Regulating Photosensitizer Metabolism with DNAzyme-Loaded Nanoparticles for Amplified Mitochondria-Targeting Photodynamic Immunotherapy

Mitochondria-specific photosensitizer accumulation is highly recommended for photodynamic therapy and mitochondrial DNA (mtDNA) oxidative damage-based innate immunotherapy but remains challenging. 5-Aminolevulinic acid (ALA), precursor of photosensitizer protoporphyrin IX (PpIX), can induce the exclusive biosynthesis of PpIX in mitochondria. Nevertheless, its photodynamic effect is limited by the intracellular biotransformation of ALA in tumors. Here, we report a photosensitizer metabolism-regulating strategy using ALA/DNAzyme-co-loaded nanoparticles (ALA&Dz@ZIF-PEG) for mitochondria-targeting photodynamic immunotherapy. The zeolitic imidazolate framework (ZIF-8) nanoparticles can be disassembled and release large amounts of zinc ions (Zn2+) within tumor cells. Notably, Zn2+ can relieve tumor hypoxia for promoting the conversion of ALA to PpIX. Moreover, Zn2+ acts as a cofactor of rationally designed DNAzyme for silencing excessive ferrochelatase (FECH; which catalyzes PpIX into photoinactive Heme), cooperatively promoting the exclusive accumulation of PpIX in mitochondria via the “open source and reduced expenditure” manner. Subsequently, the photodynamic effects derived from PpIX lead to the damage and release of mtDNA and activate the innate immune response. In addition, the released Zn2+ further enhances the mtDNA/cGAS-STING pathway mediated innate immunity. The ALA&Dz@ZIF-PEG system induced 3 times more PpIX accumulation than ALA-loaded liposome, significantly enhancing tumor regression in xenograft tumor models

Spliced sB7-H3 is expressed as a soluble form and generally exists in healthy donors.

<p>A, The schematic diagram of chimeric sB7-H3-Fc: the complete spliced <i>sb7-h3</i> gene was fused with rabbit <i>fc</i> in one opened reading frame. B, pc-sB7-H3-Fc (lane 1) and vacant vector (lane 3) were transfected into 293T cells, respectively. The supernatant of transfected cells was harvested 24 h post transfection and submitted to western blot assay. Lane 2: prestained protein ladder. C, Investigation of spliced <i>sb7-h3</i> gene in the PBMCs of healthy donors: Lane 1: negative control; Lane 2–6: randomly selected healthy donors; Lane 7: positive control; Lane 8: DNA ladder.</p

Physiologically Inspired Mucin Coated <i>Escherichia coli</i> Nissle 1917 Enhances Biotherapy by Regulating the Pathological Microenvironment to Improve Intestinal Colonization

The delivery of probiotics to the microbiota is a promising method to prevent and treat diseases. However, oral probiotics will suffer from gastrointestinal insults, especially the pathological microenvironment of inflammatory diseases such as reactive oxygen species (ROS) and the exhausted mucus layer, which can limit their survival and colonization in the intestinal tract. Inspired by the fact that probiotics colonized and grew in the mucus layer under physiological conditions, we developed a strategy for a super probiotic (EcN@TA-Ca2+@Mucin) coated with tannic acid and mucin via layer-by-layer technology. We demonstrated that mucin endows probiotics with superior resistance to the harsh environment of the gastrointestinal tract and with strong adhesiveness to the intestine through its interaction with mucus, which enhanced colonization and growth of probiotics in the mucus layer without removing the coating. Moreover, EcN@TA-Ca2+@Mucin can distinctly down-regulate inflammation with ROS scavenging and reduce the side effects of bacterial translocation in inflammatory bowel diseases, increasing the abundance and diversity of the gut microflora. We envision that it is a powerful platform to improve the colonization of probiotics by regulating the pathological microenvironment, which is expected to provide an important perspective for applying the intestinal colonization of probiotics to treat a variety of diseases

Comparison in the expression of spliced sB7-H3 between patients and healthy donors.

<p>A, Sera were obtained from healthy donors or patients with HCC. The expression level of spliced sB7-H3 was monitored with sandwich ELISA assay. B, The cDNAs produced from PBMCs of healthy donors or patients with HCC were analyzed for expression of spliced sB7-H3 by real-time PCR assay. A greater than 2-fold increase over health donors was considered clinical significant. C, The expression of spliced sB7-H3 in PBMCs of healthy donors and patients, as well as patients' tumor tissues and peritumor tissues were evaluated with comparative qPCR assay. A greater than 2-fold increase in value over healthy donors was considered clinical significant. D, Spliced sB7-H3 of individual serum from patients enrolled with HCC before and after resection was evaluated with sandwich ELISA assay. The horizontal line indicates the mean value of each group. “*” indicated P<0.05, “**” indicated P<0.01.</p

Functional analysis of spliced sB7-H3 on T cell response.

<p>CFSE-labeled purified T cells were seeded to a 96-well flat-bottomed plate precoated with 1 µg/ml anti-human CD3 (OKT3), plus either 10 µg/ml spliced sB7-H3-Fc or Fc. After culturing for 72 h, T cell proliferation and cytokines in the culture supernatant was analyzed. A, A representative plot of T cell proliferation analyzed with FACS assay. B, Statistical analysis of the mean fluorescence intensity (MFI) on T cells (mean ± s.e.m). C and D, the concentration of IL-2 and IFN-γ in the culture supernatant was analyzed with multiplex kit and analyzed by FlowCytomixPro software. “*” indicated P<0.05.</p

Physiologically Inspired Mucin Coated <i>Escherichia coli</i> Nissle 1917 Enhances Biotherapy by Regulating the Pathological Microenvironment to Improve Intestinal Colonization

The delivery of probiotics to the microbiota is a promising method to prevent and treat diseases. However, oral probiotics will suffer from gastrointestinal insults, especially the pathological microenvironment of inflammatory diseases such as reactive oxygen species (ROS) and the exhausted mucus layer, which can limit their survival and colonization in the intestinal tract. Inspired by the fact that probiotics colonized and grew in the mucus layer under physiological conditions, we developed a strategy for a super probiotic (EcN@TA-Ca2+@Mucin) coated with tannic acid and mucin via layer-by-layer technology. We demonstrated that mucin endows probiotics with superior resistance to the harsh environment of the gastrointestinal tract and with strong adhesiveness to the intestine through its interaction with mucus, which enhanced colonization and growth of probiotics in the mucus layer without removing the coating. Moreover, EcN@TA-Ca2+@Mucin can distinctly down-regulate inflammation with ROS scavenging and reduce the side effects of bacterial translocation in inflammatory bowel diseases, increasing the abundance and diversity of the gut microflora. We envision that it is a powerful platform to improve the colonization of probiotics by regulating the pathological microenvironment, which is expected to provide an important perspective for applying the intestinal colonization of probiotics to treat a variety of diseases

Nucleotide sequence analysis of spliced <i>sb7-h3</i> gene.

<p>The complete nucleotide sequence is shown. The boxed sequences are primer pair used to amplify <i>b7-h3</i> gene. The highlighted sequence is the ORF of spliced sB7-H3. The contained exons and intron are labeled on the left.</p

Physiologically Inspired Mucin Coated <i>Escherichia coli</i> Nissle 1917 Enhances Biotherapy by Regulating the Pathological Microenvironment to Improve Intestinal Colonization

The delivery of probiotics to the microbiota is a promising method to prevent and treat diseases. However, oral probiotics will suffer from gastrointestinal insults, especially the pathological microenvironment of inflammatory diseases such as reactive oxygen species (ROS) and the exhausted mucus layer, which can limit their survival and colonization in the intestinal tract. Inspired by the fact that probiotics colonized and grew in the mucus layer under physiological conditions, we developed a strategy for a super probiotic (EcN@TA-Ca2+@Mucin) coated with tannic acid and mucin via layer-by-layer technology. We demonstrated that mucin endows probiotics with superior resistance to the harsh environment of the gastrointestinal tract and with strong adhesiveness to the intestine through its interaction with mucus, which enhanced colonization and growth of probiotics in the mucus layer without removing the coating. Moreover, EcN@TA-Ca2+@Mucin can distinctly down-regulate inflammation with ROS scavenging and reduce the side effects of bacterial translocation in inflammatory bowel diseases, increasing the abundance and diversity of the gut microflora. We envision that it is a powerful platform to improve the colonization of probiotics by regulating the pathological microenvironment, which is expected to provide an important perspective for applying the intestinal colonization of probiotics to treat a variety of diseases

Physiologically Inspired Mucin Coated <i>Escherichia coli</i> Nissle 1917 Enhances Biotherapy by Regulating the Pathological Microenvironment to Improve Intestinal Colonization

The delivery of probiotics to the microbiota is a promising method to prevent and treat diseases. However, oral probiotics will suffer from gastrointestinal insults, especially the pathological microenvironment of inflammatory diseases such as reactive oxygen species (ROS) and the exhausted mucus layer, which can limit their survival and colonization in the intestinal tract. Inspired by the fact that probiotics colonized and grew in the mucus layer under physiological conditions, we developed a strategy for a super probiotic (EcN@TA-Ca2+@Mucin) coated with tannic acid and mucin via layer-by-layer technology. We demonstrated that mucin endows probiotics with superior resistance to the harsh environment of the gastrointestinal tract and with strong adhesiveness to the intestine through its interaction with mucus, which enhanced colonization and growth of probiotics in the mucus layer without removing the coating. Moreover, EcN@TA-Ca2+@Mucin can distinctly down-regulate inflammation with ROS scavenging and reduce the side effects of bacterial translocation in inflammatory bowel diseases, increasing the abundance and diversity of the gut microflora. We envision that it is a powerful platform to improve the colonization of probiotics by regulating the pathological microenvironment, which is expected to provide an important perspective for applying the intestinal colonization of probiotics to treat a variety of diseases

Physiologically Inspired Mucin Coated <i>Escherichia coli</i> Nissle 1917 Enhances Biotherapy by Regulating the Pathological Microenvironment to Improve Intestinal Colonization

The delivery of probiotics to the microbiota is a promising method to prevent and treat diseases. However, oral probiotics will suffer from gastrointestinal insults, especially the pathological microenvironment of inflammatory diseases such as reactive oxygen species (ROS) and the exhausted mucus layer, which can limit their survival and colonization in the intestinal tract. Inspired by the fact that probiotics colonized and grew in the mucus layer under physiological conditions, we developed a strategy for a super probiotic (EcN@TA-Ca2+@Mucin) coated with tannic acid and mucin via layer-by-layer technology. We demonstrated that mucin endows probiotics with superior resistance to the harsh environment of the gastrointestinal tract and with strong adhesiveness to the intestine through its interaction with mucus, which enhanced colonization and growth of probiotics in the mucus layer without removing the coating. Moreover, EcN@TA-Ca2+@Mucin can distinctly down-regulate inflammation with ROS scavenging and reduce the side effects of bacterial translocation in inflammatory bowel diseases, increasing the abundance and diversity of the gut microflora. We envision that it is a powerful platform to improve the colonization of probiotics by regulating the pathological microenvironment, which is expected to provide an important perspective for applying the intestinal colonization of probiotics to treat a variety of diseases