Automated Platform for the Plasmid Construction Process.

There is a growing need for applications capable of handling large synthesis biology experiments. At the core of synthetic biology is the process of cloning and manipulating DNA as plasmids. Here, we report the development of an application named DNAda capable of writing automation instructions for any given DNA construct design generated by the J5 DNA assembly program. We also describe the automation pipeline and several useful features. The pipeline is particularly useful for the construction of combinatorial DNA assemblies. Furthermore, we demonstrate the platform by constructing a library of polyketide synthase parts, which includes 120 plasmids ranging in size from 7 to 14 kb from 4 to 7 DNA fragments.

Improved polyketide production in C. glutamicum by preventing propionate-induced growth inhibition.

Corynebacterium glutamicum is a promising host for production of valuable polyketides. Propionate addition, a strategy known to increase polyketide production by increasing intracellular methylmalonyl-CoA availability, causes growth inhibition in C. glutamicum. The mechanism of this inhibition was unclear before our work. Here we provide evidence that accumulation of propionyl-CoA and methylmalonyl-CoA induces growth inhibition in C. glutamicum. We then show that growth inhibition can be relieved by introducing methylmalonyl-CoA-dependent polyketide synthases. With germicidin as an example, we used adaptive laboratory evolution to leverage the fitness advantage of polyketide production in the presence of propionate to evolve improved germicidin production. Whole-genome sequencing revealed mutations in germicidin synthase, which improved germicidin titer, as well as mutations in citrate synthase, which effectively evolved the native glyoxylate pathway to a new methylcitrate pathway. Together, our results show that C. glutamicum is a capable host for polyketide production and we can take advantage of propionate growth inhibition to drive titers higher using laboratory evolution or to screen for production of polyketides.

Reprogramming of Neutrophils as Non-canonical Antigen Presenting Cells by Radiotherapy-Radiodynamic Therapy to Facilitate Immune-Mediated Tumor Regression.

Ineffective antigen cross-presentation in the tumor microenvironment compromises the generation of antitumor immune responses. Radiotherapy-radiodynamic therapy (RT-RDT) with nanoscale metal-organic frameworks (nMOFs) induces robust adaptive immune responses despite modest activation of canonical antigen presenting dendritic cells. Here, using transplantable and autochthonous murine tumor models, we demonstrate that RT-RDT induces antitumor immune responses via early neutrophil infiltration and reprogramming. Intravenous or intratumoral injection of nMOFs recruited peripheral CD11b+Ly6G+CD11c- neutrophils into tumors. The activation of nMOFs by low-dose X-rays significantly increased the population of CD11b+Ly6G+CD11c+ hybrid neutrophils with upregulated expression of the co-stimulatory molecules CD80 and CD86 as well as major histocompatibility complex class II molecules. Thus, nMOF-enabled RT-RDT reshapes a favorable tumor microenvironment for antitumor immune responses by reprogramming tumor-infiltrating neutrophils to function as non-canonical antigen presenting cells for effective cross-presentation of tumor antigens.

Bifunctional Metal-Organic Layers for Tandem Catalytic Transformations Using Molecular Oxygen and Carbon Dioxide.

Tandem catalytic reactions improve atom- and step-economy over traditional synthesis but are limited by the incompatibility of the required catalysts. Herein, we report the design of bifunctional metal-organic layers (MOLs), HfOTf-Fe and HfOTf-Mn, consisting of triflate (OTf)-capped Hf6 secondary building units (SBUs) as strong Lewis acidic centers and metalated TPY ligands as metal active sites for tandem catalytic transformations using O2 and CO2 as coreactants. HfOTf-Fe effectively transforms hydrocarbons into cyanohydrins via tandem oxidation with O2 and silylcyanation whereas HfOTf-Mn converts styrenes into styrene carbonates via tandem epoxidation and CO2 insertion. Density functional theory calculations revealed the involvement of a high-spin FeIV (S = 2) center in the challenging oxidation of the sp3 C-H bond. This work highlights the potential of MOLs as a tunable platform to incorporate multiple catalysts for tandem transformations.

Nanoscale Metal-Organic Layer Isolates Phthalocyanines for Efficient Mitochondria-Targeted Photodynamic Therapy.

Zinc-phthalocyanine (ZnPc) photosensitizers (PSs) have shown great potential in photodynamic therapy (PDT) owing to their strong absorption at long wavelengths (650-750 nm), high triplet quantum yields, and biocompatibility. However, the clinical utility of ZnPc PSs is limited by their poor solubility and tendency to aggregate in aqueous environments. Here we report the design of a new nanoscale metal-organic layer (nMOL) assembly, ZnOPPc@nMOL, with ZnOPPc [ZnOPPc = zinc(II)-2,3,9,10,16,17,23,24-octa(4-carboxyphenyl)phthalocyanine] PSs supported on the secondary building units (SBUs) of a Hf12 nMOL for PDT. Upon irradiation, SBU-bound ZnOPPc PSs absorb 700 nm light and efficiently sensitize the formation of singlet oxygen by preventing aggregation-induced self-quenching of ZnOPPc excited states. With intrinsic mitochondria-targeting capability, ZnOPPc@nMOL showed exceptional PDT efficacy with >99% tumor growth inhibition and 40-60% cure rates on two mouse models of colon cancer.

Nanoscale Metal-Organic Layers Detect Mitochondrial Dysregulation and Chemoresistance via Ratiometric Sensing of Glutathione and pH.

Mitochondrial dysregulation controls cell death and survival by changing endogenous molecule concentrations and ion flows across the membrane. Here, we report the design of a triply emissive nanoscale metal-organic layer (nMOL), NA@Zr-BTB/F/R, for sensing mitochondrial dysregulation. Zr-BTB nMOL containing Zr6 secondary building units (SBUs) and 2,4,6-tris(4-carboxyphenyl)aniline (BTB-NH2) ligands was postsynthetically functionalized to afford NA@Zr-BTB/F/R by exchanging formate capping groups on the SBUs with glutathione(GSH)-selective (2E)-1-(2'-naphthyl)-3-(4-carboxyphenyl)-2-propen-1-one (NA) and covalent conjugation of pH-sensitive fluorescein (F) and GSH/pH-independent rhodamine-B (R) to the BTB-NH2 ligands. Cell imaging demonstrated NA@Zr-BTB/F/R as a ratiometric sensor for mitochondrial dysregulation and chemotherapy resistance via GSH and pH sensing.

Metal-Organic Layers Hierarchically Integrate Three Synergistic Active Sites for Tandem Catalysis.

We report the design of a bifunctional metal-organic layer (MOL), Hf12 -Ru-Co, composed of [Ru(DBB)(bpy)2 ]2+ [DBB-Ru, DBB=4,4'-di(4-benzoato)-2,2'-bipyridine; bpy=2,2'-bipyridine] connecting ligand as a photosensitizer and Co(dmgH)2 (PPA)Cl (PPA-Co, dmgH=dimethylglyoxime; PPA=4-pyridinepropionic acid) on the Hf12 secondary building unit (SBU) as a hydrogen-transfer catalyst. Hf12 -Ru-Co efficiently catalyzed acceptorless dehydrogenation of indolines and tetrahydroquinolines to afford indoles and quinolones. We extended this strategy to prepare Hf12 -Ru-Co-OTf MOL with a [Ru(DBB)(bpy)2 ]2+ photosensitizer and Hf12 SBU capped with triflate as strong Lewis acids and PPA-Co as a hydrogen transfer catalyst. With three synergistic active sites, Hf12 -Ru-Co-OTf competently catalyzed dehydrogenative tandem transformations of indolines with alkenes or aldehydes to afford 3-alkylindoles and bisindolylmethanes with turnover numbers of up to 500 and 460, respectively, illustrating the potential use of MOLs in constructing novel multifunctional heterogeneous catalysts.

Nanoscale metal-organic frameworks for x-ray activated in situ cancer vaccination.

Cancer vaccines have been actively pursued to bolster antitumor immunity. Here, we designed nanoscale metal-organic frameworks (nMOFs) as locally activable immunotherapeutics to release danger-associated molecular patterns (DAMPs) and tumor antigens and deliver pathogen-associated molecular patterns (PAMPs) for in situ personalized cancer vaccination. When activated by x-rays, nMOFs effectively generate reactive oxygen species to release DAMPs and tumor antigens while delivering CpG oligodeoxynucleotides as PAMPs to facilitate the maturation of antigen-presenting cells. Together, DAMPs, tumor antigens, and PAMPs expand cytotoxic T cells in tumor-draining lymph nodes to reinvigorate the adaptive immune system for local tumor regression. When treated in combination with an immune checkpoint inhibitor, the local therapeutic effects of nMOF-based vaccines were extended to distant tumors via attenuating T cell exhaustion. Our work demonstrates the potential of nMOFs as x-ray-activable in situ cancer vaccines to awaken the host's innate and adaptive immune systems for systemic antitumor immunity.

Nanoscale Metal-Organic Frameworks Generate Reactive Oxygen Species for Cancer Therapy.

In the past 15 years, enormous progress has been made in cancer nanotechnology, and a several nanoparticles have entered clinical testing for cancer treatment. Among these nanoparticles are nanoscale metal-organic frameworks (nMOFs), a class of organic-inorganic hybrid nanomaterials constructed from metal binding sites and bridging ligands, which have attracted significant attention for their ability to integrate porosity, crystallinity, compositional and structural tunability, multifunctionality, and biocompatibility into a singular nanomaterial for cancer therapies. This Outlook article summarizes the progress on the design of nMOFs as nanosensitizers for photodynamic therapy (PDT), radiotherapy (RT), radiotherapy-radiodynamic therapy (RT-RDT), and chemodynamic therapy (CDT) via nMOF-mediated reactive oxygen species (ROS) generated under external energy stimuli or in the presence of endogenous chemical triggers. Inflammatory responses induced by nMOF-mediated ROS generation activate tumor microenvironments to potentiate cancer immunotherapy, extending the local treatment effects of nMOF-based ROS therapy to distant tumors via abscopal effects. Future research directions in nMOF-mediated ROS therapies and the prospect of clinical applications of nMOFs as cancer therapeutics are also discussed.

Nanoscale Metal-Organic Frameworks Stabilize Bacteriochlorins for Type I and Type II Photodynamic Therapy.

Herein we report the design of a bacteriochlorin-based nanoscale metal-organic framework, Zr-TBB, for highly effective photodynamic therapy via both type I and type II mechanisms. The framework of Zr-TBB stabilizes 5,10,15,20-tetra(p-benzoato)bacteriochlorin (TBB) ligands toward oxygen and light via geometrical constraint. Upon 740 nm light irradiation, Zr-TBB efficiently generates various reactive oxygen species, including singlet oxygen, superoxide anion, hydrogen peroxide, and hydroxyl radicals, to afford superb antitumor efficacy on mouse models of breast and colon cancers, with cure rates of 40% and 60%, respectively.

Metal-Organic Layers for Synergistic Lewis Acid and Photoredox Catalysis.

We report the design of a new multifunctional metal-organic layer (MOL), Hf12-Ir-OTf, comprising triflate (OTf)-capped Hf12 secondary building units (SBUs) and photosensitizing Ir(DBB)[dF(CF3)ppy]2+ [DBB-Ir-F, DBB = 4,4'-di(4-benzoato)-2,2'-bipyridine; dF(CF3)ppy = 2-(2,4-difluorophenyl)-5-(trifluoromethyl)pyridine] bridging ligands. Hf12-Ir-OTf effectively catalyzed dehydrogenative cross-couplings of heteroarenes with ethers, amines, and unactivated alkanes with turnover numbers of 930, 790, and 950, respectively. Hf12-Ir-OTf also competently catalyzed late-stage functionalization of bioactive and drug molecules such as caffeine, Fasudil, and Metyrapone. The superior catalytic performance of Hf12-Ir-OTf over a mixture of photoredox catalyst and stoichiometric amounts of Brønsted acids or substoichiometric amounts (20 mol %) of Lewis acids is attributed to the close proximity (1.2 nm) between photoredox and Lewis acid catalysts in Hf12-Ir-OTf, which not only facilitates the reaction between the carbon radical and the activated heteroarene but also accelerates the electron transfer from the nitrogen radical intermediate to the Ir(IV) species in the catalytic cycle.

Synergistic Effect over Sub-nm Pt Nanocluster@MOFs Significantly Boosts Photo-oxidation of N-alkyl(iso)quinolinium Salts.

Quinolones and isoquinolones are of interest to pharmaceutical industry owing to their potent biological activities. Herein, we first encapsulated sub-nm Pt nanoclusters into Zr-porphyrin frameworks to afford an efficient photocatalyst Pt0.9@PCN-221. This catalyst can dramatically promote electron-hole separation and 1O2 generation to achieve synergistic effect first in the metal-organic framework (MOF) system, leading to the highest activity in photosynthesis of (iso)quinolones in >90.0% yields without any electronic sacrificial agents. Impressively, Pt0.9@PCN-221 was reused 10 times without loss of activity and can catalyze gram-scale synthesis of 1-methyl-5-nitroisoquinolinone at an activity of 175.8 g·gcat-1, 22 times higher than that of PCN-221. Systematic investigations reveal the contribution of synergistic effect of photogenerated electron, photogenerated hole, and 1O2 generation for efficient photo-oxidation, thus highlighting a new strategy to integrate multiple functional components into MOFs to synergistically catalyze complex photoreactions for exploring biologically active heterocyclic molecules.

Biomimetic nanoscale metal-organic framework harnesses hypoxia for effective cancer radiotherapy and immunotherapy.

Tumor hypoxia presents a major impediment to effective cancer therapy with ionizing radiation and immune checkpoint inhibitors. Here we report the design of a biomimetic nanoscale metal-organic-framework (nMOF), Hf-DBP-Fe, with catalase-like activity to decompose elevated levels of H2O2 in hypoxic tumors to generate oxygen and hydroxyl radical. The generated oxygen attenuates hypoxia to enable radiodynamic therapy upon X-ray irradiation and fixes DNA damage while hydroxyl radical inflicts direct damage to tumor cells to afford chemodynamic therapy. Hf-DBP-Fe thus mediates effective local therapy of hypoxic cancer with low-dose X-ray irradiation, leading to highly immunogenic tumor microenvironments for synergistic combination with anti-PD-L1 immune checkpoint blockade. This combination treatment not only eradicates primary tumors but also rejects distant tumors through systemic anti-tumor immunity. We have thus advanced an nMOF-based strategy to harness hypoxic tumor microenvironments for highly effective cancer therapy using a synergistic combination of low dose radiation and immune checkpoint blockade.

A Nanoscale Metal-Organic Framework to Mediate Photodynamic Therapy and Deliver CpG Oligodeoxynucleotides to Enhance Antigen Presentation and Cancer Immunotherapy.

Checkpoint blockade immunotherapy (CBI) awakes a host innate immune system and reactivates cytotoxic T cells to elicit durable response in some cancer patients. Now, a cationic nanoscale metal-organic framework, W-TBP, is used to facilitate tumor antigen presentation by enabling immunogenic photodynamic therapy (PDT) and promoting the maturation of dendritic cells (DCs). Comprised of dinuclear WVI secondary building units and photosensitizing 5,10,15,20-tetra(p-benzoato)porphyrin (TBP) ligands, cationic W-TBP mediates PDT to release tumor associated antigens and delivers immunostimulatory CpG oligodeoxynucleotides to DCs. The enhanced antigen presentation synergizes with CBI to expand and reinvigorate cytotoxic T cells, leading to superb anticancer efficacy and robust abscopal effects with >97 % tumor regression in a bilateral breast cancer model.

Multifunctional Nanoscale Metal-Organic Layers for Ratiometric pH and Oxygen Sensing.

As a monolayered version of nanoscale metal-organic frameworks (nMOFs), nanoscale metal-organic layers (nMOLs) represent an emerging class of highly tunable two-dimensional materials for hierarchical functionalization and with facile access to analytes. Here we report the design of the first nMOL-based biosensor for ratiometric pH and oxygen sensing in mitochondria. Cationic Hf12-Ru nMOL was solvothermally synthesized by laterally connecting Hf12 secondary building units (SBUs) with oxygen-sensitive Ru(bpy)32+-derived DBB-Ru ligands (bpy = 2,2'-bipyridine). The Hf12-Ru nMOL was then covalently functionalized with pH-sensitive fluorescein isothiocyanate and pH/oxygen-independent Rhodamine-B isothiocyanate through thiourea linkages to afford Hf12-Ru-F/R as a mitochondria-targeted ratiometric sensor for pH and O2 in live cells. High-resolution confocal microscope imaging with Hf12-Ru-F/R revealed a positive correlation between pH and local O2 concentration in mitochondria. Our work shows the potential of nMOL-based ratiometric biosensors in sensing and imaging of biologically important analytes in live cells.

Metal-Organic Layers as Multifunctional Two-Dimensional Nanomaterials for Enhanced Photoredox Catalysis.

Metal-organic layers (MOLs) have recently emerged as a novel class of molecular two-dimensional (2D) materials with significant potential for catalytic applications. Herein we report the design of a new multifunctional MOL, Hf12-Ir-Ni, by laterally linking Hf12 secondary building units (SBUs) with photosensitizing Ir(DBB)[dF(CF3)ppy]2+ [DBB-Ir-F, DBB = 4,4'-di(4-benzoato)-2,2'-bipyridine; dF(CF3)ppy = 2-(2,4-difluorophenyl)-5-(trifluoromethyl)pyridine] bridging ligands and vertically terminating the SBUs with catalytic Ni(MBA)Cl2 [MBA = 2-(4'-methyl-[2,2'-bipyridin]-4-yl)acetate] capping agents. Hf12-Ir-Ni was synthesized in a bottom-up approach and characterized by TEM, AFM, PXRD, TGA, NMR, ICP-MS, UV-vis, and luminescence spectroscopy. The proximity between photosensitizing Ir centers and catalytic Ni centers (∼0.85 nm) in Hf12-Ir-Ni facilitates single electron transfer, leading to a 15-fold increase in photoredox reactivity. Hf12-Ir-Ni was highly effective in catalytic C-S, C-O, and C-C cross-coupling reactions with broad substrate scopes and turnover numbers of ∼4500, ∼1900, and ∼450, respectively.

Nanoscale Metal-Organic Framework Hierarchically Combines High-Z Components for Multifarious Radio-Enhancement.

With tunability and porosity, nanoscale metal-organic frameworks (nMOFs) can incorporate multiple components to realize complex functions for biomedical applications. Here we report the synthesis of W18@Hf12-DBB-Ir, a new nMOF assembly hierarchically incorporating three high-Z components-Hf-based metal-oxo clusters, Ir-based bridging ligands, and W-based polyoxometalates (POMs)-as a multifarious radioenhancer. Cationic Hf12-DBB-Ir was built from Hf12 secondary building units (SBUs) and [Ir(bpy)2(ppy)]+ (bpy = 2,2'-bipyridine, ppy = 2-phenylpyridine) derived dicarboxylate ligands (DBB-Ir) and then loaded with Wells-Dawson-type [P2W18O62]6- (W18) POMs to afford W18@Hf12-DBB-Ir. Upon X-ray irradiation, W18@Hf12-DBB-Ir significantly enhances hydroxyl radical generation from Hf12 SBUs, singlet oxygen generation from DBB-Ir ligands, and superoxide generation from W18 POMs, respectively. Through synergistic cell killing by these distinct reactive oxygen species, W18@Hf12-DBB-Ir elicited superb anticancer efficacy with >98% tumor regression at a low X-ray dose of 5 × 1 Gy.

Titanium-Based Nanoscale Metal-Organic Framework for Type I Photodynamic Therapy.

Nanoscale metal-organic frameworks (nMOFs) have shown great potential as nanophotosensitizers for photodynamic therapy (PDT) owing to their high photosensitizer loadings, facile diffusion of reactive oxygen species (ROSs) through their porous structures, and intrinsic biodegradability. The exploration of nMOFs in PDT, however, remains limited to an oxygen-dependent type II mechanism. Here we report the design of a new nMOF, Ti-TBP, composed of Ti-oxo chain secondary building units (SBUs) and photosensitizing 5,10,15,20-tetra( p-benzoato)porphyrin (TBP) ligands, for hypoxia-tolerant type I PDT. Upon light irradiation, Ti-TBP not only sensitizes singlet oxygen production, but also transfers electrons from excited TBP* species to Ti4+-based SBUs to afford TBP•+ ligands and Ti3+ centers, thus propagating the generation of superoxide, hydrogen peroxide, and hydroxyl radicals. By generating four distinct ROSs, Ti-TBP-mediated PDT elicits superb anticancer efficacy with >98% tumor regression and 60% cure rate.

Nanoscale Metal-Organic Frameworks for Phototherapy of Cancer.

Phototherapy involves the irradiation of tissues with light, and is commonly implemented in the forms of photodynamic therapy (PDT) and photothermal therapy (PTT). Photosensitizers (PSs) are often needed to improve the efficacy and selectivity of phototherapy via enhanced singlet oxygen generation in PDT and photothermal responses in PTT. In both cases, efficient and selective delivery of PSs to the diseased tissues is of paramount importance. Nanoscale metal-organic frameworks (nMOFs), a new class of hybrid materials built from metal connecting points and bridging ligands, have been examined as nanocarriers for drug delivery due to their compositional and structural tunability, highly porous structures, and good biocompatibility. This review summarizes recent advances on using nMOFs as nanoparticle PSs for applications in PDT and PTT.