. Herein, to make full use of this feature of PMCP, we synthesized the diblock copolymer poly（2-（methacryloyloxy）ethyl choline phosphate）-b-poly（2- （diisopropylamino）ethyl methacrylate） （PMCP-b-PDPA）, which could self-assemble into polymersomes with hydrophilic PMCP corona and hydrophobic membrane wall in mild conditions when the pH value is 〉 6.4. It has been found that these polymersomes can be successfully used to load anticancer drug Dox with the loading content of about 11.30 wt%. After the polymersome is rapidly internalized by the cell with the aid of PMCP, the loaded drug can be burst-released in endosomes since PDPA segment is protonated at low pH environment, which renders PDPA to transfer from hydrophobic to hydrophilic, and the subsequent polymersomes collapse thoroughly. Ultimately, the ＂proton sponge＂ effect of PDPA chain can further accelerate the Dox to escape from endosome to cytoplasm to exert cytostatic effects. Meanwhile, the cell viability assays showed that the Dox-loaded polymersomes exhibited significant inhibitory effect on tumor cells, indicating its great potential as a targeted intracellular delivery system with high efficiency.

Micelle or polymersome formation by PCL-PEG-PCL copolymers as drug delivery systems

Polye-caprolactone）-b-poly（ethylene glycol）-b-poly（e-caprolactone）（PCL-b-PEG-b-PCL,PCEC） triblock copolymers have been widely investigated in last several decades.Here,by altering the weight ratio of monomers in ring-opening polymerization,a series of PCEC triblock copolymers with varying hydrophobicity were synthesized,which were characterized by FTIR,1 H NMR,GPC and DSC.When PCEC copolymers with different weight ratios of PCL/PEG were dispersed in different aqueous solutions,they could self-assemble and form two distinctive nanoparticular structures：micelles or polymersomes.We then chose paclitaxel（PTX） as the model drug and encapsulate PTX into PCEC polymeric micelles and polymersomes.The physicochemical characterizations of the nanoparticles such as morphology,the size and distribution,zeta potential,drug loading content,and encapsulation efficiency were also performed.Our results showed that polymeric micelles or polymersomes from PCEC both displayed narrow size distributions and could achieve high drug loading efficiencies.In vitro cellular uptake results suggested that Nile Red loaded polymeric micelles or polymersomes displayed more internalization after 24 h incubation than those after 4 h incubation.These findings suggest that polymeric micelles and polymersomes based on PCL-b-PEG-b-PCL copolymers have great potential to effectively delivery hydrophobic drugs.

A Three-drug co-Delivery System Based on Reduction-sensitive Polymeric Prodrug to Effectively Reverse Multi-drug Resistance

In the present study, we prepared a multi-drug delivery system based on reduction-sensitive paclitaxel （PTX） polymeric prodrug（PEG-b-PMPMC-g-PTX, PMP） polymersomes to co-deliver PTX, doxorubicin hydrochlo- ride（DOX.HC1） and the P-glycoprotein（P-gp） inhibitor Tariquidar（TQR） to effectively reverse drug resistance by inhibiting the expression of P-gp and improving the accumulation of the encapsulated anticancer drugs. The PTX was linked to the backbone by reduction-sensitive disulphide, making the polymersomes prone to collapse in the reductive environment and to release the drugs. Transmission electron microscope（TEM） was used to confirm the morphology of polymeric assemblies. Moreover, the rupture process of polymersomes was verified by dynamic light scattering （DLS）. The results of confocal laser scanning microscopy（CLSM） and flow cytometry indicate that the PMP/DOX.HCl/TQR three-drug-loaded polymersomes show the strongest fluorescence intensity for DOX-HC1 compared with PMP/DOX-HC1 polymersomes and free DOX-HCl in drug-resistant MCF-7/ADR cells. More importantly, the PMP/DOX.HCl/TQR multi-drug co-delivery system shows a greater growth-inhibitory effect on tumour cells than the other two samples, including PMP/DOX.HC1 nanoparticles without the TQR component and free DOX-HCl, when co-incubated with either nonresistant HeLa cells or drug-resistant MCF-7/ADR cells. This growth-inhibitory effect was especially evident in drug-resistant cells. These results imply that the co-delivery of PTX, DOX-HCl and TQR based on reduction-sensitive polymeric prodrug may be promising for overcoming multi-drug resistance in tumour treatments.

Polymersome formation by solvent annealing-induced structural reengineering under 3D soft confinement

A solvent annealing-induced structural reengineering approach is exploited to fabricate polymersomes from block copolymers that are hard to form vesicles through the traditional solution self-assembly route.More specifically,polystyrene-b-poly(4-vinyl pyridine)(PS-b-P4VP)particles with sphere-within-sphere structure(SS particles)are prepared by three-dimensional(3D)soft-confined assembly through emulsion-solvent evaporation,followed by 3D soft-confined solvent annealing upon the SS particles in aqueous dispersions for structural engineering.A water-miscible solvent(e.g.,THF)is employed for annealing,which results in dramatic transitions of the assemblies,e.g.,from SS particles to polymersomes.This approach works for PS-b-P4VP in a wide range of block ratios.Moreover,this method enables effective encapsulation/loading of cargoes such as fluorescent dyes and metal nanoparticles,which offers a new route to prepare polymersomes that could be applied for cargo release,diagnostic imaging,and nanoreactor,etc.

A pH-responsive polymersome depleting regulatory T cells and blocking A2A receptor for cancer immunotherapy

The immunosuppressive tumor microenvironment(ITM)and low immunogenicity of tumors greatly limit cancer immunotherapy efficacy.The approach of solely depleting regulatory T cells(Tregs)cannot ameliorate ITM,but possibly worsen it since the produced apoptotic Tregs will activate the A2A signaling pathway and cause more severe immune suppression.To address it,in this work a pH-responsive polymersome(CY/ZM@CS-BPA)based on chondroitin sulfate(CS)-poly(β-amino ester)is rationally developed.In the acidic tumor microenvironment,the tertiary amine groups in the polymersome will reverse from hydrophobic to hydrophilic due to protonation,which leads to the disintegration of nanostructures and the release of cyclophosphamide(CY)and A2A receptor(A2AR)antagonist ZM241385(ZM).CY can selectively deplete Tregs.Additionally,CY can induce immunogenic cell death(ICD)of tumor cells,which results in the proapoptotic translocation of calreticulin to the cell surface,further initiating the antitumor immune responses.ZM can inhibit the activation of the adenosine A2A pathway,subsequently preventing the differentiation of CD4^(+)T cells into Tregs and enhancing the cytotoxicity of CD8+T cells.As a result,the combination of depleting regulatory T cells and blocking the A2A receptor can enhance cancer immunotherapy efficacy.