A dew-responsive pectin-based herbicide for enhanced photodynamic inactivation.

5-aminolevulinic acid (5-ALA) has been fully demonstrated as a biodegradable, without resistance, and pollution-free pesticide. However, the lack of targeting and the poor adhesion result in a low utilization rate, limiting its practical application. Herein, a dew-responsive polymer pro-pesticide Pec-hyd-ALA was successfully synthesized by grafting 5-ALA onto the pectin (PEC) backbone via acid-sensitive acylhydrazone bonds. When the pro-pesticide is exposed to acid dew on plant surfaces at night, 5-ALA is released and subsequently converted to photosensitize (Protoporphyrin IX, PpIX)in plant cells, leading to its accumulation and promoting photodynamic inactivation (PDI). An inverted fluorescence microscope has verified the accumulation of tetrapyrrole in plant cells. In addition, the highly bio-adhesive PEC backbone effectively improved the wetting and retention of 5-ALA on leaves. The pot experiment also demonstrated the system's control effect on barnyard grass. This work provides a promising approach to improving the herbicidal efficacy of 5-ALA.

Photodynamic anticancer activity evaluation of novel 5-aminolevulinic acid and 3-hydroxypyridinone conjugates.

5-Aminolevulinic acid (ALA) and its derivatives, serving as the endogenous precursor of the photosensitizer (PS) protoporphyrin IX (PpIX), successfully applied in tumor imaging and photodynamic therapy (PDT). ALA and its derivatives have been used to treat actinic keratosis (AK), basal cell carcinoma (BCC), and improve the detection of superficial bladder cancer. However, the high hydrophilicity of ALA and the conversion of PpIX to heme have limited the accumulation of PpIX, hindering the efficiency and potential application of ALA-PDT. This study aims to evaluate the PDT activity of three rationally designed series of ALA-HPO prodrugs, which were based on enhancing the lipophilicity of the prodrugs and reducing the labile iron pool (LIP) through HPO iron chelators to promote PpIX accumulation. Twenty-four ALA-HPO conjugates, incorporating amide, amino acid, and ester linkages, were synthesized. Most of the conjugates, exhibited no dark-toxicity to cells, according to bioactivity evaluation. Ester conjugates 19a-g showed promoted phototoxicity when tested on tumor cell lines, and this increased phototoxicity was strongly correlated with elevated PpIX levels. Among them, conjugate 19c emerged as the most promising (HeLa, IC50 = 24.25 ± 1.43 µM; MCF-7, IC50 = 43.30 ± 1.76 µM; A375, IC50 = 28.03 ± 1.00 µM), displaying superior photodynamic anticancer activity to ALA (IC50 > 100 µM). At a concentration of 80 µM, the fluorescence intensity of PpIX induced by compound 19c in HeLa, MCF-7, and A375 cells was 18.9, 5.3, and 2.8 times higher, respectively, than that induced by ALA. In conclusion, cellular phototoxicity showed a strong correlation with intracellular PpIX fluorescence levels, indicating the potential application of ALA-HPO conjugates in ALA-PDT.

Revisiting catalytic His and Glu residues in coproporphyrin ferrochelatase - unexpected activities of active site variants.

The identification of the coproporphyrin-dependent heme biosynthetic pathway, which is used almost exclusively by monoderm bacteria in 2015 by Dailey et al. triggered studies aimed at investigating the enzymes involved in this pathway that were originally assigned to the protoporphyrin-dependent heme biosynthetic pathway. Here, we revisit the active site of coproporphyrin ferrochelatase by a biophysical and biochemical investigation using the physiological substrate coproporphyrin III, which in contrast to the previously used substrate protoporphyrin IX has four propionate substituents and no vinyl groups. In particular, we have compared the reactivity of wild-type coproporphyrin ferrochelatase from the firmicute Listeria monocytogenes with those of variants, namely, His182Ala (H182A) and Glu263Gln (E263Q), involving two key active site residues. Interestingly, both variants are active only toward the physiological substrate coproporphyrin III but inactive toward protoporphyrin IX. In addition, E263 exchange impairs the final oxidation step from ferrous coproheme to ferric coproheme. The characteristics of the active site in the context of the residues involved and the substrate binding properties are discussed here using structural and functional means, providing a further contribution to the deciphering of this enigmatic reaction mechanism.

Water extractability of the zinc protoporphyrin IX-myoglobin complex from Parma ham is pH-dependent.

The bright red color of Parma ham is mainly derived from zinc protoporphyrin IX (ZnPP), which exists in both water-soluble and insoluble states. Water-soluble ZnPP mainly binds to hemoglobin, however, the presence of water-insoluble ZnPP remains unexplained. Therefore, we aimed to elucidate how ZnPP exists in a water-insoluble state by focusing on its binding substance. Depending on the skeletal muscle, water-insoluble ZnPP comprised 30-50% of total ZnPP. The ZnPP water extractability was positively correlated with muscle pH. Water-insoluble ZnPP was extractable with a high-pH solution and existed as a complex with myoglobin or hemoglobin; nevertheless, myoglobin-binding ZnPP was more abundant. Furthermore, the water solubility of the myoglobin globin moiety at pH 5.5-6.0 was reduced by ZnPP binding. These results suggest that water-insoluble ZnPP mainly exists as a ZnPP-Mb complex, with low solubility attributed to the low pH of the ham.

The formation of complexes with albumin increases the stability of the protoporphyrin IX spectra.

Photodynamic therapy is a high-target, low-invasive treatment utilized to manage a variety of malignant diseases and precancerous lesions. Protoporphyrin IX (PpIX) is one of the most important photosensitizers used in photodynamic therapy, carried to the cancer tissue by serum albumin. Its delivery by transport protein is one of the major factors in determining the efficacy of photodynamic therapy. The distribution of the albumin-PpIX complexes to the target tissue enables the accomplishment of an optimal PDT effect. This study aimed to assess in vitro the stability of spectrofluorimetric spectra of albumin-PpIX complexes. The experiment used three chemicals: PpIX, human serum albumin (HSA), and bovine serum albumin (BSA). Spectral data was recorded using a Kontron SFM-25 Instrument AG, at two excitation wavelengths λex=280 nm and 295 nm. A concentration of 1x10-5M of PpIX, in combination with 1.25x10-6M of HSA and 4x10-7M of BSA, have been recorded repetitively for ten days and compared to the initial spectrum. The maximum of PpIX fluorescence changed significantly on the first day following sample preparation. The maximum of PpIX - serum albumin complex was stable 10 and 4 days for HSA and 5 and 2 days for BSA for λex=280 nm and 295 nm, respectively. The formation of a complex between PpIX and serum albumin was seen to extend the stability of the spectrofluorimetric spectrum. However, a less significant effect was observed in the case of BSA, which could most plausibly be attributed to the variations in primary structure between HSA and BSA, leading to discernible variations in spectroscopic measurements.

Protoporphyrin-sensitized degradable bismuth nanoformulations for enhanced sonodynamic oncotherapy.

Decreasing the scavenging capacity of reactive oxygen species (ROS) and enhancing ROS production are the two principal objectives in the development of novel sonosensitizers for sonodynamic therapy (SDT). Herein, we designed a protoporphyrin-sensitized bismuth-based semiconductor (P-NBOF) as a sonosensitizer to generate ROS and synergistically depleted glutathione for enhanced SDT against tumors. The bismuth-based nanomaterial (NBOF) is a wide-bandgap semiconductor. Sensitization by protoporphyrin made it easier to excite electrons under ultrasonic stimulation, and the energy of the lowest unoccupied electron orbital in protoporphyrin was higher than the conduction-band energy of NBOF. Under ultrasound excitation, the excited electrons in the protoporphyrin were injected into the conduction band of the NBOF, increasing its reducing ability leading to the production of more superoxide anion radicals and also helping to increase the charge separation of protoporphyrin leading to the production of more singlet oxygen. Meanwhile, P-NBOF continuously depleted glutathione, which was not only conducive to breaking the redox balance of the tumor microenvironment to enhance the therapeutic efficacy of SDT, but also promoted its degradation and metabolism. The construction of this P-NBOF sonosensitizer thus provided an effective strategy to enhance SDT for tumors. STATEMENT OF SIGNIFICANCE: To enhance the efficacy of sonodynamic tumor therapy, we developed a degradable protoporphyrin-sensitized bismuth-based nano-semiconductor (P-NBOF) by optimizing the band structure and glutathione-depletion ability. Protoporphyrin in P-NBOF under excitation preferentially generates free electrons, which are then injected into the conduction band of NBOF, improving the reducing ability of NBOF and promoting the separation of electron-hole pairs, thereby enhancing the production capacity of reactive oxygen species. Furthermore, P-NBOF can deplete glutathione, reduce the scavenging of reactive oxygen species, and reactivate and amplify the effect of sonodynamic therapy. The construction of the nanotherapeutic platform provides an option for enhancing sonodynamic therapy.

Radical Mediated Rapid In Vitro Formation of c-Type Cytochrome.

A cytochrome c552 mutant from Thermus thermophilus HB8 (rC552 C14A) was reported, where the polypeptide with replaced Cys14 by alanine, overexpressed in the cytosol of E. coli. The apo-form of the C14A mutant (apo-C14A) without the original prosthetic group was obtained by simple chemical treatments that retained compact conformation amenable to reconstitution with heme b and zinc(II)-protoporphyrin(IX), gradually followed by spontaneous formation of a covalent bond between the polypeptide and porphyrin ring in the reconstituted apo-C14A. Further analysis suggested that the residual Cys11 and vinyl group of the porphyrin ring linked through the thiol-ene reaction promoted by light under ambient conditions. In this study, we describe the kinetic improvement of the covalent bond formation in accordance with the mechanism of the photoinduced thiol-ene reaction, which involves a thiyl radical as a reaction intermediate. Adding a radical generator to the reconstituted C14A mutant with either heme-b or zinc(II) porphyrin accelerated the bond-forming reaction, which supported the involvement of a radical species in the reaction. Partial observation of the reconstituted C14A in a dimer form and detection of sulfuryl radical by EPR spectroscopy indicated a thiyl radical on Cys11, a unique cysteinyl residue in rC552 C14A. The covalent bond forming mediated by the radical generator was also adaptable to the reconstituted apo-C14A with manganese(II)-protoporphyrin(IX), which also exhibits light-mediated covalent linkage formation. Therefore, the radical generator extends the versatility of producing c-type-like cytochrome starting from a metallo-protoporphyrin(IX) and the apo-C14A instantaneously.

Zinc protoporphyrin IX predominantly exists as a complex non-enzymatically bound to apo-hemoglobin in Parma ham.

Most of the water-soluble zinc protoporphyrin IX (ZnPP) in Parma ham mainly exists as complexes with hemoglobin and myoglobin (ZnPP-Hb and ZnPP-Mb). To elucidate the formation mechanism of these complexes, a new experimental model to produce higher amount of water-soluble ZnPP complexes was established. ZnPP-Hb was detected as the main water-soluble ZnPP complex in this model, which is the same as that in Parma ham. Adding exogenous Hb into this model promoted higher ZnPP formation than with Mb added, indicating that Hb was the superior substrate for generating ZnPP compared to Mb. The increase in non-heme iron content with ZnPP formation in both the Hb- and Mb-added groups indicated that the release of iron ion from heme was a crucial step in ZnPP formation. ZnPP-Hb was formed when ZnPP non-enzymatically bound with apo-Hb. These results revealed the mechanism of why ZnPP-Hb is more dominant in Parma ham than to ZnPP-Mb.

Protoporphyrin IX-Chitosan Oligosaccharide Conjugate with Potent Antifungal Photodynamic Activity.

A new chemical conjugate between protoporphyrin IX (PPIX) and chitosan oligosaccharides (CH) was prepared and evaluated in vitro as an antifungal agent against Penicillium digitatum. Chemical characterization and photophysical/photochemical studies were conducted. The antifungal effect of the CH-PPIX conjugate was compared to its components (PPIX and CH) and a physical mixture of both, under dark and illuminated conditions. The CH-PPIX conjugate was photostable and inhibited fungal growth with 100% efficiency at a dose of 0.005% w/v under visible light irradiation, while no antifungal activity was observed in the dark. Under the same conditions, CH and PPIX did not display any fungicidal activity, demonstrating the improved properties of the conjugate. Insights into the mechanism of fungal inactivation revealed an efficient spore uptake and photoinduced membrane damage through singlet oxygen generation. This new bioconjugate, which is based on natural components, represents a promising agent for fungicidal formulations based on antimicrobial photodynamic therapy.

Photodynamic Activity of Protoporphyrin IX-Immobilized Cellulose Monolith for Nerve Tissue Regeneration.

The development of nerve conduits with a three-dimensional porous structure has attracted great attention as they closely mimic the major features of the natural extracellular matrix of the nerve tissue. As low levels of reactive oxygen species (ROS) function as signaling molecules to promote cell proliferation and growth, this study aimed to fabricate protoporphyrin IX (PpIX)-immobilized cellulose (CEPP) monoliths as a means to both guide and stimulate nerve regeneration. CEPP monoliths can be fabricated via a simple thermally induced phase separation method and surface modification. The improved nerve tissue regeneration of CEPP monoliths was achieved by the activation of mitogen-activated protein kinases, such as extracellular signal-regulated kinases (ERKs). The resulting CEPP monoliths exhibited interconnected microporous structures and uniform morphology. The results of in vitro bioactivity assays demonstrated that the CEPP monoliths with under 0.54 ± 0.07 µmol/g PpIX exhibited enhanced photodynamic activity on Schwann cells via the generation of low levels of ROS. This photodynamic activation of the CEPP monoliths is a cell-safe process to stimulate cell proliferation without cytotoxic side effects. In addition, the protein expression of phospho-ERK increased considerably after the laser irradiation on the CEPP monoliths with low content of PpIX. Therefore, the CEPP monoliths have a potential application in nerve tissue regeneration as new nerve conduits.

miRNA-Guided Imaging and Photodynamic Therapy Treatment of Cancer Cells Using Zn(II)-Protoporphyrin IX-Loaded Metal-Organic Framework Nanoparticles.

An analytical platform for the selective miRNA-21-guided imaging of breast cancer cells and miRNA-221-guided imaging of ovarian cancer cells and the selective photodynamic therapy (PDT) of these cancer cells is introduced. The method is based on Zn(II)-protoporphyrin IX, Zn(II)-PPIX-loaded UiO-66 metal-organic framework nanoparticles, NMOFs, gated by two hairpins Hi/Hj through ligation of their phosphate residues to the vacant Zr4+-ions associated with the NMOFs. The hairpins are engineered to include the miRNA recognition sequence in the stem domain of Hi, and in the Hi and Hj, partial locked stem regions of G-quadruplex subunits. Intracellular phosphate-ions displace the hairpins, resulting in the release of the Zn(II)-PPIX and intracellular miRNAs open Hi, and this triggers the autonomous cross-opening of Hi and Hj. This activates the interhairpin hybridization chain reaction and leads to the assembly of highly fluorescent Zn(II)-PPIX-loaded G-quadruplex chains. The miRNA-guided fluorescent chains allow selective imaging of cancer cells. Moreover, PDT with visible light selectively kills cancer cells and tumor cells through the formation of toxic reactive oxygen species.

Theoretical and experimental study of interaction of macroheterocyclic compounds with ORF3a of SARS-CoV-2.

The pandemic infectious disease (Covid-19) caused by the coronavirus (SARS-CoV2) is spreading rapidly around the world. Covid-19 does an irreparable harm to the health and life of people. It also has a negative financial impact on the economies of most countries of the world. In this regard, the issue of creating drugs aimed at combating this disease is especially acute. In this work, molecular docking was used to study the docking of 23 compounds with QRF3a SARS-CoV2. The performed in silico modeling made it possible to identify leading compounds capable of exerting a potential inhibitory and virucidal effect. The leading compounds include chlorin (a drug used in PDT), iron(III)protoporphyrin (endogenous porphyrin), and tetraanthraquinone porphyrazine (an exogenous substance). Having taken into consideration the localization of ligands in the QRF3a SARS-CoV2, we have made an assumption about their influence on the pathogenesis of Covid-19. The interaction of chlorin, iron(III)protoporphyrin and protoporphyrin with the viral protein ORF3a were studied by fluorescence and UV-Vis spectroscopy. The obtained experimental results confirm the data of molecular docking. The results showed that a viral protein binds to endogenous porphyrins and chlorins, moreover, chlorin is a competitive ligand for endogenous porphyrins. Chlorin should be considered as a promising drug for repurposing.

Characterization of autofluorescence and quantitative protoporphyrin IX biomarkers for optical spectroscopy-guided glioma surgery.

5-Aminolevulinic acid (5-ALA)-mediated fluorescence does not effectively depict low grade gliomas (LGG) or the infiltrative tumor portion of high-grade gliomas (HGG). While spectroscopy improves sensitivity and precision, this is currently limited by autofluorescence and a second protoporphyrin IX (PpIX) fluorescence state at 620 nm. We investigated the autofluorescence to better characterize the present spectra and thus increase PpIX quantification precision and sensitivity. This study included 128 patients undergoing surgery for malignant glioma. 5-ALA (Gliolan) was administered before anesthesia, and fluorescence was measured using a hyperspectral device. It was found that all 2692 measured spectra consisted of contributions from 620 to 634 nm PpIX, NADH, lipofuscin, and flavins. The basis spectra were characterized and their use in spectral unmixing led to 82.4% lower fitting error for weakly fluorescing areas (p < 0.001), and 92.3% fewer false positive tumor identifications in control measurements (p = 0.0065) compared to previous works. They also decreased the PpIX620 contribution, thus halving the mean Ratio620/634 (p < 0.001). The ratio was approximately 0 for HGGs and increasing for LGGs, as demonstrated previously. Additionally, the Ratio620/634, the MIB-1/Ki-67 proliferation index, and the PpIX peak blue-shift were found to be significantly related to WHO grade, fluorescence visibility, and PpIX contribution (p < 0.001), and the value of these three as quantitative biomarkers is discussed.

A Versatile Nanoplatform for Broad-Spectrum Immunotherapy by Reversing the Tumor Microenvironment.

Immunotherapy is currently an important adjuvant therapy for malignant tumors besides surgical treatment. However, the heterogeneity and low immunogenicity of the tumor are two main challenges of the immunotherapy. Here, we have constructed a nanoplatform (CP@mRBC-PpIX) to realize reversion of the tumor acidosis and hypoxia through alkali and oxygen generation triggered by tumor acidosis. By targeting tumor universal features other than endogenous biomarkers, it was found that CP@mRBC-PpIX could polarize tumor-associated macrophages to anti-tumor M1 phenotype macrophages to enhance tumor immune response. Furthermore, under regional light irradiation, the reactive oxygen species produced by photosensitizers located in CP@mRBC-PpIX could increase the immunogenicity of tumors, so that tumor changes from an immunosuppressive "cold tumor" to an immunogenic "hot tumor," thereby increasing the infiltration and response of T cells, further amplifying the effect of immunotherapy. This strategy circumvented the problem of tumor heterogeneity to realize a kind of broad-spectrum immunotherapy, which could effectively prevent tumor metastasis and recurrence.

Imaging of Cancer Cells and Dictated Cytotoxicity Using Aptamer-Guided Hybridization Chain Reaction (HCR)-Generated G-Quadruplex Chains.

DNA nanotechnology provides powerful tools for developing cancer theranostics. Here we introduce the autonomous surface-nucleolin-guided HCR that leads to the polymerization of G-quadruplex polymer chains, in which the ZnII -protoporphyrin IX is intercalated. We demonstrate that MDA-MB-231 (Triple Negative Breast Cancer cells, TNBC) with overexpressed surface nucleolin were able to induce HCR leading to the formation of the ZnII PPIX-loaded G-quadruplex polymer chains, while the M10 epithelial breast cells served as control. The ZnII PPIX-loaded nanowires allow the selective imaging of TNBC, and their permeation into the TNBC leads to selective cytotoxicity and guided photodynamic therapy toward the cancer cells due to structural perturbation of the membranes. The aptamer-guided HCR-generated G-quadruplex polymer chains may serve as a versatile tool to target TNBC featuring poor prognosis and high pathological risk of recurrence, thus offering a promising theranostic platform.

Gated Dissipative Dynamic Artificial Photosynthetic Model Systems.

Gated dissipative artificial photosynthetic systems modeling dynamically modulated environmental effects on the photosynthetic apparatus are presented. Two photochemical systems composed of a supramolecular duplex scaffold, a photosensitizer-functionalized strand (photosensitizer is Zn(II)protoporphyrin IX, Zn(II)PPIX, or pyrene), an electron acceptor bipyridinium (V2+)-modified strand, and a nicking enzyme (Nt.BbvCI) act as functional assemblies driving transient photosynthetic-like processes. In the presence of a fuel strand, the transient electron transfer quenching of the photosensitizers, in each of the photochemical systems, is activated. In the presence of a sacrificial electron donor (mercaptoethanol) and continuous irradiation, the resulting electron transfer process in the Zn(II)PPIX/V2+ photochemical module leads to the transient accumulation and depletion of the bipyridinium radical-cation (V·+) product, and in the presence of ferredoxin-NADP+ reductase and NADP+, to the kinetically modulated photosynthesis of NADPH. By subjecting the mixture of two photochemical modules to one of two inhibitors, the gated transient photoinduced electron transfer in the two modules is demonstrated. Such gated dissipative process highlights its potential as an important pathway to protect artificial photosynthetic module against overdose of irradiance and to minimize photodamage.

REAP (Rapid Elimination of Active Plasmodium): A photodynamic strategy exploiting intrinsic kinetics of the parasite to combat severe malaria.

Plasmodium falciparum, the causative organism of Malaria is a mosquito-borne parasitic disease which infects red blood cells (RBCs), where it multiplies rapidly and goes through different stages of its life cycle. When the parasite load exceeds >3% in the blood, malaria transforms into severe malaria which requires immediate attention as death occurs within hours to days. The increase in people traveling to malaria-endemic areas and resistance/partial resistance to most known antimalarial drugs has put the current management scheme in jeopardy. To improve the patient outcome at this point, the physician may opt to perform exchange transfusions from another individual as an adjunct therapy to reduce parasitized RBCs, but the strategy has many drawbacks, including chances of infection. These limitations can be mitigated if the patient's own blood is withdrawn/extracted, sterilized from the parasitic load and then re-transfused almost similar to what is done in extracorporeal blood treatment for sepsis, poisoning and graft versus host disease. Thus, in the present study a light-based photochemical approach, Photodynamic Therapy (PDT) built on delta-aminolevulinic acid-protoporphyrin IX (ALA-PpIX) synthesis is exploited. This modality was effective at destruction of both resistant and susceptible strains of parasites, including at a high load mimicking severe drug resistant malaria. The current findings have set the stage for concept of an ALA-PpIX based PDT platform, "the REAP (Rapid Elimination of Active Plasmodium) strategy". This approach provides an additional tool towards the defense against multi-drug resistant severe malaria, and other intracellular blood pathogens, dependent on heme-synthesis.

Understanding delayed fluorescence and triplet decays of Protoporphyrin IX under hypoxic conditions.

Photosensitizers of singlet oxygen exhibit three main types of reverse intersystem-crossing (RISC): thermally activated, triplet-triplet annihilation, and singlet oxygen feedback. RISC can be followed by delayed fluorescence (DF) emission, which can provide important information about the excited state dynamics in the studied system. An excellent model example is a widely used clinical photosensitizer Protoporphyrin IX, which manifests all three mentioned types of RISC and DF. Here, we estimated rate constants of individual RISC and DF processes in Protoporphyrin IX in dimethylformamide, and we showed how these affect triplet decays and DF signals under diverse experimental conditions, such as a varying oxygen concentration or excitation intensity. This provided a basis for a general discussion on guidelines for a more precise analysis of long-lived signals. Furthermore, it has been found that PpIX photoproducts and potential transient excited complexes introduce a new overlapping delayed luminescence spectral band with a distinct lifetime. These findings are important for design of more accurate biological oxygen sensors and assays based on DF and triplet lifetime.

An MSN-based synergistic nanoplatform for root canal biofilm eradication via Fenton-enhanced sonodynamic therapy.

The validity and biocompatibility of irrigating agents are imperative for the success of root canal therapy. The imperfections in the currently available irrigants highlight the fact that more advanced technologies and strategies are required for complete disinfection in endodontic treatments. In the present study, a Fenton reaction-enhanced antimicrobial sonodynamic therapy (SDT) platform was fabricated for root canal disinfection. Firstly, mesoporous silica nanoparticles (MSNs) were synthesized, grafted with an amino group and then conjugated with sonosensitizer protoporphyrin IX (PpIX). Iron ions were then anchored (M@P-Fe) to initiate a Fenton reaction. Nanoparticle characterization by size and zeta potential measurements, scanning electron microscopy, transmission electron microscopy and thermogravimetric analysis confirmed that the platform was successfully developed. Reactive oxygen species (ROS) generation assessment, methylene blue degradation and electron spin resonance assays illustrated upon ultrasound (US) irradiation, that augmented ROS, can be produced by US activated PpIX and iron mediated Fenton reactions from low concentration H2O2 (0.01%). In vitro anti-Enterococcus faecalis efficacy was demonstrated by growth curve and colony forming unit measurements. Confocal laser scanning microscopy and scanning electron microscopy observations illustrated the effectiveness of the platform on in situ biofilm eradication in root canal. Owing to the stronger oxidizing capability and short lifetime of ROS, the Fenton reaction-enhanced SDT can induce detrimental oxidative damage to bacteria upon activation of US while avoiding nonspecific toxicity to cells, which was verified by cytotoxicity evaluations using CCK-8 assay and morphology observation of MC3T3-E1 cells. Compared to commonly used NaClO, this nanoplatform displayed desirable anti-bacterial, anti-biofilm abilities and better biocompatibility. These results highlight that the integrated M@P-Fe + US + H2O2 platform is a promising candidate for US enhanced root canal irrigation and disinfection.

Oxygen-producing proenzyme hydrogels for photodynamic-mediated metastasis-inhibiting combinational therapy.

Photodynamic therapy (PDT) has provided a promising approach for the treatment of solid tumors, while the therapeutic efficacy is often limited due to the hypoxic tumor microenvironment, resulting in tumor metastasis. Herein, we report an oxygen-producing proenzyme hydrogel (OPeH) with photoactivatable enzymatic activity for PDT enabled metastasis-inhibiting combinational therapy of breast cancer. This OPeH based on alginate is composed of protoporphyrin IX (PpIX) conjugated manganese oxide (MnO2) nanoparticles, which act as both the photosensitizer and oxygen-producing agent, and singlet oxygen (1O2)-responsive proenzyme nanoparticles. In the hypoxic and acidic tumor microenvironment, MnO2 can generate 1O2 to promote PpIX-mediated PDT with an amplified 1O2 generation efficiency, which also triggers the cleavage of 1O2-responsive linkers and cascade activation of proenzymes for cancer cell death. This combinational therapy upon photoactivation not only greatly inhibited the tumor growth, but also suppressed lung metastasis in a mouse xenograft breast tumor model, which is impossible in the case of PDT alone. This study thus provides a proenzyme hydrogel platform with photoactivatable activity for metastasis-inhibiting cancer therapy with high efficacy and safety.