Intracellular Gelation-Mediated Living Bacteria for Advanced Biotherapeutics in Mouse Models.

Despite its significant potential in various disease treatments and diagnostics, microbiotherapy is consistently plagued by multiple limitations ranging from manufacturing challenges to in vivo functionality. Inspired by the strategy involving nonproliferating yet metabolically active microorganisms, we report an intracellular gelation approach that can generate a synthetic polymer network within bacterial cells to solve these challenges. Specifically, poly(ethylene glycol dimethacrylate) (PEGDA, 700 Da) monomers are introduced into the bacterial cytosol through a single cycle of freeze-thawing followed by the initiation of intracellular free radical polymerization by UV light to create a macromolecular PEGDA gel within the bacterial cytosol. The molecular crowding resulting from intracytoplasmic gelation prohibits bacterial division and confers robust resistance to simulated gastrointestinal fluids and bile acids while retaining the ability to secrete functional proteins. Biocompatibility assessments demonstrate that the nondividing gelatinized bacteria are effective in alleviating systemic inflammation triggered by intravenous Escherichia coli injection. Furthermore, the therapeutic efficacy of gelatinized Lactobacillus rhamnosus in colitis mice provides additional support for this approach. Collectively, intracellular gelation indicates a universal strategy to manufacture next-generation live biotherapeutics for advanced microbiotherapy.

In Situ Sprayed Exosome-Cross-Linked Gel as Artificial Lymph Nodes for Postoperative Glioblastoma Immunotherapy.

One key challenge in postoperative glioblastoma immunotherapy is to guarantee a potent and durable T-cell response, which is restricted by the immunosuppressive microenvironment within the lymph nodes (LNs). Here, we develop an in situ sprayed exosome-cross-linked gel that acts as an artificial LN structure to directly activate the tumor-infiltrating T cells for prevention of glioma recurrence. Briefly, this gel is generated by a bio-orthogonal reaction between azide-modified chimeric exosomes and alkyne-modified alginate polymers. Particularly, these chimeric exosomes are generated from dendritic cell (DC)-tumor hybrid cells, allowing for direct and robust T-cell activation. The gel structure with chimeric exosomes as cross-linking points avoids the quick clearance by the immune system and thus prolongs the durability of antitumor T-cell immunity. Importantly, this exosome-containing immunotherapeutic gel provides chances for ameliorating functions of antigen-presenting cells (APCs) through accommodating different intracellular-acting adjuvants, such as stimulator of interferon genes (STING) agonists. This further enhances the antitumor T-cell response, resulting in the almost complete elimination of residual lesions after surgery. Our findings provide a promising strategy for postsurgical glioma immunotherapy that warrants further exploration in the clinical arena.

Chimeric Exosomes Functionalized with STING Activation for Personalized Glioblastoma Immunotherapy.

A critical challenge of existing cancer vaccines is to orchestrate the demands of antigen-enriched furnishment and optimal antigen-presentation functionality within antigen-presenting cells (APCs). Here, a complementary immunotherapeutic strategy is developed using dendritic cell (DC)-tumor hybrid cell-derived chimeric exosomes loaded with stimulator of interferon genes (STING) agonists (DT-Exo-STING) for maximized tumor-specific T-cell immunity. These chimeric carriers are furnished with broad-spectrum antigen complexes to elicit a robust T-cell-mediated inflammatory program through direct self-presentation and indirect DC-to-T immunostimulatory pathway. This chimeric exosome-assisted delivery strategy possesses the merits versus off-the-shelf cyclic dinucleotide (CDN) delivery techniques in both the brilliant tissue-homing capacity, even across the intractable blood-brain barrier (BBB), and the desired cytosolic entry for enhanced STING-activating signaling. The improved antigen-presentation performance with this nanovaccine-driven STING activation further enhances tumor-specific T-cell immunoresponse. Thus, DT-Exo-STING reverses immunosuppressive glioblastoma microenvironments to pro-inflammatory, tumoricidal states, leading to an almost obliteration of intracranial primary lesions. Significantly, an upscaling option that harnesses autologous tumor tissues for personalized DT-Exo-STING vaccines increases sensitivity to immune checkpoint blockade (ICB) therapy and exerts systemic immune memory against post-operative glioma recrudesce. These findings represent an emerging method for glioblastoma immunotherapy, warranting further exploratory development in the clinical realm.

Immunoadjuvant-Modified Rhodobacter sphaeroides Potentiate Cancer Photothermal Immunotherapy.

Photothermal immunotherapy has become a promising strategy for tumor treatment. However, the intrinsic drawbacks like light instability, poor immunoadjuvant effect, and poor accumulation of conventional inorganic or organic photothermal agents limit their further applications. Based on the superior carrying capacity and active tumor targeting property of living bacteria, an immunoadjuvant-intensified and engineered tumor-targeting bacterium was constructed to achieve effective photothermal immunotherapy. Specifically, immunoadjuvant imiquimod (R837)-loaded thermosensitive liposomes (R837@TSL) were covalently decorated onto Rhodobacter sphaeroides (R.S) to obtain nanoimmunoadjuvant-armed bacteria (R.S-R837@TSL). The intrinsic photothermal property of R.S combined R837@TSL to achieve in situ near-infrared (NIR) laser-controlled release of R837. Meanwhile, tumor immunogenic cell death (ICD) caused by photothermal effect of R.S-R837@TSL, synergizes with released immunoadjuvants to promote maturation of dendritic cells (DCs), which enhance cytotoxic T lymphocytes (CTLs) infiltration for further tumor eradication. The photosynthetic bacteria armed with immunoadjuvant-loaded liposomes provide a strategy for immunoadjuvant-enhanced cancer photothermal immunotherapy.

Mineralized Porphyrin Metal-Organic Framework for Improved Tumor Elimination and Combined Immunotherapy.

Calcium ion therapy is a potential anticancer treatment. However, the cellular calcium-buffering mechanism limited the effectiveness of calcium ion therapy. Here, we constructed a mineralized porphyrin metal-organic framework (PCa) to produce calcium ions and reactive oxygen species (ROS), which destroyed cell calcium buffering capacity and amplified the cell damage caused by calcium overload. In addition, PCa could induce cell immunogenic death to release tumor-associated antigen (TAA) and be used as an adjuvant. Thus, PCa could increase DC maturation and promote the antitumor activity of CD8+ T cells. For mice experiment, PCa not only showed excellent tumor elimination on the subcutaneous breast tumor but also achieved obvious antimetastasis effect in the metastatic tumor model. This nanosystem could eliminate the primary tumor and boost effective antitumor immunotherapy for comprehensive anticancer treatment.

A H2O2-responsive theranostic platform for chemiluminescence detection and synergistic therapy of tumors.

Chemiluminescence substances that respond to hydrogen peroxide (H2O2) in a tumor microenvironment have the potential to achieve accurate tumor imaging. Here, Pluronic F-127 (PF127) and polymers containing oxalate ester (POE) were assembled by hydrophilic and hydrophobic forces to form nanoparticles to load the anti-tumor drug lapachone (Lapa) and rubrene. The Lapa-loaded H2O2-responsive nanoparticles (L-HPOX) could track tumors in vivo through H2O2-related chemiluminescence. With the presence of H2O2 in the tumor microenvironment, L-HPOX would collapse and release the loaded drug for anti-tumor therapy. After treatment with 5,6-dimethylxanthenone-4-acetic acid (DMXAA), the inflammatory level and H2O2 content increased. Thus, L-HPOX exhibited good capabilities of tumor imaging and treatment. Importantly, the immune system was also activated for anti-metastatic activity. This intelligent and efficient chemiluminescent tumor theranostic nanoplatform will find great potential for precise and efficient tumor treatment.

Multifunctionalized Gold Sub-Nanometer Particles for Sensitizing Radiotherapy against Glioblastoma.

Glioblastoma is the most common lethal malignant intracranial tumor with a low 5-year survival rate. Currently, the maximal safe surgical resection, followed by high-dose radiotherapy (RT), is a standard treatment for glioblastoma. However, high-dose radiation to the brain is associated with brain injury and results in a high fatality rate. Here, integrated pharmaceutics (named D-iGSNPs) composed of gold sub-nanometer particles (GSNPs), blood-brain barrier (BBB) penetration peptide iRGD, and cell cycle regulator α-difluoromethylornithine is designed. In both simulated BBB and orthotopic murine GL261 glioblastoma models, D-iGSNPs are proved to have a beneficial effect on the BBB penetration and tumor targeting. Meanwhile, data from cell and animal experiments reveal that D-iGSNPs are able to sensitize RT. More importantly, the synergy of D-iGSNPs with low-dose RT can exhibit an almost equal therapeutic effect with that of high-dose RT. This study demonstrates the therapeutic advantages of D-iGSNPs in boosting RT, and may provide a facile approach to update the current treatment of glioblastoma.

Integration of a porous coordination network and black phosphorus nanosheets for improved photodynamic therapy of tumor.

Selectively attenuating the protection offered by heat shock protein 90 (HSP90), which is indispensable for the stabilization of the essential regulators of cell survival and works as a cell guardian under oxidative stress conditions, is a potential approach to improve the efficiency of cancer therapy. Here, we designed a biodegradable nanoplatform (APCN/BP-FA) based on a Zr(iv)-based porphyrinic porous coordination network (PCN) and black phosphorus (BP) sheets for efficient photodynamic therapy (PDT) by enhancing the accumulation of the nanoplatforms in the tumor area and attenuating the protection of cancer cells. Owing to the favorable degradability of BP, the nanosystem exhibited accelerated the release of the HSP90 inhibitor tanespimycin (17-AAG) and an apparent promotion in the reactive oxygen species (ROS) yield of PCN as well as expedited the degradation of the PCN-laden BP nanoplatforms. Both in vitro and in vivo results revealed that the elevated amounts of ROS and reduced cytoprotection in tumor cells were caused by the nanoplatforms. This strategy may provide a promising method for attenuating cytoprotection to aid efficient photodynamic therapy.

Biomimetic carbon monoxide nanogenerator ameliorates streptozotocin induced type 1 diabetes in mice.

Diabetes is an increasing health problem and associated with inflammatory complications that seriously affects the quality of life and survival of patients. Carbon monoxide (CO), owing to its anti-inflammatory and anti-apoptotic properties, has become a potential therapeutic molecule for the treatment of autoimmune diseases. Here, we constructed a mesoporous silica-based biomimetic CO nanogenerator (mMMn), which was loaded with manganese carbonyl and camouflaged with macrophage membrane. Driven by the active targeting of macrophage membrane to inflammatory sites, the as-designed mMMn could effectively accumulate in pancreatic tissue of type 1 diabetic mice, which was established by consecutive administration of streptozotocin (STZ). It was found that the local reactive oxygen species (ROS) within pancreas could trigger the continuous CO release from mMMn, which greatly ameliorated diabetes in mice with improved blood glucose homeostasis by alleviating inflammatory responses and inhibiting ß-cells apoptosis. The exogenous CO targeting to pancreatic tissue paves a novel way for the treatment of type 1 diabetes.

A MSN-based tumor-targeted nanoplatform to interfere with lactate metabolism to induce tumor cell acidosis for tumor suppression and anti-metastasis.

Lactate, the main contributor to the acidic tumor microenvironment, not only promotes the proliferation of tumor cells, but also closely relates to tumor invasion and metastasis. Here, a tumor targeting nanoplatform, designated as Me&Flu@MSN@MnO2-FA, was fabricated for effective tumor suppression and anti-metastasis by interfering with lactate metabolism of tumor cells. Metformin (Me) and fluvastatin sodium (Flu) were incorporated into MnO2-coated mesoporous silicon nanoparticles (MSNs), the synergism between Me and Flu can modulate the pyruvate metabolic pathway to produce more lactate, and concurrently inhibit lactate efflux to induce intracellular acidosis to kill tumor cells. As a result of the restricted lactate efflux, the extracellular lactate concentration is reduced, and the ability of the tumor cells to migrate is also weakened. This ingenious strategy based on Me&Flu@MSN@MnO2-FA showed an obvious inhibitory effect on tumor growth and resistance to metastasis.

Hydrogen gas improves photothermal therapy of tumor and restrains the relapse of distant dormant tumor.

Inflammation during photothermal therapy (PTT) of tumor usually results in adverse consequences. Here, a biomembrane camouflaged nanomedicine (mPDAB) containing polydopamine and ammonia borane was designed to enhance PTT efficacy and mitigate inflammation. Polydopamine, a biocompatible photothermal agent, can effectively convert light into heat for PTT. Ammonia borane was linked to the surface of polydopamine through the interaction of hydrogen bonding, which could destroy redox homoeostasis in tumor cells and reduce inflammation by H2 release in tumor microenvironment. Owing to the same origin of outer biomembranes, mPDAB showed excellent tumor accumulation and low systemic toxicity in a breast tumor model. Excellent PTT efficacy and inflammation reduction made the mPDAB completely eliminate the primary tumors, while also restraining the outgrowth of distant dormant tumors. The biomimetic nanomedicine shows potentials as a universal inflammation-self-alleviated platform to ameliorate inflammation-related disease treatment, including but not limited to PTT for tumor.

A hybrid nanomaterial with NIR-induced heat and associated hydroxyl radical generation for synergistic tumor therapy.

Although photothermal therapy (PTT) and photodynamic therapy (PDT) are widely commended for tumor treatment recently, they still suffer severe challenges due to the non-specificity of photothermal agents (PTAs)/photosensitizers (PSs) and hypoxic tumor microenvironment. Here, an oxygen independent biomimetic nanoplatform based on carbon sphere dotted with cerium oxide and coated by cell membrane (MCSCe) was designed and synthesized with good biocompatibility, homologous targeting ability, and improved photophysical activity. Notably, MCSCe could realize accumulation of hydrogen peroxide (H2O2) in tumor cells and hyperthermia under single laser (808 nm) irradiation, which were simultaneously utilized by itself to produce more toxic hydroxyl radical (OH). Resultantly, the synergistic therapeutic effect against tumor cells was obtained under near infrared (NIR) laser irradiation.

A tungsten nitride-based degradable nanoplatform for dual-modal image-guided combinatorial chemo-photothermal therapy of tumors.

An innovative tungsten-based multifunctional nanoplatform composed of polyethylene glycol (PEG)-modified tungsten nitride nanoparticles (WN NPs) is constructed for tumor treatment. The PEG-WN NPs not only possess strong near-infrared (NIR) absorbance, high photothermal conversion efficiency, and excellent photothermal stability, but also effectively inhibit tumor cells upon 808 nm laser irradiation. After coating with thiolated (2-hydroxypropyl)-ß-cyclodextrin (MUA-CD) on the surface, such a nanoplatform can also be used for drug delivery (such as DOX) and presents a synergistic tumor inhibition effect both in vitro and in vivo. Furthermore, the PEG-WN NPs present good contrasting capability for X-ray computed tomography (CT) and photoacoustic (PA) imaging. With PA/CT imaging, the tumor can be accurately positioned for precise treatment. It is worth mentioning that PEG-WN NPs are biodegradable and could be effectively excreted from the body with no appreciable toxicity in vivo. It is expected that this biocompatible multifunctional nanoplatform can serve as a potential candidate for tumor treatment in future clinical applications.

The influence of amine structures on the stability and catalytic activity of gold nanoparticles stabilized by amine-modified hyperbranched polymers.

Amine-modified amphiphilic hyperbranched polymers (MePEG-H104-Nx) were prepared from hyperbranched 2,2-bis(methylol)propionic acid polyester (H104) by decoration with polyethylene glycol monomethyl ether (MePEG) and different classes of oligo(ethylenimine)s. By using the MePEG-H104-Nx polymers as stabilizers, gold nanoparticles (AuNPs) were prepared in an aqueous medium by the reduction of HAuCl4 with NaBH4. The AuNPs were sphere-like with diameters of 2-4 nm, which were dependent on the structure of the amines. Further, the catalytic activity of these AuNPs was evaluated by monitoring the reduction reaction of 4-nitrophenol by sodium borohydride. The results demonstrate that the longer chain length and the branched structure of the amine moieties are beneficial for the stability and catalytic activity of the AuNPs. The AuNPs stabilized by MePEG-H104-N4 and MePEG-H104-Nb3 showed high catalytic activity for the reduction of 4-nitrophenol to 4-aminophenol.

Injectable hyaluronic acid/poly(ethylene glycol) hydrogels crosslinked via strain-promoted azide-alkyne cycloaddition click reaction.

This paper reports injectable hyaluronic acid (HA)-based hydrogels crosslinked with azide-modified poly(ethylene glycol) (PEG) via the strain-promoted azide-alkyne cycloaddition (SPAAC) between cyclooctyne and azide groups. Cyclooctyne-modified HA (Cyclooctyne-HA) is prepared by the reaction of HA with 2-(aminoethoxy)cyclooctyne. To crosslink the modified HA, quadruply azide-terminated poly(ethylene glycol) (Azide-PEG) is designed and prepared. The mixture of Cyclooctyne-HA and Azide-PEG gelates in a few minutes to form a strong HA-PEG hydrogel. The hydrogel has fast gelation time, good strength, and slow degradation rate, because of the high reactivity of SPAAC, high crosslinking density originated from the quadruply-substituted Azide-PEG, and the good stability of the crosslinking amide bonds. In vitro cell culturing within the hydrogel demonstrated an excellent cell-compatibility. The bioorthogonality of SPAAC makes the hydrogel injectable. With good mechanical properties and biocompatibility, the hydrogel would be useful in a wide range of applications such as injection filling materials for plastic surgery.

Redox-Responsive Micelles with Cores Crosslinked via Click Chemistry.

Redox-responsive micelles with cores crosslinked via click chemistry are developed to improve the stability of polymer micelles. Amphiphilic block copolymer mPEG-b-P(DTC-ADTC) with pendant azido groups on the hydrophobic chains is synthesized by the ring-opening polymerization of 2,2-bis(azidomethyl)trimethylene carbonate (ADTC) and 2,2-dimethyltrimethylene carbonate (DTC) with monomethoxy poly(ethylene glycol) (mPEG) as an initiator. mPEG-b-P(DTC-ADTC) self-assemble to form the micelles in aqueous solution and the cores of the micelles are crosslinked via click chemistry to afford redox-responsive core-crosslinked micelles. Core-crosslinking enhances the stability of the micelles in aqueous solution and improve the drug-loading property. The redox-responsive core-crosslinked micelles can be reduced by the addition of reducing agents such as dithiothreitol (DTT), and thus release the loaded drug quickly in the presence of DTT.

An injectable and fast-degradable poly(ethylene glycol) hydrogel fabricated via bioorthogonal strain-promoted azide-alkyne cycloaddition click chemistry.

Biocompatible and degradable injectable materials prepared via bioorthogonal reactions are highly promising for biomedical applications because they can be formed in situ and administered in a minimally invasive way. In this work, a PEG-based injectable hydrogel was fabricated via a copper-free, strain-promoted azide-alkyne cycloaddition (SPAAC) click chemistry. Azide and cyclooctyne moieties on the PEG backbones underwent a rapid click reaction to trigger the formation of the hydrogel within several minutes. Resulting from the introduction of ester groups into the cross-linked network, the hydrogel presented pH-dependent hydrolysis and biological fast degradability. Good biocompatibility of the hydrogel was verified by in vitro cytotoxicity assay and in vivo studies. The hydrogel formed in situ after subcutaneously injecting the gel precursors into Kungming (KM) mice. The implanted hydrogel caused a mild inflammatory response in vivo, and the surrounding tissues fully recovered a week after the injection. The injectable and fast-degradable hydrogel fabricated by the bioorthogonal click reaction may be useful as biomaterials such as embolic agents for interventional therapy.

Phenylboronic acid-functionalized polymeric micelles with a HepG2 cell targetability.

Phenylboronic acid-functionalized amphiphilic block copolymer Pluronic-PMCC-BA was synthesized via ring-opening polymerization of 5-methyl-5-benzyloxycarbonyl-1,3-dioxan-2-one (MBC) with fumaric acid as a catalyst followed by the deprotection of carboxyl groups by catalyzed hydrogenation and the condensation of 3-aminophenylboronic acid with the copolymer side groups. Pluronic-PMCC-BA can form stable micelle solution by self-assembly in water. The phenylboronic acid groups are located at the shell of micelle as proved by (1)H NMR. The diameter of drug-free micelles is approximate 60 nm. Nano-spheres with narrow size distribution could be observed in the TEM image. MTT assay results show that Pluronic-PMCC-BA exhibits slight cytotoxicity when the polymer concentration is higher than 25 µg mL(-1). The toxicities of DOX@Pluronic-PMCC and DOX@Pluronic-PMCC-BA to COS7, HeLa, and HepG2 cell lines are similar with those of free DOX. Interestingly, phenylboronic acid groups located at the surface of Pluronic-PMCC-BA micelles can recognize HepG2 cells and promote the drug uptake of the cells, which are observed by confocal laser scanning microscopy (CLSM). The results imply that Pluronic-PMCC-BA would be a promising material for targeted drug delivery to the cancer cells.

Nanotoxicity comparison of four amphiphilic polymeric micelles with similar hydrophilic or hydrophobic structure.

BACKGROUND: Nanocarriers represent an attractive means of drug delivery, but their biosafety must be established before their use in clinical research. OBJECTIVES: Four kinds of amphiphilic polymeric (PEG-PG-PCL, PEEP-PCL, PEG-PCL and PEG-DSPE) micelles with similar hydrophilic or hydrophobic structure were prepared and their in vitro and in vivo safety were evaluated and compared. METHODS: In vitro nanotoxicity evaluations included assessments of cell morphology, cell volume, inflammatory effects, cytotoxicity, apoptosis and membrane fluidity. An umbilical vein cell line (Eahy.926) and a kind of macrophages (J774.A1) were used as cell models considering that intravenous route is dominant for micelle delivery systems. In vivo analyses included complete blood count, lymphocyte subset analysis, detection of plasma inflammatory factors and histological observations of major organs after intravenous administration to KM mice. RESULTS: All the micelles enhanced inflammatory molecules in J774.A1 cells, likely resulting from the increased ROS levels. PEG-PG-PCL and PEEP-PCL micelles were found to increase the J774.A1 cell volume. This likely correlated with the size of PEG-PG-PCL micelles and the polyphosphoester structure in PEEP-PCL. PEG-DSPE micelles inhibited the growth of Eahy.926 cells via inducing apoptosis. This might relate to the structure of DSPE, which is a type of phospholipid and has good affinity with cell membrane. No evidence was found for cell membrane changes after treatment with these micelles for 24 h. In the in vivo study, during 8 days of 4 time injection, each of the four nanocarriers altered the hematic phase differently without changes in inflammatory factors or pathological changes in target organs. CONCLUSIONS: These results demonstrate that the micelles investigated exhibit diverse nanotoxicity correlated with their structures, their biosafety is different in different cell model, and there is no in vitro and in vivo correlation found. We believe that this study will certainly provide more scientific understandings on the nanotoxicity of amphiphilic polymeric micelles.

Phenylboronic-acid-modified amphiphilic polyether as a neutral gene vector.

A phenylboronic-acid-modified amphiphilic block polyether is prepared via reaction of polyglycidol-block-poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide)-block-polyglycidol (Pluronic-PG) with 2-(N,N-dimethylaminomethyl)-5-aminomethyl phenylboronic acid using phosgene as a coupling reagent. The boronic-acid-modified non-cationic polymer binds plasmid pGL3 effectively, forms sub-µm polymer/DNA complex particles, and greatly facilitates the cell uptake of the plasmid. The efficiency of the polymer as a gene vector is evaluated in vitro by transfection of pGL3 to HeLa, COS-7 and HepG2 cells. Pluronic-PG-BA enhances the transfection efficiency by 100 to 1000 times compared with Pluronic-PG. The presence of serum does not significantly affect the transfection efficiency.