Surface programmed bacteria as photo-controlled NO generator for tumor immunological and gas therapy.

The use of bacteria as living vehicles has attracted increasing attentions in tumor therapy field. The combination of functional materials with bacteria dramatically facilitates the antitumor effect. Here, we presented a rationally designed living system formed by programmed Escherichia Coli MG1655 cells (Ec) and black phosphorus (BP) nanoparticles (NPs). The bacteria were genetically engineered to express tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), via an outer membrane YiaT protein (Ec-T). The Ec-T cells were associated with BP NPs on their surface to acquire BP@Ec-T. The designed living system could transfer the photoelectrons produced by BP NPs after laser irradiation and triggered the reductive metabolism of nitrate to nitric oxide for the in situ release at tumor sites, facilitating the therapeutic efficacy and the polarization of tumor associated macrophages to M1 phenotype. Meanwhile, the generation of reactive oxygen species induced the immunogenic cell death to further improve the antitumor efficacy. Additionally, the living system enhanced the immunological effect by promoting the apoptosis of tumor cells, activating the effect of T lymphocytes and releasing the pro-inflammatory cytokines. The integration of BP NPs, MG1655 cells and TRAIL led to an effective tumor therapy. Our work established an approach for the multifunctional antitumor living therapy.

Degradable Multifunctional Porphyrin-Based Porous Organic Polymer Nanosonosensitizer for Tumor-Specific Sonodynamic, Chemo- and Immunotherapy.

Sonodynamic therapy (SDT) benefiting from its intrinsic merits, such as noninvasiveness and deep tissue penetrability, is receiving increasing considerable attention in reactive oxygen species (ROS)-based tumor treatment. However, current sonosensitizers usually suffer from low tumor lesion accumulation, insufficient ROS generation efficiency under ultrasound, and non-biodegradability, which seriously impede the therapeutic outcomes. Additionally, it is difficult that SDT alone can completely eradicate tumors because of the complex and immunosuppressive tumor microenvironment (TME). Herein, we simultaneously employ sonosensitive porphyrin building blocks and glutathione (GSH)-responsive disulfide bonds to construct a novel degradable multifunctional porphyrin-based hollow porous organic polymer (POP) nanosonosensitizer (H-Pys-HA@M/R), which combine SDT, "on-demand" chemotherapy, and immunotherapy. Taking the unique advantages of POPs with designable structures and high specific surface area, this H-Pys-HA@M/R nanosonosensitizer can achieve tumor target accumulation, GSH-triggered drug release, and low-frequency ultrasound-activating ROS generation with encouraging results. Furthermore, this multifunctional nanosonosensitizer can effectively evoke immunogenic cell death (ICD) response through the combination of SDT and chemotherapy for both primary and distal tumor growth suppression. Meanwhile, H-Pys-HA@M/R exhibits favorable biodegradation and biosafety. Therefore, this study provides a new strategy for reasonably designing and constructing POP-related sonosensitizers combining SDT/chemotherapy/immunotherapy triple treatment modalities to eradicate malignant tumors.

Organizing Enzymes on Self-Assembled Protein Cages for Cascade Reactions.

Cells use self-assembled biomaterials such as lipid membranes or proteinaceous shells to coordinate thousands of reactions that simultaneously take place within crowded spaces. However, mimicking such spatial organization for synthetic applications in engineered systems remains a challenge, resulting in inferior catalytic efficiency. In this work, we show that protein cages as an ideal scaffold to organize enzymes to enhance cascade reactions both in vitro and in living cells. We demonstrate that not only enzyme-enzyme distance but also the improved Km value contribute to the enhanced reaction rate of cascade reactions. Three sequential enzymes for lycopene biosynthesis have been co-localized on the exterior of the engineered protein cages in Escherichia coli, leading to an 8.5-fold increase of lycopene production by streamlining metabolic flux towards its biosynthesis. This versatile system offers a powerful tool to achieve enzyme spatial organization for broad applications in biocatalysis.

Programmable living assembly of materials by bacterial adhesion.

The field of engineered living materials aims to construct functional materials with desirable properties of natural living systems. A recent study demonstrated the programmed self-assembly of bacterial populations by engineered adhesion. Here we use this strategy to engineer self-healing living materials with versatile functions. Bacteria displaying outer membrane-anchored nanobody-antigen pairs are cultured separately and, when mixed, adhere to each other to enable processing into functional materials, which we term living assembled material by bacterial adhesion (LAMBA). LAMBA is programmable and can be functionalized with extracellular moieties up to 545 amino acids. Notably, the adhesion between nanobody-antigen pairs in LAMBA leads to fast recovery under stretching or bending. By exploiting this feature, we fabricated wearable LAMBA sensors that can detect bioelectrical or biomechanical signals. Our work establishes a scalable approach to produce genetically editable and self-healable living functional materials that can be applied in biomanufacturing, bioremediation and soft bioelectronics assembly.

Multifunctional FeS2@SRF@BSA nanoplatform for chemo-combined photothermal enhanced photodynamic/chemodynamic combination therapy.

Combination therapy has been widely studied due to its promising applications in tumor therapy. However, a sophisticated nanoplatform and sequential irradiation with different laser sources for phototherapy complicate the treatment process. Unlike the integration of therapeutic agents, we report a FeS2@SRF@BSA nanoplatform for the combination of chemo-combined photothermal therapy (PTT) enhanced photodynamic therapy (PDT) and chemodynamic therapy (CDT) to achieve an "all-in-one" therapeutic agent. Ultrasmall FeS2 nanoparticles (NPs) with a size of 7 nm exhibited higher Fenton reaction rates due to their large specific surface areas. A photodynamic reaction could be triggered and could generate 1O2 to achieve PDT under 808 nm irradiation. FeS2 NPs also exhibited the desired photothermal properties under the same wavelength of the laser. The Fenton reaction and photodynamic reaction were both significantly improved to accumulate more reactive oxygen species (ROS) with an increase of temperature under laser irradiation. Besides, loading of the chemotherapeutic drug sorafenib (SRF) further improved the efficacy of tumor treatment. To realize long blood circulation, bovine serum albumin (BSA) was used as a carrier to encapsulate FeS2 NPs and SRF, remarkably improving the biocompatibility and tumor enrichment ability of the nanomaterials. Additionally, the tumors on mice treated with FeS2@SRF@BSA almost disappeared under 808 nm irradiation. To sum up, FeS2@SRF@BSA NPs possess good biocompatibility, stability, and sufficient therapeutic efficacy in combination therapy for cancer treatment. Our study pointed out a smart design of the nanoplatform as a multifunctional therapeutic agent for combination cancer therapy in the near future.

Cationic cell penetrating peptide modified SNARE protein VAMP8 as free chains for gene delivery.

Previously, our group carried out a series of studies using branched polyethyleneimine with 25 000 g mol-1 molar mass (bPEI-25k) as a gene delivery vector and came up with the theory that free cationic chains un-complexed with plasmid DNA (pDNA) can greatly increase the gene transfection efficiency and influence the intracellular delivery process. These free chains can penetrate the membrane quickly, with some of them embedded inside the lipid bi-layers. The "stuck-out" cationic chain ends would shield the signal protein, prevent/delay the development of the later endolysosomes and enhance the efficiency of gene delivery. To mimic the effect of cationic polymers, we selected to use vesicle associated membrane protein-8 (VAMP8) and modified its N-terminus with different cationic cell penetrating peptides (CPPs). The modified fusion proteins are expressed in an Escherichia coli system and purified after extraction. These modified VAMP8 proteins are used as free chains for gene transfection, while using bPEI-25k to condense the pDNA. The results show that the gene transfection efficiency of bPEI-25k/pDNA polyplexes is obviously enhanced in the 293 T cell line. Furthermore, the gene sequences encoding these modified VAMP8 proteins are sub-cloned to pcDNA-3.1 vector and then transferred to 293 T before the treatment with bPEI-25k/pDNA polyplexes. From the result, the transfection efficiency of bPEI-25k/pDNA complexes is enhanced at a similar level to that using modified VAMP8 as free chains. Our current results prove that free cationic chains are probably embedded with the membrane and influence intracellular trafficking, pointing out a new idea to design an effective non-viral gene delivery system.

Design of Free Triblock Polylysine- b-Polyleucine- b-Polylysine Chains for Gene Delivery.

Mixing cationic polymer chains with anionic DNA chains in solution results in the polymer/DNA complexes (also known as polyplexes). We recently confirmed that it is those noncomplexed cationic chains free in the mixture that promote the gene transfection, leading to a hypothesis: free cationic chains adsorbed on various anionic membranes interfere with the signal protein interaction, disrupt the intervesicular fusion, and block the endolysosome pathway so that the plasmid DNA (pDNA) chains have a higher chance to enter the nucleus. Accordingly, we design and synthesize linear cationic-hydrophobic-cationic triblock polylysine (K)- b-polyleucine (L)- b-polylysine (K) as free cationic chains by using natural protamine to condense the pDNA. The hydrophobic middle L-block helps its insertion into the membrane, while the interaction of the two cationic side K-blocks with the signal proteins helps the escape of the polyplexes from the lysosome entrapment. We studied the transfection efficiency of these copolymers with different block lengths. We found the optimal length of blocks K and L that allows the free triblock cationic copolymer chains to effectively enhance the gene transfection process. A combination of copolypeptides and protamine provides a new kind of biocompatible and nontoxic gene vectors made of only nontoxic peptides.

Characterization of complexes made of polylysine-polyleucine-polylysine and pDNA.

Polylysine shows unique physical and biological abilities in its application. In this study, different kinds of tri-block co-polypeptides are used as gene delivery vectors with hydrophobic polyleucine (L) in the middle and polylysine (K) on both sides. We explored their physical properties in aqueous solution as well as their biological effects toward gene transfection efficiency. After reaching critical micelle concentration, it is found that these tri-block polypeptides could form vesicles, and the size of each sample is independent of concentration. The self-assembly of each kind of tri-block co-polypeptide could obtain higher molecular weight vectors for gene delivery. After measuring the transfection efficiency and cell viability, these samples showed a high gene transfer ability, together with similar or lower cytotoxicity. Additionally, pDNA binding strength tests and zeta-potential assays reveal that the addition of an L part would reduce the charge density of the vector chains and thus lower the binding strength, leading to the easier release of pDNA from the complexes and an increase in transfection efficiency.

Revisiting complexation between DNA and polyethylenimine: does the disulfide linkage play a critical role in promoting gene delivery?

Despite its cytotoxicity, polyethylenimine (PEI) is still used as a golden reference in gene transfection. Long PEI chains are more effective but also more cytotoxic. To solve this problem, an alternative strategy is to link short PEI chains into a longer PEI with disulfide bonds because they are degradable in the cell. However, how PEI promotes gene transfection is still unclear. Also, the chain length of PEI is also increased as disulfide bonds are formed. Therefore, it is important to investigate whether the increase in transfection efficiency is attributable to the disulfide linkage, chain size, or both. To distinguish between such factors, a novel method is developed here to make longer linear PEI with disulfide bonds (lPEI(s-s)) by linking the mercapto groups of short linear PEI (lPEI(i)). By comparing the physiochemical properties and the transfection efficiencies of short lPEI(i), long lPEI(s-s), and un-degradable long PEI, it is found that introducing disulfide bonds instead of directly using longer PEI chains has less effect on gene transfection, and it is the chain length that plays a key role in promoting gene transfection.