Gene therapy by virus-like self-spooling toroidal DNA condensates for revascularization of hindlimb ischemia.

Peripheral arterial diseases (PAD) have been reported to be the leading cause for limb amputations, and the current therapeutic strategies including antiplatelet medication or intervene surgery are reported to not clinically benefit the patients with high-grade PAD. To this respect, revascularization based on angiogenetic vascular endothelial growth factor (VEGF) gene therapy was attempted for the potential treatment of critical PAD. Aiming for transcellular delivery of VEGF-encoding plasmid DNA (pDNA), we proposed to elaborate intriguing virus-like DNA condensates, wherein the supercoiled rigid micrometer-scaled plasmid DNA (pDNA) could be regulated in an orderly fashion into well-defined nano-toroids by following a self-spooling process with the aid of cationic block copolymer poly(ethylene glycol)-polylysine at an extraordinary ionic strength (NaCl: 600 mM). Moreover, reversible disulfide crosslinking was proposed between the polylysine segments with the aim of stabilizing these intriguing toroidal condensates. Pertaining to the critical hindlimb ischemia, our proposed toroidal VEGF-encoding pDNA condensates demonstrated high levels of VEGF expression at the dosage sites, which consequently contributed to the neo-vasculature (the particularly abundant formation of micro-vessels in the injected hindlimb), preventing the hindlimb ischemia from causing necrosis at the extremities. Moreover, excellent safety profiles have been demonstrated by our proposed toroidal condensates, as opposed to the apparent immunogenicity of the naked pDNA. Hence, our proposed virus-like DNA condensates herald potentials as gene therapy platform in persistent expressions of the therapeutic proteins, and might consequently be highlighted in the management of a variety of intractable diseases.

An ER-targeted, Viscosity-sensitive Hemicyanine Dye for the Diagnosis of Nonalcoholic Fatty Liver and Photodynamic Cancer Therapy by Activating Pyroptosis Pathway.

The concept of molecular design, integrating diagnostic and therapeutic functions, aligns with the general trend of modern medical advancement. Herein, we rationally designed the smart molecule ER-ZS for endoplasmic reticulum (ER)-targeted diagnosis and treatment in cell and animal models by combining hemicyanine dyes with ER-targeted functional groups (p-toluenesulfonamide). Owing to its ability to target the ER with a highly specific response to viscosity, ER-ZS demonstrated substantial fluorescence turn-on only after binding to the ER, independent of other physiological environments. In addition, ER-ZS, being a small molecule, allows for the diagnosis of nonalcoholic fatty liver disease (NAFLD) via liver imaging based on high ER stress. Importantly, ER-ZS is a type I photosensitizer, producing O2 ⋅- and ⋅OH under light irradiation. Thus, after irradiating for a certain period, the photodynamic therapy inflicted severe oxidative damage to the ER of tumor cells in hypoxic (2 % O2 ) conditions and activated the unique pyroptosis pathway, demonstrating excellent antitumor capacity in xenograft tumor models. Hence, the proposed strategy will likely shed new light on integrating molecular optics for NAFLD diagnosis and cancer therapy.

Lifestyle-based nomogram for identifying the Chaoshan inhabitants of China at high risk of Helicobacter pylori infection.

BACKGROUND: Helicobacter pylori (HP) infection is associated with various diseases. Early detection can prevent the onset of illness. We constructed a nomogram to predict groups at high risk of HP infection. METHODS: Patients who underwent regular medical check-ups at hospital in Chaoshan, China from March to September 2022 were randomly allocated to the training and validation cohorts. Risk factors including basic characteristics and lifestyle habits associated with HP infection were analyzed by logistic regression analyses. The independent varieties were calculated and plotted into a nomogram. The nomogram was internally validated by receiver operating characteristic curve, calibration, and decision curve analyses (DCAs). RESULTS: Of the 945 patients, 680 were included in the training cohort and 265 in the validation cohort. 356 patients in training cohort with positive 13 C-UBT results served as the infected group, and 324 without infection were the control group. The multivariate regression analyses showed that the risk factors for HP infection included alcohol consumption (OR = 1.29, 95%CI = 0.78-2.13, P = 0.03), family history of gastric disease (OR = 4.35, 95%CI = 1.47-12.84, P = 0.01), living with an HP-positive individual (OR = 18.09, 95%CI = 10.29-31.82, P < 0.0001), drinking hot tea (OR = 1.58, 95%CI = 1.05-2.48, P = 0.04), and infection status of co-drinkers unknown (OR = 2.29, 95%CI = 1.04-5.06, P = 0.04). However, drinking tea > 3 times per day (OR = 0.56, 95%CI = 0.33-0.95, P = 0.03), using serving chopsticks (OR = 0.30, 95%CI = 0.12-0.49, P < 0.0001) were protective factors for HP infection. The nomogram had an area under the curve (AUC) of 0.85 in the training cohort. The DCA was above the reference line within a large threshold range, indicating that the model was better. The calibration analyses showed the actual occurrence rate was basically consistent with the predicted occurrence rate. The model was validated in the validation cohort, and had a good AUC (0.80), DCA and calibration curve results. CONCLUSIONS: This nomogram, which incorporates basic characteristics and lifestyle habits, is an efficient model for predicting those at high risk of HP infection in the Chaoshan region.

Reversible Chemical Protein Modification to Endogenous Glutathione and Its Utilities in the Manufacture of Transcellular Pro-Enzymes.

Proteins have been perceived as being an intriguing modality of therapeutics for the treatment of intractable diseases in view of their superlative precision and versatility. Nonetheless, proteins' intrinsic characters, particularly their being hydrophilic macromolecules with unmethodical charges, have imposed the exceeding challenge of seeking transcellular trafficking into cells' interiors. To circumvent this drawback, we have attempted to employ triple-functional amine-reactive 4-(2-((2-(((4-nitrophenoxy)carbonyl)oxy)ethyl)disulfaneyl)ethoxy)-4-oxobutanoic acid for the efficient incorporation of the anionic carboxyl moiety into amine-enriched enzymes, resulting in overall negatively charged pro-enzymes. The resulting pro-enzymes could be readily electrostatically assembled with cationic species [for instance: block copolymers of poly(ethylene glycol)-polylysine] into core-shell architectural delivery nanoparticles for their facilitated endocytosis into cells. Noteworthy is the aforementioned carboxylation chemistry designed to allow facile reversal of the pro-enzymes to the original amine groups due to the thiolysis of intermediate disulfide linkage for subsequent cascade reactions in response to the cytosol-enriched glutathione. Therefore, cytosol-selective structural disassembly for the liberation and activation of the pro-enzymes was accomplished. Our subsequent investigations utilizing ribonuclease A and catalase as the model enzymes demonstrated appreciable transcellular transportation of the active enzymes to the cell interiors, exerting overwhelming cytotoxic potencies and H2O2 scavenging capacities, respectively. Hence, we reported an unprecedented redox-stimulated charge reversal strategy in engineering cytosol-activatable pro-enzymes, manifesting a simple and efficient approach in the manufacture of transcellular proteinic therapeutics, which should be highlighted to promote their wide availability for use with diverse functional proteins as molecular biological tools and precision therapeutics.

Bioresorbable Depot for Sustained Release of Immunostimulatory Resiquimod in Suppressing Both Primary Triple-Negative Breast Tumors and Metastatic Occurrence.

In light of immune facilities trafficking toward the pathological sites along upward gradient of immunostimulatory cytokines, a localized resiquimod (Toll-like receptor 7/8 agonist) release depot was manufactured for pursuit of precision immunostimulation toward intractable triple-negative breast carcinoma. In principle, resiquimod/poly(lactic-co-glycolic acid) microspheres were fabricated and embedded into injectable and biodegradable poly(ethylene glycol) (PEG)-based hydrogel. The subsequent investigations approved persistent retention of immunostimulatory resiquimod in tumors upon peritumoral administration, which consequently led to localized and consistent secretion of immunostimulatory cytokines. Initially, not only innate tumor phagocytosis but also adaptive antitumor immunities were successfully cultivated for in situ suppression of the growth of primary solid tumors, more importantly, capable of inhibiting distant pulmonary metastasis, as evidenced by observation of enormous lymphocytes selectively gathering in the pulmonary artery. Hence, our presented study provided an important clinical indication of using immunostimulatory drugs to activate potent innate and adaptive antitumor immunities for precision antitumor therapy. Further immunomodulatory strategies, such as checkpoint blockage and tumor immunogenicity, could also be complementary for development of advanced antitumor immunotherapeutics in treatment of a number of intractable tumors.

Polymeric RNAi Constructs Tailored with Appreciable Transcellular Trafficking Functions for Potential Suppression of Parathyroid Hormone Production.

Polymeric small interfering RNA (siRNA) conjugate was elaborated to sequentially circumvent the predefined biological barriers encountered in the journey of transcellular delivery of siRNA into cytosol. Herein, classic ring-opening polymerization was employed for synthesis of well-defined poly(amino acid) derivatives possessing an array of carboxyl groups in an attempt to resemble the structural characteristics of hyaluronan. Furthermore, the hyaluronan-like synthetic was conjugated with a multiple of siRNA through a glutathione (GSH)-responsive disulfide linkage. The siRNA conjugate appeared to utilize the hyaluronan-specific receptors of CD44 for cell internalization, indicating similar functionalities to our hyaluronan-mimicking synthetic. Furthermore, the carboxyl groups of hyaluronan-like synthetics were designed to be selectively detached in subcellular acidic endosomes/lysosomes and transform into the cytomembrane-disruptive flanking ethylenediamine moieties, which appeared to be crucial in facilitating translocation of siRNA payloads from entrapment and degradation in lysosomes toward the cytosol. Eventually, active siRNA could be smoothly released from the synthetic due to the GSH cleavage disulfide linkage (disulfide), consequently accounting for potent RNA knockdown activities (>90%) toward cancerous cells. In addition, appreciable knockdown of parathyroid hormone was also achieved from our proposed siRNA conjugates in parathyroid cells. Hence, the elaborated siRNA conjugate showed tremendous potential in treatment of hyperparathyroidism, and could be developed further for systemic RNA interference (RNAi) therapeutics. Moreover, this study could also be the first example of a synthetic mimic to hyaluronan acquiring its functionalities, which could have important implications for further development of biomimic materials in pursuit of biomedical applications.

Redox-Responsive Polymeric RNAi Based on Multivalent Conjugation of siRNA for Improved Intracellular Delivery.

Learning from the design concept of antibody-drug conjugates (ADCs), we attempted to construct siRNA conjugated polymer brush by attaching a multiple of siRNA to the units of poly(amino acids) [poly(lysine) derivatives] through an intracellular cleavable disulfide bond. Note that the disulfide linkage is stable at extracellular milieu yet subjected to cleavage into free thiol residues at the intracellular reducing compartments. Consequently, ready release of arrays of active siRNA was achieved selectively in the intracellular compartments. Furthermore, tumor-targeted cyclic Asp-Gly-Arg (RGD) was conjugated to the aforementioned polymer brush in view that the RGD receptors (αVß3 and αVß5 integrins) were overexpressed over a wide spectrum of cancerous cells. Our subsequent results have achieved potent gene silencing in cultured cancerous cells from our proposed siRNA delivery construct. To our best knowledge, our proposed conjugate should be the first example of using an ADC platform in successful intracellular transportation of larger macromolecular biological payloads rather than small molecular chemotherapeutic drugs. Hence, the proposed strategy may serve as a promising avenue for targeted delivery of macromolecular pharmaceutical payloads.

Ultrasound-Responsive Nanoparticulate for Selective Amplification of Chemotherapeutic Potency for Ablation of Solid Tumors.

Precision medicine requests preferential transportation of the pharmaceutical substances to the pathological site and impartation of localized therapeutic activities to the targeted cells. To accomplish this goal, we attempted a facile nanoscaled ultrasound-responsive delivery system, characterized by doxorubicin assembled with an amphiphilic copolymer (multiple of hydrophobic stearic segments tethered onto the hydrophilic pullulan backbone through ultrasound-labile oxyl-alkylhydroxylamine linkage). As a consequence of the strategically installed ultrasound-labile oxyl-alkylhydroxylamine linkage to elicit the tailored segregation of the hydrophilic pullulan and the hydrophobic stearic segments upon ultrasound impetus, the constructed nanoscaled self-assembly presented distinctive structural destabilization behaviors and afforded spatiotemporal controlled liberation of the cytotoxic drugs. It is worthy to note that the ultrasound was determined to markedly lower the IC50 of the proposed system from over 10 µg/mL to 2.33 µg/mL (approximate 4-fold), thereby serving as a facile impetus to amplify the cytotoxic potency of the proposed drug delivery vehicles. Furthermore, drastic tumor ablation was validated by dosage of the proposed doxorubicin delivery system to T41 tumor-bearing mice accompanied by the tumor-localized ultrasound impetus, while no observable adverse side effect was confirmed. Therefore, the results advocated our ultrasound-responsive delivery vehicle as a tempting strategy for precise spatiotemporal control of the release of the drug cargo, thus affording selectively amplified cytotoxic potency to the ultrasound-imposed site, which should be highlighted as important progress toward precision medicine.

Rational Design of Multifunctional Polymeric Nanoparticles Based on Poly(l-histidine) and d-α-Vitamin E Succinate for Reversing Tumor Multidrug Resistance.

A multifunctional nanoparticulate system composed of methoxy poly(ethylene glycol)-poly(l-histidine)-d-α-vitamin E succinate (MPEG-PLH-VES) copolymers for encapsulation of doxorubicin (DOX) was elaborated with the aim of circumventing the multidrug resistance (MDR) in breast cancer treatment. The MPEG-PLH-VES nanoparticles (NPs) were subsequently functionalized with biotin motif for targeted drug delivery. The MPEG-PLH-VES copolymer exerts no obvious effect on the P-gp expression level of MCF-7/ADR but exhibited a significant influence on the loss of mitochondrial membrane potential, the reduction of intracellular ATP level, and the inhibition of P-gp ATPase activity of MCF-7/ADR cells. The constructed MPEG-PLH-VES NPs exhibited an acidic pH-induced increase on particle size in aqueous solution. The DOX-encapsulated MPEG-PLH-VES/biotin-PEG-VES (MPEG-PLH-VES/B) NPs were characterized to possess high drug encapsulation efficiency of approximate 90%, an average particle size of approximately 130 nm, and a pH-responsive drug release profile in acidic milieu. Confocal laser scanning microscopy (CLSM) investigations revealed that the DOX-loaded NPs resulted in an effective delivery of DOX into MCF-/ADR cells and a notable carrier-facilitated escape from endolysosomal entrapment. Pertaining to the in vitro cytotoxicity evaluation, the DOX-loaded MPEG-PLH-VES/B NPs resulted in more pronounced cytotoxicity to MCF-/ADR cells compared with DOX-loaded MPEG-PLH-VES NPs and free DOX solution. In vivo imaging study in MCF-7/ADR tumor-engrafted mice exhibited that the MPEG-PLH-VES/B NPs accumulated at the tumor site more effectively than MPEG-PLH-VES NPs due to the biotin-mediated active targeting effect. In accordance with the in vitro results, DOX-loaded MPEG-PLH-VES/B NPs showed the strongest inhibitory effect against the MCF-7/ADR xenografted tumors with negligible systemic toxicity, as evidenced by the histological analysis and change of body weight. The multifunctional MPEG-PLH-VES/B nanoparticulate system has been demonstrated to provide a promising strategy for efficient delivery of DOX into MCF-7/ADR cancerous cells and reversing MDR.

A Targeted and Stable Polymeric Nanoformulation Enhances Systemic Delivery of mRNA to Tumors.

The high vulnerability of mRNA necessitates the manufacture of delivery vehicles to afford adequate protection in the biological milieu. Here, mRNA was complexed with a mixture of cRGD-poly(ethylene glycol) (PEG)-polylysine (PLys) (thiol) and poly(N-isopropylacrylamide) (PNIPAM)-PLys(thiol). The ionic complex core consisting of opposite-charged PLys and mRNA was crosslinked though redox-responsive disulfide linkage, thereby avoiding structural disassembly for exposure of mRNA to harsh biological environments. Furthermore, PNIPAM contributed to prolonged survival in systemic circulation by presenting a spatial barrier in impeding accessibility of nucleases, e.g., RNase, due to the thermo-responsive hydrophilic-hydrophobic transition behavior upon incubation at physiological temperature enabling translocation of PNIPAM from shell to intermediate barrier. Ultimately, the cRGD ligand attached to the formulation demonstrated improved tumor accumulation and potent gene expression, as manifested by virtue of facilitated cellular uptake and intracellular trafficking. These results indicate promise for the utility of mRNA as a therapeutic tool for disease treatment.

Polyplex Micelle with pH-Responsive PEG Detachment and Functional Tetraphenylene Incorporation to Promote Systemic Gene Expression.

Tetraphenylene (TPE), characterized as a lipophilic and aggregation-induced-emissive fluorophore, was used to incorporate into an electrostatic self-assembled polyethylenimine-poly(ethylene glycol) (PEI-PEG)/plasmid DNA (pDNA) complexed micelle. The hydrophobic character of TPE appeared to drive a higher degree of condensation of the pDNA payload, which consequently resulted in not only strengthened colloidal stability of the constructed polyplex micelle but also improved biocompatibility by virtue of the elevated PEG crowdedness owing to the TPE-induced collapse of pDNA. These beneficial consequences potentially permitted a larger number of polyplex micelles to be internalized into the cells. PEG segments were designed to enable selective detachment from polyplex micelles in acidic milieu, e.g., the tumor microenvironment, and intracellular endosome compartment, based on the strategic arrangement of acid-responsive cleavable linkage between PEG and PEI. Upon PEG detachment, the exposure of cationic PEI/TPE polyplex was allowed to directly interact with the cell membrane, endosome membrane, and charged intracellular species, thus promoting cell internalization, endosome escape, and the release of the pDNA payload. Of note, this association of cationic PEI/TPE polyplex with the endosomal membrane could be further facilitated with the aid of lipophilic TPE, thereby eliciting pronounced destabilization potency to the endosome membrane and exerting an endosomal escape function. Eventually, the proposed system of these facile strategies, including responsive PEG detachment and functional TPE incorporation, was proven to provide efficient gene expression in the targeted tumors with an appreciable safety profile via systemic administration.

Poly(ethylene glycol) Crowding as Critical Factor To Determine pDNA Packaging Scheme into Polyplex Micelles for Enhanced Gene Expression.

A critical role of polyethylene glycol (PEG) crowding in the packaging of plasmid DNA (pDNA) into polyplex micelles (PMs) was investigated using a series of PEG-b-poly(l-lysine) (PEG-PLys) block copolymers with varying molecular weights of both PEG and PLys segments. Rod-shaped PMs preferentially formed when the tethered PEG chains covering pDNA in a precondensed state were dense enough to overlap one another (reduced tethering density (RTD) > 1), whereas globular PMs were obtained when they were not overlapped (RTD < 1). These results submitted a scheme that steric repulsive effect of PEG regulated packaging pathways of pDNA either through folding into rod-shape or collapsing into globular depending on whether the PEG chains are overlapped or not. The rod-shaped PMs gave significantly higher gene expression efficacies in a cell-free system compared to the globular PMs, demonstrating the practical relevance of regulating packaging structure of pDNA for developing efficient gene delivery systems.

Bundling potent natural toxin cantharidin within platinum (IV) prodrugs for liposome drug delivery and effective malignant neuroblastoma treatment.

Neuroblastoma (NB) is one of the most commonly seen malignancies in childhood and infancy. Cantharidin is a highly potent natural toxin that possesses potent anti-tumor properties on various cancers including NB. However, exposure to cantharidin can cause severe chemical burns and application of cantharidin for cancer therapy is limited. Here we report a strategy of bundling cantharidin within a hybrid platinum (IV) prodrug conjugate. This hydrophobic drug conjugate, ie, CanPt can be further formulated into liposome for drug delivery to minimize the exposure of cantharidin to normal cells for efficient chemotherapeutic agent against NB.

Rod-to-Globule Transition of pDNA/PEG-Poly(l-Lysine) Polyplex Micelles Induced by a Collapsed Balance Between DNA Rigidity and PEG Crowdedness.

The role of poly(ethylene-glycol) (PEG) in rod-shaped polyplex micelle structures, having a characteristic core of folded plasmid DNA (pDNA) and a shell of tethered PEG chains, is investigated using PEG-detachable polyplex micelles. Rod shapes undergo change to compacted globule shapes by removal of PEG from polyplex micelles prepared from block copolymer with acid-labile linkage between PEG and poly(l-lysine) (PLys) through exposure to acidic milieu. This structural change supports the previous investigation on the rod shapes that PEG shell prevents the DNA structure from being globule shaped as the most favored structure in minimizing surface area. Noteworthy, despite the PEG is continuously depleted, the structural change does not occur in gradual shortening manner but the rod shapes keep their length unchanged and abruptly transform into globule shapes. Analysis of PEG density reveals the transition occurred when tethered PEG of rod shapes has decreased to a critical crowdedness, i.e., discontacted with neighboring PEG, which eventually illuminates another contribution, rigidity of DNA packaged as bundle in the rod shapes, in addition to the steric repulsion of PEG, in sustaining rod shapes. This investigation affirms significant role of PEG and also DNA rigidity as bundle in the formation of rod-shaped structures enduring the quest of compaction of charge-neutralized DNA in the polyplex micelles.

Shedding PEG Palisade by Temporal Photostimulation and Intracellular Reducing Milieu for Facilitated Intracellular Trafficking and DNA Release.

The dilemma of poly(ethylene glycol) surface modification (PEGylation) inspired us to develop an intracellularly sheddable PEG palisade for synthetic delivery systems. Here, we attempted to conjugate PEG to polyethylenimine (PEI) through tandem linkages of disulfide-bridge susceptible to cytoplasmic reduction and an azobenzene/cyclodextrin inclusion complex responsive to external photoirradiation. The subsequent investigations revealed that facile PEG detachment could be achieved in endosomes upon photoirradiation, consequently engendering exposure of membrane-disruptive PEI for facilitated endosome escape. The liberated formulation in the cytosol was further subjected to complete PEG detachment relying on disulfide cleavage in the reductive cytosol, thus accelerating dissociation of electrostatically assembled PEI/DNA polyplex to release DNA by means of polyion exchange reaction with intracellularly charged species, ultimately contributing to efficient gene expression.

Toroidal Packaging of pDNA into Block Ionomer Micelles Exerting Promoted in Vivo Gene Expression.

Selectively spooling single plasmid DNA (pDNA), as a giant polyelectrolyte, into a nanosized toroidal structure or folding it into a rod-like structure has been accomplished by polyion complexation with block catiomers to form polymeric micelles in varying NaCl concentrations. The interactive potency between the pDNA and block catiomers was determined to play a critical role in defining the ultimate structure of the pDNA; the formation of toroidal or rod-like structures was achieved by complexation in 600 or 0 mM NaCl solutions, respectively. Compared with the rod-like structure, the toroidal structure possessed superior biological functions capable not only of elevating in vitro transcription but also of elevating in vivo gene transduction efficiency. This demonstrated the great utility of the toroidal pDNA packaging as a distinct structured gene carrier. Furthermore, the fact that the NaCl concentration at which the toroidal structure was specifically formed corresponds to seawater stimulates interest in this ordered nanostructure as a possible inherent structure for DNA.

A tadpole-shaped gene carrier with distinct phase segregation in a ternary polymeric micelle.

A distinct tadpole-shaped nanostructure characterized by a spherical head and an extended shaft was identified in a single plasmid DNA (pDNA)-based polymeric micelle. The tadpole-shaped structure was constructed by adding anionic chondroitin sulfate (CS) to the rod-shaped polyplex micelle containing a single pDNA molecule packaged by the PEG-polycation block copolymer through their electrostatic self-assembly. The complex consequently developed a novel structure composed of segregated domains of the CS-rich inflated head and CS-poor folded DNA tail. Hence, this tadpole structure can be regarded as evidence that distinct phase segregation occurred in a single polymeric micelle containing pDNA.

Polyplex micelles with thermoresponsive heterogeneous coronas for prolonged blood retention and promoted gene transfection.

Adequate retention in blood circulation is a prerequisite for construction of gene delivery carriers for systemic applications. The stability of gene carriers in the bloodstream requires them to effectively resist protein adsorption and maintain small size in the bloodstream avoiding dissociation, aggregation, and nuclease digestion under salty and proteinous medium. Herein, a mixture of two block catiomers consisting of the same cationic block, poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (PAsp(DET)), but varying shell-forming blocks, poly[2-(2-methoxyethoxy) ethyl methacrylate] (PMEO2MA), and poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMA), was used to complex with plasmid DNA (pDNA) to fabricate polyplex micelles with mixed shells (MPMs) at 20 °C. The thermoresponsive property of PMEO2MA allows distinct phase transition from hydrophilic to hydrophobic by increasing incubation temperature from 20 to 37 °C, which results in a distinct heterogeneous corona containing hydrophilic and hydrophobic regions at the surface of the MPMs. Subsequent study verified that this transition promoted further condensation of pDNA, thereby giving rise to improved complex and colloidal stability. The proposed system has shown remarkable stability in salty and proteinous solution and superior tolerance to nuclease degradation. As compared with polyplex micelles formed from single POEGMA-b-PAsp(DET) block copolymer, in vivo circulation experiments in the bloodstream further confirmed that the retention time of MPMs was prolonged significantly. Moreover, the proposed system exhibited remarkably high cell transfection activity especially at low N/P ratios and negligible cytotoxicity and thus portends promising utility for systemic gene therapy applications.

A gene delivery system with autophagy blockade for enhanced anti-angiogenic therapy against Fusobacterium nucleatum-associated colorectal cancer.

Anti-angiogenesis has emerged a promising strategy against colorectal cancer (CRC). However, the efficacy of anti-angiogenic therapy is greatly compromised by the up-regulated autophagy levels resulting from the evolutionary resistance mechanism and the presence of Fusobacterium nucleatum (F. nucleatum) in CRC. Herein, we report a cationic polymer capable of blocking autophagic flux to deliver plasmid DNA (pDNA) encoding soluble FMS-like tyrosine kinase-1 (sFlt-1) for enhanced anti-angiogenic therapy against F. nucleatum-associated CRC. The autophagy-inhibiting cationic polymer, referred to as PNHCQ, is synthesized by conjugating hydroxychloroquine (HCQ) into 3,3'-diaminodipropylamine-pendant poly(ß-benzyl-L-aspartate) (PAsp(Nors)), which can be assembled and electrostatically interacted with sFlt-1 plasmid to form PNHCQ/sFlt-1 polyplexes. Hydrophobic HCQ modification not only boosts transfection efficiency but confers autophagy inhibition activity to the polymer. Hyaluronic acid (HA) coating is further introduced to afford PNHCQ/sFlt-1@HA for improved tumor targeting without compromising on transfection. Consequently, PNHCQ/sFlt-1@HA demonstrates significant anti-tumor efficacy in F. nucleatum-colocalized HT29 mouse xenograft model by simultaneously exerting anti-angiogenic effects through sFlt-1 expression and down-regulating autophagy levels exacerbated by F. nucleatum challenge. The combination of anti-angiogenic gene delivery and overall autophagy blockade effectively sensitizes CRC tumors to anti-angiogenesis, providing an innovative approach for enhanced anti-angiogenic therapy against F. nucleatum-resident CRC. STATEMENT OF SIGNIFICANCE: Up-regulated autophagy level within tumors is considered responsible for the impaired efficacy of clinic antiangiogenic therapy against CRC colonized with pathogenic F. nucleatum. To tackle this problem, an autophagy-inhibiting cationic polymer is developed to enable efficient intracellular delivery of plasmid DNA encoding soluble FMS-like tyrosine kinase-1 (sFlt-1) and enhance anti-angiogenic therapy against F. nucleatum-associated CRC. HA coating that can be degraded by tumor-enriching hyaluronidase is further introduced for improved tumor targeting without compromising transfection efficiency. The well-orchestrated polyplexes achieve considerable tumor accumulation, efficient in vivo transfection, and effectively reinforce the sensitivity of CRC to the sFlt-1-derived anti-angiogenic effects by significantly blocking overall autophagy flux exacerbated by F. nucleatum challenge, thus harvesting robust antitumor outcomes against F. nucleatum-resident CRC.

Amphiphilic polymeric nanodrug integrated with superparamagnetic iron oxide nanoparticles for synergistic antibacterial and antitumor therapy of colorectal cancer.

Colorectal cancer (CRC) is one of the most prevalent and deadly malignancies that can be influenced by Fusobacterium nucleatum (Fn), a bacterium that promotes tumor development and chemoresistance, resulting in limited therapeutic efficacy. Traditional antibiotics cannot effectively eliminate Fn at tumor site due to issues like biofilm formation, while chemotherapy alone fails to suppress tumor progression. Therefore, the development of new methods to eliminate Fn and promote antitumor efficacy is of great significance for improving the outcome of CRC treatment. Herein, we developed a nanodrug (OPPL) that integrates oleic acid-modified superparamagnetic iron oxide nanoparticles (O-SPIONs) and an amphiphilic polymer (PPL) to deliver the platinum prodrug and antimicrobial lauric acid (LA) for enhancing the treatment of CRC. We demonstrated that OPPL can synergistically enhance antibacterial and biofilm disruption activities against Fn along with the antimicrobial LA by producing reactive oxygen species (ROS) through its peroxidase-like activity. Furthermore, the OPPL nanodrug can increase intracellular ROS, promote lipid peroxides and deplete glutathione, leading to ferroptosis. By combining chemotherapy and induced ferroptosis, the OPPL nanodrug exhibited high cytotoxicity against CRC cells. In vivo studies showed that the OPPL nanodrug could enhance tumor accumulation, enable magnetic resonance imaging, suppresse tumor growth, and inhibit growth of intratumor Fn. These results suggest that OPPL is an effective and promising candidate for the treatment of Fn-infected CRC. STATEMENT OF SIGNIFICANCE: The enrichment of Fusobacterium nucleatum (Fn) in colorectal cancer is reported to exacerbate tumor malignancy and is particularly responsible for chemoresistance. To this respect, we strategically elaborated multifaceted therapeutics, namely OPPL nanodrug, combining oleic acid-modified superparamagnetic iron oxide nanoparticles (O-SPIONs) with a polymer containing a platinum prodrug and antimicrobial lauric acid. The O-SPION components exert distinctive peroxidase-like activity, capable of stimulating Fenton reactions selectively in the tumor microenvironment, consequently accounting for the progressive production of reactive oxygen species. Hence, O-SPIONs have been demonstrated to not only supplement the antimicrobial activities of lauric acid in overcoming Fn-induced chemoresistance but also stimulate potent tumor ferroptosis. Our proposed dual antimicrobial and chemotherapeutic nanodrug provides an appreciable strategy for managing challenging Fn-infected colorectal cancer.