Spatiotemporally Controlled T-Cell Combination Therapy for Solid Tumor.

Due to multidimensional complexity of solid tumor, development of rational T-cell combinations and corresponding formulations is still challenging. Herein, a triple combination of T cells are developed with Indoleamine 2,3-dioxygenase inhibitors (IDOi) and Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i). To maximize synergism, a spatiotemporally controlled T-cell engineering technology to formulate triple drugs into one cell therapeutic, is established. Specifically, a sequentially responsive core-shell nanoparticle (SRN) encapsulating IDOi and CDK4/6i is anchored onto T cells. The yielded SRN-T cells migrated into solid tumor, and achieved a 1st release of IDOi in acidic tumor microenvironment (TME). Released IDOi restored tryptophan supply in TME, which activated effector T cells and inhibited Tregs. Meanwhile, 1st released core is internalized by tumor cells and degraded by glutathione (GSH), to realize a 2nd release of CDK4/6i, which induced up-regulated expression of C-X-C motif chemokine ligand 10 (CXCL10) and C-C motif chemokine ligand 5 (CCL5), and thus significantly increased tumor infiltration of T cells. Together, with an enhanced recruitment and activation, T cells significantly suppressed tumor growth, and prolonged survival of tumor-bearing mice. This study demonstrated rationality and superiority of a tri-drug combination mediated by spatiotemporally controlled cell-engineering technology, which provides a new treatment regimen for solid tumor.

Light-mediated intracellular polymerization.

The synthesis of synthetic intracellular polymers offers groundbreaking possibilities in cellular biology and medical research, allowing for novel experiments in drug delivery, bioimaging and targeted cancer therapies. These macromolecules, composed of biocompatible monomers, are pivotal in manipulating cellular functions and pathways due to their bioavailability, cytocompatibility and distinct chemical properties. This protocol details two innovative methods for intracellular polymerization. The first one uses 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959) as a photoinitiator for free radical polymerization under UV light (365 nm, 5 mW/cm2). The second method employs photoinduced electron transfer-reversible addition-fragmentation chain-transfer polymerization with visible light (470 nm, 100 mW/cm2). We further elaborate on isolating these intracellular polymers by streptavidin/biotin interaction or immobilized metal ion affinity chromatography for polymers tagged with biotin or histidine. The entire process, from polymerization to isolation, takes ~48 h. Moreover, the intracellular polymers thus generated demonstrate significant potential in enhancing actin polymerization, in bioimaging applications and as a novel avenue in cancer treatment strategies. The protocol extends to animal models, providing a comprehensive approach from cellular to systemic applications. Users are advised to have a basic understanding of organic synthesis and cell biology techniques.

Trap & kill: a neutrophil-extracellular-trap mimic nanoparticle for anti-bacterial therapy.

Fenton chemistry-mediated antimicrobials have demonstrated great promise in antibacterial therapy. However, the short life span and diffusion distance of hydroxyl radicals dampen the therapeutic efficiency of these antimicrobials. Herein, inspired by the neutrophil extracellular trap (NET), in which bacteria are trapped and agglutinated via electronic interactions and killed by reactive oxygen species, we fabricated a NET-mimic nanoparticle to suppress bacterial infection in a "trap & kill" manner. Specifically, this NET-mimic nanoparticle was synthesized via polymerization of ferrocene monomers followed by quaternization with a mannose derivative. Similar to the NET, the NET-mimic nanoparticles trap bacteria through electronic and sugar-lectin interactions between their mannose moieties and the lectins of bacteria, forming bacterial agglutinations. Therefore, they confine the spread of the bacteria and restrict the bacterial cells to the destruction range of hydroxyl radicals. Meanwhile, the ferrocene component of the nanoparticle catalyzes the production of highly toxic hydroxyl radicals at the H2O2 rich infection foci and effectively eradicates the agglutinated bacteria. In a mouse model of an antimicrobial-resistant bacteria-infected wound, the NET-mimic nanoparticles displayed potent antibacterial activity and accelerated wound healing.

Cell-surface photochemistry mediated calcium overload for synergistic tumor therapy.

Calcium (Ca2+) is essential for mitochondrial homeostasis and function coordination, particularly in cancer cells that metabolize frequently to sustain their growth. Photochemistry mediated calcium overload has attracted lots of attention as an effective way to achieve tumor suppression. Herein, we developed a photonanomedicine to synergistically induce calcium overload via cell-surface photochemistry and thus tumor suppression. Specifically, the photosensitizer, protoporphyrin IX (PpIX) was loaded onto upconversion nanoparticles (UCNP), which was subsequently modified by a polymer bearing photo-crosslinking cinnamate (CA) groups. The resulting nanoparticle was further functionalized by anti-CD20 aptamers (Apt), to give photonanomedicine. The interaction between CD20 receptors and anti-CD20 aptamers allowed photonanomedicine to accurately attach onto the Raji cell surface after an intravenous injection. Following the local application of a 980 nm NIR laser, the photonanomedicine was able to capture the NIR light and convert it into ultraviolet (UV) light. On one hand, the converted UV light led the crosslinking of cinnamate groups in photonanomedicine, further stimulating the clustering of CD20 receptors and causing Ca2+ influx. On the other hand, the UV light could simultaneously excited PpIX to generate reactive oxygen species (ROS) in situ to break down the integrity of cell membrane and lead to an influx of Ca2+. The synergistic Ca2+ overload mediated by photonanomedicine exhibited an enhanced and superior anti-tumor efficacy. We believe this photonanomedicine expands the toolbox to manipulate intracellular Ca2+ concentration and holds a great potential as an anti-tumor therapy.

A photo-activable nano-agonist for the two-signal model of T cell in vivo activation.

The two-signal model of T cell activation has helped shape our understanding of the adaptive immune response for over four decades. According to the model, activation of T cells requires a stimulus through the T cell receptor/CD3 complex (signal 1) and a costimulatory signal 2. Stimulation of activatory signals via T cell agonists has thus emerged. However, for a robust T cell activation, it necessitates not only the presence of both signal 1 and signal 2, but also a high signaling strength. Herein, we report a photo-activable nano-agonist for the two-signal model of T cell in vivo activation. A UV-crosslinkable polymer is coated onto upconversion nanoparticles with satisfactory NIR-to-UV light conversion efficiency. Then dual signal molecules, i.e., signal 1 and signal 2, are conjugated to the polymer end to yield the photo-activable T cell nano-agonist. In melanoma and breast cancer models, photo-activable nano-agonist could bind onto corresponding activatory receptors on the surface of T cells, but has limited activity without the application of NIR light (absence of photo-crosslinking of receptors and consequently a poor signaling strength). While when the NIR light is switched on locally, T cells in tumor are remarkably activated and kill tumor cells effectively. Moreover, we do not observe any detectable toxicities related to the photo-activable nano-agonist. We believe with two activatory signals being simultaneously strengthened by local photo-switched crosslinking, T cells realize a robust and selective activation in tumor and, consequently contribute to an enhanced and safe tumor immunotherapy.

Achieving tunable Kerker-type invisibility for a radiation-enhanced electrically small antenna.

Low-temperature gaseous plasmas exhibit great potential in designing tunable and reconfigurable electromagnetic devices. In this paper, based on an overdense-underdense core-shell plasma structure, tunable Kerker-type invisibility for a radiation-enhanced electrically small antenna is achieved, where dominant scattering direction can be mutated between backward and forward while omnidirectional invisibility and signal enhancement are maintained. Moreover, by electromagnetic multipole decompositions, it is shown that the underdense outer plasma with a negative polarizability is able to weaken the strength and modulate the phase of the electric dipolar scattering component (a_{1}), while the magnetic dipolar term (b_{1}) nearly remains unchanged. Consequently, quasi-first and -second Kerker conditions are fulfilled near the cutoff band of a_{1}.

An EPR-Independent extravasation Strategy: Deformable leukocytes as vehicles for improved solid tumor therapy.

Effective delivery of therapeutic modality throughout the tumorous nidus plays a crucial role in successful solid tumor treatment. However, conventional nanomedicines based on enhanced permeability and retention (EPR) effect have yielded limited delivery/therapeutic efficiency, due mainly to the heterogeneity of the solid tumor. Leukocytes, which could intrinsically migrate across the vessel wall and crawl through tissue interstitium in a self-deformable manner, have currently emerged as an alternative drug delivery vehicle. In this review, we start with the intrinsic properties of leukocytes (e.g., extravasation and crawling inside tumor), focusing on unveiling the conceptual rationality of leveraging leukocytes as EPR-independent delivery vehicles. Then we discussed various cargoes-loading/unloading strategies for leukocyte-based vehicles as well as their promising applications. This review aims to serve as an up-to-date compilation, which might provide inspiration for scientists in the field of drug delivery.

Polypyrrole Percolation Network Gas Sensors: Improved Reproducibility through Conductance Monitoring during Polymer Growth.

Conducting-polymer-based electrical percolation networks are promising materials for use in high-sensitivity chemiresistive devices. An ongoing challenge is to create percolation networks that have consistent properties, so that devices based on these materials do not have to be individually calibrated. Here, an in situ conductance technique is used during the electrochemical growth of polypyrrole (PPy) percolation networks. The drain current (i d) across the interdigitated electrodes (IDEs) is a measure of the conductance of the PPy network during electrochemical polymerization. The i d curve is used to determine the percolation region. To improve the reproducibility of PPy percolation networks, an in situ conductance monitoring method based on the value of i d is used. A set of optimal ammonia gas percolation sensors was created using this method with an average sensitivity of ΔR/R 0 × 100% ppm-1 = 11.3 ± 1.2% ppm-1 and an average limit of detection of 15.0 ± 3.6 ppb.

Controlled Intracellular Polymerization for Cancer Treatment.

Numerous prodrugs have been developed and used for cancer treatments to reduce side effects and promote efficacy. In this work, we have developed a new photoactivatable prodrug system based on intracellular photoinduced electron transfer-reversible addition-fragmentation chain-transfer (PET-RAFT) polymerization. This unique polymerization process provided a platform for the synthesis of structure-predictable polymers with well-defined structures in living cells. The intracellularly generated poly(N,N-dimethylacrylamide)s were found to induce cell cycle arrest, apoptosis, and necroptosis, inhibit cell proliferation, and reduce cancer cell motilities. This polymerization-based "prodrug" system efficiently inhibits tumor growth and metastasis both in vitro and in vivo and will promote the development of targeted and directed cancer chemotherapy.

Neutrophil Cyto-Pharmaceuticals Suppressing Tumor Metastasis via Inhibiting Hypoxia-Inducible Factor-1α in Circulating Breast Cancer Cells.

Circulating tumor cells (CTCs) are reported as the precursor of tumor metastases, implying that stifling CTCs would be beneficial for metastasis prevention. However, challenges remain for the application of therapies that aim at CTCs due to lack of effective CTC-targeting strategy and sensitive therapeutic agents. Herein, a general CTC-intervention strategy based on neutrophil cyto-pharmaceuticals is proposed for suppressing CTC colonization and metastasis formation. Breast cancer 4T1 cells are infused as the mimic CTCs, and 4T1 cells trapped are first elucidated in neutrophil extracellular traps (NETs) expressing high levels of hypoxia-inducible factor-1α (HIF-1α) due to NET formation and thus promoting tumor cell colonization through enhanced migration, invasion and stemness. After verifying HIF-1α as a potential target for metastasis prevention, living neutrophil cyto-pharmaceuticals (CytPNEs) loaded with HIF-1α inhibitor are fabricated to therapeutically inhibit HIF-1α. It is demonstrated that CytPNEs can specially convey the HIF-1α inhibitor to 4T1 cells according to the inflammatory chemotaxis of neutrophils and down-regulate HIF-1α, thereby inhibiting metastasis and prolonging the median survival of mice bearing breast cancer lung metastasis. The research offers a new perspective for understanding the mechanism of CTC colonization, and puts forward the strategy of targeted intervention of CTCs as a meaningful treatment for tumor metastasis.

Simultaneous blockage of contextual TGF-ß by cyto-pharmaceuticals to suppress breast cancer metastasis.

It remains challenging to treat tumor metastasis currently in the light of multiple cascade processes of tumor metastasis. Additionally, multiple clinical drugs for metastasis have quite limited therapeutic potential and even facilitate metastasis in preclinical models. Thus, potential metastasis targets and novel metastasis-directed drugs are urgently needed to be further developed. Herein, transforming growth factor-ß (TGF-ß) is verified to contribute to lung metastasis in a context-dependent manner in the 4T1 orthotopic tumor-bearing mice model, which induces epithelial-mesenchymal-transition (EMT) to promote tumor dissemination from the primary site and dampens the anti-tumor response of neutrophils to support tumor colonization at the metastatic niche. In view of neutrophils' superior tropism towards both inflammatory primary tumor and metastatic niche, SB525334, a TGF-ß receptor inhibitor, is loaded into cationic liposome (SBLP) which is subsequently incorporated into neutrophils to yield the cyto-pharmaceuticals (SBLP/NE). The systemically infused SBLP/NE can simultaneously migrate into both primary and metastatic sites, then release SB525334 in response to tumor stimuli, and contextually inhibit TGF-ß-mediated-EMT and phenotype reversal of infiltrated neutrophils, showing substantial metastasis suppression efficacy without causing any detectable toxicities. This project shifts the paradigm for metastasis suppression therapy by simultaneous blockage of contextual TGF-ß using metastatic-cascades-targeting neutrophil cyto-pharmaceuticals.

Combination of metabolic intervention and T cell therapy enhances solid tumor immunotherapy.

Treatment of solid tumors with T cell therapy has yielded limited therapeutic benefits to date. Although T cell therapy in combination with proinflammatory cytokines or immune checkpoints inhibitors has demonstrated preclinical and clinical successes in a subset of solid tumors, unsatisfactory results and severe toxicities necessitate the development of effective and safe combinatorial strategies. Here, the liposomal avasimibe (a metabolism-modulating drug) was clicked onto the T cell surface by lipid insertion without disturbing the physiological functions of the T cell. Avasimibe could be restrained on the T cell surface during circulation and extravasation and locally released to increase the concentration of cholesterol in the T cell membrane, which induced rapid T cell receptor clustering and sustained T cell activation. Treatment with surface anchor-engineered T cells, including mouse T cell receptor transgenic CD8+ T cells or human chimeric antigen receptor T cells, resulted in superior antitumor efficacy in mouse models of melanoma and glioblastoma. Glioblastoma was completely eradicated in three of the five mice receiving surface anchor-engineered chimeric antigen receptor T cells, whereas mice in other treatment groups survived no more than 64 days. Moreover, the administration of engineered T cells showed no obvious systemic side effects. These cell-surface anchor-engineered T cells hold translational potential because of their simple generation and their safety profile.

Cytopharmaceuticals: An emerging paradigm for drug delivery.

Cytopharmaceuticals, in which drugs/nanomedicines are loaded into/onto autologous patient- or allogeneic donor-derived living cells ex vivo, have displayed great promise for targeted drug delivery in terms of improved biocompatibility, superior targeting, and prolonged circulation. Despite certain impressive therapeutic benefits in preclinical studies, several obstacles retard their clinical application, such as the lack of facile and convenient methods of carrier cell acquisition, technologies for preparing cytopharmaceuticals at scale with undisturbed carrier cell viability, and modalities for monitoring the in vivo fate of cytopharmaceuticals. To comprehensively understand cytopharmaceuticals and thereby accelerate their clinical translation, this review covers the main sources of various cytopharmaceuticals, technologies for preparing cytopharmaceuticals, the in vivo fate of cytopharmaceuticals including carrier cells and loaded drugs/nanomedicines, and the application prospects of cytopharmaceuticals. It is our hope that this review will elucidate the bottlenecks associated with cytopharmaceutical preparation, leading to the acceleration of future industrialization of cell-based formulations.

Cyto-friendly polymerization at cell surfaces modulates cell fate by clustering cell-surface receptors.

Lots of strategies, e.g. using multivalent synthetic polymers, have been developed to control the spatial distribution of cell-surface receptors, thus modulating the cell function and fate in a custom-tailored manner. However, clustering cell-surface receptors via multivalent synthetic polymers is highly dependent on the structure as well as the ligand-density of the polymers, which may impose difficulties on the synthesis of polymers with a high density of ligands. Here, we pioneered the utilization of a cyto-friendly polymerization at the cell surface to cluster cell-surface receptors. As a proof of concept, an anti-CD20 aptamer conjugated macromer was initially synthesized, which was then efficiently and stably introduced onto the Raji cell surface via ligand-receptor interaction. With the assistance of an initiator, i.e. ammonium peroxysulfate (APS), the macromer bound onto the Raji cell surface polymerized, inducing the clustering of CD20 receptors, and thereby triggering cell apoptosis. This cell-surface polymerization induced cell-surface receptor crosslinking could alternatively be applied in modulating the fates and functions of other cells, especially those mediated by the spatial distribution of cell-surface receptors, such as T cell activation. Our work opens new possibilities in the area of chemical biology to some extent.

Iron chelated melanin-like nanoparticles for tumor-associated macrophage repolarization and cancer therapy.

Tumor-associated macrophages (TAMs) are abundant in many cancers, and predominately display an immunosuppressive M2-like function that fosters tumor progression and promotes malignant metastasis. Current TAMs repolarization strategies mainly focused on harnessing the direct cancer cell killing property of M1-like macrophages repolarized from TAMs. However, the latent role of M1-like macrophages as professional antigen-presenting cells (APCs) also needs to be explored. Here, iron chelated melanin-like nanoparticles (Fe@PDA-PEG) were developed for M2-to-M1 TAMs repolarization and photothermal therapy (PTT) induced tumor-associated antigens (TAAs) releasing, which would exploit the potential of M1-like macrophages acquired as professional APCs for TAAs presentation. The results showed that M1 macrophages repolarized from TAMs by Fe@PDA-PEG could capture, process and present TAAs released by PTT through the major histocompatibility complex class II (MHC II) pathway, recruiting T-helper cells and effector T cells in tumor site, which leads to the controlled tumor growth and limited malignant metastasis.

pH and Thermal Dual-Sensitive Nanoparticle-Mediated Synergistic Antitumor Effect of Immunotherapy and Microwave Thermotherapy.

Cationic anticancer peptides, which can induce tumor cell immunogenic death and further promote systemic tumor-specific immune responses, have offered a promising solution to relieve the tumor immunosuppressive microenvironment. However, peptide drugs are easily degraded and lack of targeting ability when administered systemically, leading to limitations in their applications. Herein, we report a pH and thermal dual-sensitive bovine lactoferricin-loaded (one of the most widely studied cationic anticancer peptides) nanoparticles, which simultaneously exhibited antitumor and immune cell activated effects when applied with microwave thermotherapy, an auxiliary method of immunotherapy. The bovine lactoferricin could be delivered to the tumor site by nanoparticles, be immediately released from nanoparticles in the acidic environment of lysosomes and the thermal condition caused by microwave radiation, and ultimately induce tumor apoptosis with the release of damage-associated molecular patterns (DAMPs). It is worth noting that the strategy of bovine lactoferricin-loaded nanoparticles intravenous injection combined with local microwave thermotherapy not only showed excellent efficacy in relieving tumor growth but also resulted in strong antitumor immunities, which was due to the released bovine lactoferricin under stimulating conditions, and the pool of tumor-associated antigens generated by tumor destruction. In conclusion, this work presents a strategy for tumor treatment based on dual-sensitive bovine lactoferricin-loaded nanoparticles combined with microwave thermotherapy, which may provide a solution for cationic anticancer peptides delivery and improving antitumor immune responses.

Tocopherol polyethylene glycol succinate-modified hollow silver nanoparticles for combating bacteria-resistance.

Multiple drug resistance and the increase in the appearance of superbugs together with the exceedingly scant development of new potent antibiotic drugs pose an urgent global medical threat and imminent public security crisis. In the present study, we fabricated well-dispersed tocopherol polyethylene glycol succinate (TPGS)-capped silver nanoparticles (AgNPs) of about 10 nm in size. The hollow structure of the TPGS-capped AgNPs (TPGS/AgNPs) was confirmed and applied to load antibiotics. The TPGS/AgNPs proved to be able to cross the bacterial cell wall and penetrate into bacteria, thereby delivering more of the antibiotic to the interior of bacteria and thus enhancing the in vitro antibacterial effect of the antibiotic, even overcoming the drug-resistance in drug-resistant E. coli and Acinetobacter baumannii. It was found that the TPGS modification in the TPGS/AgNPs could decrease the activity of the efflux pumps AdeABC and AdeIJK in drug-resistant Acinetobacter baumannii via inhibiting the efflux pump genes adeB and adeJ, thus increasing the accumulation of the delivered antibiotic and overcoming the drug-resistance. Tigecycline delivered by TPGS/AgNPs could effectively antagonize drug-resistance in an acute peritonitis model mice, thereby increasing the survival rate and alleviating the inflammatory response. TPGS/AgNPs were developed as a novel and effective antibiotic delivery system and TPGS was demonstrated to have great potential as a pharmaceutical excipient for use in drug-resistant infection therapy.

Radical polymerization inside living cells.

Polymerization reactions conducted inside cells must be compatible with the complex intracellular environment, which contains numerous molecules and functional groups that could potentially prevent or quench polymerization reactions. Here we report a strategy for directly synthesizing unnatural polymers in cells through free radical photopolymerization using a number of biocompatible acrylic and methacrylic monomers. This offers a platform to manipulate, track and control cellular behaviour by the in cellulo generation of macromolecules that have the ability to alter cellular motility, label cells by the generation of fluorescent polymers for long-term tracking studies, as well as generate a variety of nanostructures within cells. It is remarkable that free radical polymerization chemistry can take place within such complex cellular environments. This demonstration opens up a multitude of new possibilities for how chemists can modulate cellular function and behaviour and for understanding cellular behaviour in response to the generation of free radicals.

Polycaprolactone/polysialic acid hybrid, multifunctional nanofiber scaffolds for treatment of spinal cord injury.

Scaffold-based tissue engineering is widely used for spinal cord injury (SCI) treatment by creating supporting and guiding neuronal tissue regeneration. However, how to enhance the axonal regeneration capacity following SCI still remains a challenge. Polysialic acid (PSA), a natural, biodegradable polysaccharide, has been increasingly explored for controlling central nervous system (CNS) development by regulating cell adhesive properties and promoting axonal growth. Here, a polycaprolactone (PCL)/PSA hybrid nanofiber scaffold encapsulating glucocorticoid methylprednisolone (MP) is developed for SCI treatment. Rat models with spinal cord transection is established and the PCL/PSA/MP scaffold is transplanted into lesion area. PCL/PSA/MP scaffold decreases tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) release by inhibiting ionized calcium-binding adapter molecule 1 (Iba1) positive microglia/macrophage activation and reduces apoptosis-associated Caspase-3 protein expression. In addition, the PCL/PSA/MP scaffold inhibits axonal demyelination and glial fibrillary acidic protein (GFAP) expression, increases neurofilament 200 (NF-200) expression and improves functional outcome by Basso, Beattie and Bresnahan (BBB) test. These results demonstrate the therapeutic potential of PSA hybrid nanofiber scaffold in promoting axonal growth and enhancing the functional recovery following SCI. STATEMENT OF SIGNIFICANCE: Scaffold-based tissue engineering is widely used for spinal cord injury (SCI) treatment by creating supporting and guiding neuronal tissue regeneration. And how to enhance the axonal regeneration capacity following SCI still remains a challenge. Polysialic acid (PSA), a natural, biodegradable polysaccharide, has been increasingly explored for controlling central nervous system (CNS) development by regulating cell adhesive properties and promoting axonal growth. However, in vivo therapeutic effect of PSA scaffolds towards SCI is still lack of evidence and needs to be further explored. In this study, a novel electrospun polycaprolactone/PSA scaffold loaded with methylprednisolone (MP) was developed to achieve efficient therapeutic effects towards SCI. And we believe that it broadens the application of PSA for SCI treatment.