Striking Back against Fungal Infections: The Utilization of Nanosystems for Antifungal Strategies.

Fungal infections have become a major health concern, given that invasive infections by Candida, Cryptococcus, and Aspergillus species have led to millions of mortalities. Conventional antifungal drugs including polyenes, echinocandins, azoles, allylamins, and antimetabolites have been used for decades, but their limitations include off-target toxicity, drug-resistance, poor water solubility, low bioavailability, and weak tissue penetration, which cannot be ignored. These drawbacks have led to the emergence of novel antifungal therapies. In this review, we discuss the nanosystems that are currently utilized for drug delivery and the application of antifungal therapies.

Exploring emerging drug responses in Cryptococcus.

Cryptococcosis imposes a considerable burden on public health, and emerging drug responses to anticryptococcal drugs remain to be addressed. In this forum article we discuss the emerging drug responses of Cryptococcus, focusing on the critical nature of understanding such responses in order to improve the effectiveness of anticryptococcal therapeutics.

The function and regulation of heat shock transcription factor in Cryptococcus.

Cryptococcus species are opportunistic human fungal pathogens. Survival in a hostile environment, such as the elevated body temperatures of transmitting animals and humans, is crucial for Cryptococcus infection. Numerous intriguing investigations have shown that the Hsf family of thermotolerance transcription regulators plays a crucial role in the pathogen-host axis of Cryptococcus. Although Hsf1 is known to be a master regulator of the heat shock response through the activation of gene expression of heat shock proteins (Hsps). Hsf1 and other Hsfs are multifaceted transcription regulators that regulate the expression of genes involved in protein chaperones, metabolism, cell signal transduction, and the electron transfer chain. In Saccharomyces cerevisiae, a model organism, Hsf1's working mechanism has been intensively examined. Nonetheless, the link between Hsfs and Cryptococcus pathogenicity remains poorly understood. This review will focus on the transcriptional regulation of Hsf function in Cryptococcus, as well as potential antifungal treatments targeting Hsf proteins.

A thermo-responsive hydrogel loaded with an ionic liquid microemulsion for transdermal delivery of methotrexate.

Systemic administration of methotrexate (MTX), the gold standard for the treatment of psoriasis, can cause adverse side effects. To address this issue, a thermally responsive hydrogel loaded with an ionic liquid microemulsion (IL-ME) was developed for the transdermal delivery of MTX. The microemulsion was prepared using water, Tween 20 and choline and geranic acid (CAGE) ionic liquid. The solubility of methotrexate in the IL-ME was 9-fold higher than that in phosphate buffer (PBS). The hydrogel (Gel) based on isopropylacrylamide and silk fibroin acts as a drug reservoir to achieve temperature-responsive drug release in the human epidermis. The loading of the IL-ME or MTX-containing IL-ME on the Gel produced a ME@Gel or MTX/ME@Gel. In vitro permeation experiments showed that the MTX/ME@Gel exhibited a 27.6% increase in MTX permeation. In addition, IL-ME exhibits strong antibacterial activity. In vivo studies indicated that when compared with the results obtained in PBS medium, the MTX/ME@Gel could effectively treat skin redness, swelling and scaling caused by imiquimod (IMQ). In general, the present study demonstrated that the MTX/ME@Gel provides vast potential to serve as a biocompatible drug carrier for the efficient delivery of poorly soluble drugs, i.e., MTX in this particular case.

An ionic gel incorporating copper nanodots with antibacterial and antioxidant dual functions for deep tissue penetration treatment of periodontitis in rats.

The local treatment of periodontitis has recently received extensive attention due to the advantages of mild systemic side effects and a high local drug concentration. But the local drug delivery systems are not widely used in clinical practice, and a major obstacle is the poor tissue permeability. In the present study, we report a copper nanodot based ionic gel (Cu-NDs/IL gel) with favorable tissue penetration capability, and it concurrently exhibits antibacterial and anti-inflammatory functions. A Cu-NDs/IL gel was prepared by loading copper nanodots (Cu-NDs) with triple enzyme-like activities into a multifunctional gel. The derived Cu-NDs/IL gel possesses remarkable antibacterial properties attributed to the peroxidase-like activity of Cu-NDs. In addition, Cu-NDs mimic superoxide dismutase and catalase activities, which endow the gel with excellent free radical scavenging capability in a neutral environment to relieve periodontal inflammation. More importantly, the IL moiety in the Cu-NDs/IL gel promotes the penetration of Cu-NDs into the gingival tissue, wherein the triple enzymatic activity of Cu-NDs may function. In short, the Cu-NDs/IL gel has promising potential to serve as a topical drug for periodontitis by promoting penetration, killing bacteria, and scavenging ROS.

Advances of ionic liquid-based nanohybrids for biomedical applications.

Ionic liquids (ILs) are composed of asymmetric cationic and anionic moieties and are used as green solvents. Their non-toxic nature, favorable biocompatibility and adjustable structure facilitate wide biomedical applications. ILs promote the generation of various nanohybrids that exhibit multiple functions and novel/improved properties with respect to their precursors. Generally, nanostructures have a large specific surface area and abundant functional groups which enable loading and incorporation of ILs through physical interactions or chemical bonding. According to their main skeleton structures, IL-based nanohybrids may be divided into five categories, i.e., poly(ionic liquid)s (PILs), IL-inorganic nanohybrids, IL-metal organic framework nanohybrids (IL-MOF nanohybrids), ILs/carbon materials and ionic materials. These IL-based nanohybrids exhibit various specific features, including thermal responsive behavior, metal chelating, photothermal conversion and antibacterial capabilities. Taking advantage of these characteristics, IL-based nanohybrids may overcome the shortcomings of conventional medicines/drugs and exhibit promising prospects in biomedicine to facilitate controlled drug release, bactericidal treatment and thermotherapy. The present review presents the state-of-the-art progress made in the studies of IL-based nanohybrids in terms of their classifications, structure characteristics, versatile functionalities and biomedical and pharmaceutical applications. The challenges and future perspectives in the developments and applications of IL-based nanohybrids in biomedicine are discussed.

GSH-depleting metal-polyphenol-network nanoparticles with dual enzyme activities induce enhanced ferroptosis.

Ferroptosis is a non-apoptotic form of regulated cell death. The efficiency of ferroptosis is restrained in the tumor microenvironment (TME) by overexpression of glutathione (GSH) and insufficient production of hydrogen peroxide (H2O2). In this work, theranostic nanoparticles Ce-aMOFs@Fe3+-EGCG, termed MEFs, are developed by coating uniform Ce-based amorphous metal-organic frameworks (Ce-aMOFs) with epigallocatechin gallate (EGCG) and Fe3+. Fe3+ is chelated by the adjacent phenol hydroxyl groups in EGCG. In the tumor cell interior, overexpressed GSH and weak acidic medium degrade the coating to release Fe3+ and EGCG accompanied by exposure of Ce-aMOFs. Fe3+ and EGCG consume GSH along with turning Fe3+ into Fe2+. Ce-aMOFs act as a nanozyme possessing dual-enzymatic activities, i.e. superoxide dismutase (SOD)- and phosphatase-like activities. In the TME, Ce-aMOFs catalyze the conversion of endogenous superoxide (O2˙-) into H2O2, and Fe2+ catalyzes H2O2 to generate toxic hydroxyl radicals (˙OH), which may further induce tumor cell death through ferroptosis. In addition, the phosphatase-like activity of Ce-aMOFs may sustainably dephosphorylate NADPH and effectively inhibit intracellular biosynthesis of GSH. Therefore, MEFs ensure down-regulation of intracellular GSH levels and up-regulation of oxidative pressure, which enhance the ferroptosis effect.

Immunization with a heat-killed prm1 deletion strain protects the host from Cryptococcus neoformans infection.

Systemic infection with Cryptococcus neoformans, a dangerous and contagious pathogen found throughout the world, frequently results in lethal cryptococcal pneumonia and meningoencephalitis, and no effective treatments and vaccination of cryptococcosis are available. Here, we describe Prm1, a novel regulator of C. neoformans virulence. C. neoformans prm1Δ cells exhibit extreme sensitivity to various environmental stress conditions. Furthermore, prm1Δ cells show deficiencies in the biosynthesis of chitosan and mannoprotein, which in turn result in impairment of cell wall integrity. Treatment of mice with heat-killed prm1Δ cells was found to facilitate the host immunological defence against infection with wild-type C. neoformans. Further investigation demonstrated that prm1Δ cells strongly promote pulmonary production of interferon-γ, leading to activation of macrophage M1 differentiation and inhibition of M2 polarization. Therefore, our findings suggest that C. neoformans Prm1 may be a viable target for the development of anti-cryptococcosis medications and, cells lacking Prm1 represent a promising candidate for a vaccine.

Ionic liquids enable the preparation of a copper-loaded gel with transdermal delivery function for wound dressings.

Antibacterial hydrogel dressings play an important role in wound healing and infection treatment. The majority of hydrogels are obtained through chemical cross-linking and complex synthesis or processing. Copper ions (Cu2+) have been involved in sterilization; however, their direct use may lead to high local concentrations and heavy metal toxic side effects. Herein, dopamine (DA) was polymerized in situ along a polyvinyl alcohol (PVA) chain and chelated copper ions (Cu2+) to form a mixture. Ionic liquid (IL) choline-glycolate (CGLY) was added to the mixture to form an ionic gel. CGLY promotes gel formation through intermolecular hydrogen bonds with the polymer chains and avoids the use of toxic chemical crosslinking agents. Meanwhile, CGLY can also promote the release of Cu2+ and generate hydrogel free radicals (˙OH) in the wound through chemodynamic therapy to kill drug-resistant bacteria. In addition, the excellent transdermal property of CGLY enables the released Cu2+ to stimulate cell migration and accelerate wound healing. The gel exhibits favorable biocompatibility and its use has been demonstrated in skin infection therapy of mice.

Correction: Photodynamic antimicrobial chemotherapy with cationic phthalocyanines against Escherichia coli planktonic and biofilm cultures.

[This corrects the article DOI: 10.1039/C7RA06073D.].

Cryptococcal Hsf3 controls intramitochondrial ROS homeostasis by regulating the respiratory process.

Mitochondrial quality control prevents accumulation of intramitochondrial-derived reactive oxygen species (mtROS), thereby protecting cells against DNA damage, genome instability, and programmed cell death. However, underlying mechanisms are incompletely understood, particularly in fungal species. Here, we show that Cryptococcus neoformans heat shock factor 3 (CnHsf3) exhibits an atypical function in regulating mtROS independent of the unfolded protein response. CnHsf3 acts in nuclei and mitochondria, and nuclear- and mitochondrial-targeting signals are required for its organelle-specific functions. It represses the expression of genes involved in the tricarboxylic acid cycle while promoting expression of genes involved in electron transfer chain. In addition, CnHsf3 responds to multiple intramitochondrial stresses; this response is mediated by oxidation of the cysteine residue on its DNA binding domain, which enhances DNA binding. Our results reveal a function of HSF proteins in regulating mtROS homeostasis that is independent of the unfolded protein response.

Gold nanoclusters exert antibacterial effects against gram-negative bacteria by targeting thiol-redox homeostasis.

Low-molecular-weight thiols play a central role in preventing oxidative damage caused by reactive oxygen species to maintain a reductive intracellular environment in bacteria. Therefore, targeting thiol-redox homeostasis is considered a promising antimicrobial strategy. Here, we synthesize histidine-stabilized gold nanoclusters (Au NCs), in which the histidine ligand has a weak affinity for gold, thus constructing an effective thiol scavenger by employing metal-thiol depletion chemicals. Au NCs exert excellent antimicrobial effects by consuming thiols and simultaneously causing the accumulation of reactive oxygen species in bacteria, resulting in severe oxidative stress. In a mouse skin wound model infected with ampicillin-resistant Escherichia coli pHSP70-EGFP, a low dose of Au NCs exerts a strong therapeutic effect on bacterial clearance and wound healing, indicating the effectiveness of this antimicrobial strategy for clinical application.

Mutual Benefit between Cu(II) and Polydopamine for Improving Photothermal-Chemodynamic Therapy.

Combination therapy has attracted extensive interest in alleviating the shortcomings of monotherapy and enhancing the treatment efficacy. In this work, hollow mesoporous silica nanoparticles (HMSNs) play the role of nanocarriers in the delivery of Cu(II)-doped polydopamine (PDA), termed as HMSNs@PDA-Cu, for synergistic therapy. PDA acts as a traditional photothermal agent to realize photothermal treatment (PTT). Chemodynamic therapy (CDT) is realized by the reaction of Cu(II) with intracellular glutathione (GSH), and subsequently, the generated Cu(I) reacts with H2O2 to produce toxic hydroxyl radical (•OH) through a Fenton-like reaction. The photothermal performance of PDA is improved after its coordination with Cu(II). On the other hand, PDA exhibits superoxide dismutase (SOD)-mimicking activity. PDA converts O2•- to H2O2 and improves the production of H2O2, which promotes the therapeutic effect of CDT. Moreover, the high temperature caused by PTT further enhances the yield of •OH for CDT. This nanotheranostic platform perfectly applied to the tumor depletion of mice, presenting great potential for cancer metastasis therapy in vitro and in vivo.

Tailoring the cationic lipid composition of lipo-DVDMS augments the phototherapy efficiency of burn infection.

Increase in infections with Gram-negative Pseudomonas aeruginosa (P. aeruginosa) is a serious global challenge in healthcare. Sinoporphyrin sodium (DVDMS) combined with photodynamic antimicrobial chemotherapy (PACT) can effectively eradicate Gram-positive organisms. However, the poor penetration of DVDMS into the Gram-negative bacterial cell membrane and bacterial biofilm greatly limits the photo-inspired antimicrobial activity. This study optimized the cationic lipid-mediated nano-DVDMS delivery to improve the cellular uptake, and evaluated the antimicrobial efficacy of cationic DVDMS-liposome (CDL)-provoked PACT in both P. aeruginosa and its multidrug resistant strain. The results showed that the positively charged liposome modification promoted the enrichment of DVDMS in Gram-negative bacteria. CDL-PACT-produced ROS and caused bacterial death, accompanied by the decreased expression levels of virulence factor-related genes. The P. aeruginosa-infected burn model indicated satisfactory bacterial eradication and accelerated wound healing after CDL-PACT, in addition to gradually increasing bFGF, VEGF, TGF-ß1 and Hyp levels and reducing TNF-α and IL-6, with no detectable side-effects. Overall, these findings provide fundamental knowledge that enables the design of feasible and efficient PACT treatments, including biophysical membrane permeabilization and photodynamic eradication, which are promising to overcome the infection and resistance of highly opportunistic Gram-negative bacteria.

State-of-the-art advances of copper-based nanostructures in the enhancement of chemodynamic therapy.

Chemodynamic therapy (CDT) is a new emerging strategy for the in situ treatment of tumors. In the microenvironment of tumor cells, CDT may be achieved through the generation of reactive oxygen species (ROS), e.g., hydroxyl radicals (˙OH) and singlet oxygen (1O2), which induce the death of tumor cells. Copper (Cu) or other transition-metal ions catalyze the production of ˙OH by hydrogen peroxide (H2O2) through Fenton or Fenton-like reactions. With the development of advanced nanotechnology, nanotherapeutic systems with Cu-based nanostructures have received extensive attention and have been demonstrated for their wide applications in the design and construction of nanotherapeutic systems for CDT, along with multimodal synergistic therapy. Herein, the cutting-edge developments of Cu-based nanostructures in CDT are reviewed and discussed, by focusing on the monotherapy of CDT as well as synergistic treatments by hyphenating CDT with various therapeutic protocols, e.g., photothermal therapy (PTT), photodynamic therapy (PDT), sonodynamic therapy (SDT), and so on. In addition, the potential challenges and future perspectives are described in the improvement of CDT therapeutic efficacy, the enhancement of targeting capability, and mechanistic investigations on CDT therapy.

Smart Hydrogel-Based DVDMS/bFGF Nanohybrids for Antibacterial Phototherapy with Multiple Damaging Sites and Accelerated Wound Healing.

Burn infection is one of the commonest causes of death in severely burned patients. Developing multifunctional biological nanomaterials has a great significance for the comprehensive treatment of burn infection. In this paper, we developed a hydrogel-based nanodelivery system with antibacterial activity and skin regeneration function, which was used for photodynamic antimicrobial chemotherapy (PACT) in the treatment of burns. The treatment system is mainly composed of porphyrin photosensitizer sinoporphyrin sodium (DVDMS) and poly(lactic-co-glycolic acid) (PLGA)-encapsulated basic fibroblast growth factor (bFGF) nanospheres that are embedded in carboxymethyl chitosan (CMCS)-sodium alginate to form CSDP hybrid hydrogel. We systematically evaluated the inherent antibacterial performance, rheological properties, fluorescence imaging, and biocompatibility of the CSDP nanosystem. Under mild photoirradiation (30 J/cm2, 5 min), 10 µg/mL CSDP showed excellent antibacterial and anti-biofilm activities, which eradicated almost 99.99% of Staphylococcus aureus and multidrug-resistant (MDR) S. aureus in vitro. KEGG analysis identified that multiple signaling pathways were changed in MDR S. aureus after PACT. In the burn-infection model, CSDP-PACT successfully inhibited bacteria growth and concurrently promoted wound healing. Moreover, several regenerative factors were increased and some proinflammatory factors were reduced in the burn wounds of CSDP hydrogel treatment. These results suggest that the multifunctional CSDP hydrogel is a portable, light-triggered, antibacterial theranostic-platform and CSDP-PACT provides a promising strategy or the mechanically based synergistic treatment of burn infections.

Development of nose-to-brain delivery of ketoconazole by nanostructured lipid carriers against cryptococcal meningoencephalitis in mice.

Cryptococcus neoformans-mediated meningoencephalitis is a critical infectious disorder of the human central nervous system. However, efficient treatment for the disease is limited due to the poor penetration across the blood brain barrier (BBB). Here, we develop a nose-to-brain drug delivery system utilizing nanostructured lipid carriers (NLCs). We demonstrated that fluorescent-dye-loaded NLCs efficiently uptake into the cytoplasm of encapsulated C. neoformans cells. In comparison with current antifungal drugs, the ketoconazole (keto)-NLCs show significantly increased antifungal activity against C. neoformans in vivo under various growth conditions. The NLCs show enhanced tissue colonization properties. Importantly, using animal imaging analyses, NLCs are able to enter brain tissues via the olfactory bulb region by intranasal administration, bypassing the BBB. In addition, NLCs maintain prolonged residence in tissues. In mouse brain tissue, keto-NLCs showed significantly enhanced antifungal activity when administered intranasally, drastically dampening the C. neoformans burden. Taken together, NLCs not only improve the ketoconazole penetration efficiency against capsulated C. neoformans cells, but also boost the efficacy of antifungal drugs. Most importantly, keto-NLCs significantly contribute to the treatment of cryptococcal meningoencephalitis in mice by bypassing the BBB via the olfactory system.

Antimicrobial properties of a new type of photosensitizer derived from phthalocyanine against planktonic and biofilm forms of Staphylococcus aureus.

BACKGROUND: Bacterial infection is a common clinical problem. Community-associated Staphylococcus aureus (S. aureus) infections can cause extensive tissue damage and necrosis. Photodynamic antimicrobial chemotherapy (PACT) has recently attracted attention as a feasible bacterial therapy. Octa-cationic zinc phthalocyanines are newly identified photosensitizers derived from phthalocyanines bearing 1, 2-ethanediamine groups and quaternized derivatives with different numbers of positive charges (ZnPcn+, n = 4 or 8). Here we report the antimicrobial effects of ZnPcn+-mediated PACT on planktonic and biofilm cultures of S. aureus. METHODS: ZnPcn+ uptake was detected by photometry after alkaline lysis. Dark-toxicity and light-mediated antimicrobial effects of the drug was determined by the plate count method. The production of intracellular reactive oxygen species (ROS) was detected by flow cytometry. SYTO 9 and propidium iodide (PI) were used to detect the bacterial cell membrane permeability. DNA damage after ZnPcn+-PACT was analyzed by flow cytometry and PI staining. The destruction of biofilm was evaluated by scanning electron microscope (SEM). RESULTS: The study of uptake showed that the relative fluorescence intensity of ZnPcn+ in S. aureus peaked at 15 min. Generation of reactive oxygen species (ROS) by ZnPcn+ was enhanced in PACT treatment groups. SYTO 9 and PI staining indicated that cell membrane was damaged. Flow cytometry and PI staining revealed DNA damage. Biofilms were damaged in PACT treatment groups. CONCLUSIONS: Our results suggest that light-activated ZnPcn+ can efficiently inhibit planktonic and biofilm cultures of S. aureus.

Photodynamic antimicrobial chemotherapy for Staphylococcus aureus and multidrug-resistant bacterial burn infection in vitro and in vivo.

BACKGROUND AND OBJECTIVES: Antibiotic resistance has emerged as one of the most important determinants of outcome in patients with serious infections, along with the virulence of the underlying pathogen. Photodynamic antimicrobial chemotherapy (PACT) has been proposed as an alternative approach for the inactivation of bacteria. This study aims to evaluate the antibacterial effect of sinoporphyrin sodium (DVDMS)-mediated PACT on Staphylococcus aureus and multidrug resistant S. aureus in vitro and in vivo. MATERIALS AND METHODS: Bacteria were incubated with DVDMS and exposed to treatment with light. After PACT treatment, colony-forming units were counted to estimate the bactericidal effect. Intracellular reactive oxygen-species production was detected by flow cytometry. Flow cytometry and fluorescence-microscopy detection of bacterial cell-membrane permeability. Enzyme-linked immunosorbent assays were used to determine expression of VEGF, TGFß1, TNFα, IL6, and bFGF factors in burn infection. RESULTS: DVDMS-PACT effectively killed bacterial proliferation. Intracellular ROS levels were enhanced obviously in the PACT-treatment group. SYTO 9 and propidium iodide staining showed a decrease in the ratio of green:red fluorescence intensity in the PACT-treatment group in comparison to the control group. Enzyme-linked immunosorbent-assay results revealed that in the healing process, the expression of bFGF, TGFß1, and VEGF in the treatment group were higher than in the control group, which inhibited inflammation-factor secretion. In addition, skin-tissue bacteria were reduced after treatment. CONCLUSION: These results indicate that DVDMS-PACT presents significant bactericidal activity and promotes wound healing after burn infections.