Applications of Biomolecular Nanostructures for Anti-Angiogenic Theranostics.

Angiogenesis is a physiological process of forming new blood vessels that has pathological importance in seemingly unrelated illnesses like cancer, diabetes, and various inflammatory diseases. Treatment targeting angiogenesis has shown promise for these types of diseases, but current anti-angiogenic agents have critical limitations in delivery and side-effects. This necessitates exploration of alternative approaches like biomolecule-based drugs. Proteins, lipids, and oligonucleotides have recently become popular in biomedicine, specifically as biocompatible components of therapeutic drugs. Their excellent bioavailability and potential bioactive and immunogenic properties make them prime candidates for drug discovery or drug delivery systems. Lipid-based liposomes have become standard vehicles for targeted nanoparticle (NP) delivery, while protein and nucleotide NPs show promise for environment-sensitive delivery as smart NPs. Their therapeutic applications have initially been hampered by short circulation times and difficulty of fabrication but recent developments in nanofabrication and NP engineering have found ways to circumvent these disadvantages, vastly improving the practicality of biomolecular NPs. In this review, we are going to briefly discuss how biomolecule-based NPs have improved anti-angiogenesis-based therapy.

Injective hydrogel loaded with liposomes-encapsulated MY-1 promotes wound healing and increases tensile strength by accelerating fibroblast migration via the PI3K/AKT-Rac1 signaling pathway.

Failed skin wound healing, through delayed wound healing or wound dehiscence, is a global public health issue that imposes significant burdens on individuals and society. Although the application of growth factor is an effective method to improve the pace and quality of wound healing, the clinically approved factors are limited. Parathyroid hormone (PTH) demonstrates promising results in wound healing by promoting collagen deposition and cell migration, but its application is limited by potentially inhibitory effects when administered continuously and locally. Through partially replacing and repeating the amino acid domains of PTH(1-34), we previously designed a novel PTH analog, PTH(3-34)(29-34) or MY-1, and found that it avoided the inhibitory effects of PTH while retaining its positive functions. To evaluate its role in wound healing, MY-1 was encapsulated in liposomes and incorporated into the methacryloyl gelatin (GelMA) hydrogel, through which an injectable nanocomposite hydrogel (GelMA-MY@Lipo, or GML) was developed. In vitro studies revealed that the GML had similar properties in terms of the appearance, microstructure, functional groups, swelling, and degradation capacities as the GelMA hydrogel. In vitro drug release testing showed a relatively more sustainable release of MY-1, which was still detectable in vivo 9 days post-application. When the GML was topically applied to the wound areas of rat models, wound closure as well as tensile strength were improved. Further studies showed that the effects of GML on wound repair and tensile strength were closely related to the promotion of fibroblast migration to the wound area through the controlled release of MY-1. Mechanically, MY-1 enhanced fibroblast migration by activating PI3K/AKT signaling and its downstream molecule, Rac1, by which it increased fibroblast aggregation in the early stage and resulting in denser collagen deposition at a later time. Overall, these findings demonstrated that the nanocomposite hydrogel system promoted skin wound healing and increased tensile strength, thus offering new potential in the treatment of wound healing.

Chikusetsu Saponin IVa liposomes modified with a retro-enantio peptide penetrating the blood-brain barrier to suppress pyroptosis in acute ischemic stroke rats.

BACKGROUND: The therapeutic strategies for acute ischemic stroke were faced with substantial constraints, emphasizing the necessity to safeguard neuronal cells during cerebral ischemia to reduce neurological impairments and enhance recovery outcomes. Despite its potential as a neuroprotective agent in stroke treatment, Chikusetsu saponin IVa encounters numerous challenges in clinical application. RESULT: Brain-targeted liposomes modified with THRre peptides showed substantial uptake by bEnd. 3 and PC-12 cells and demonstrated the ability to cross an in vitro blood-brain barrier model, subsequently accumulating in PC-12 cells. In vivo, they could significantly accumulate in rat brain. Treatment with C-IVa-LPs-THRre notably reduced the expression of proteins in the P2RX7/NLRP3/Caspase-1 pathway and inflammatory factors. This was evidenced by decreased cerebral infarct size and improved neurological function in MCAO rats. CONCLUSION: The findings indicate that C-IVa-LPs-THRre could serve as a promising strategy for targeting cerebral ischemia. This approach enhances drug concentration in the brain, mitigates pyroptosis, and improves the neuroinflammatory response associated with stroke.

Studying lipid flip-flop in asymmetric liposomes using 1H NMR and TR-SANS.

The specific spatial and temporal distribution of lipids in membranes play a crucial role in determining the biochemical and biophysical properties of the system. In nature, the asymmetric distribution of lipids is a dynamic process with ATP-dependent lipid transporters maintaining asymmetry, and passive transbilayer diffusion, that is, flip-flop, counteracting it. In this chapter, two probe-free techniques, 1H NMR and time-resolved small angle neutron scattering, are described in detail as methods of investigating lipid flip-flop rates in synthetic liposomes that have been generated with an asymmetric bilayer composition.

Probing the structural evolution on the surface of cardiolipin vesicles with an amphiphilic second harmonic generation and fluorescence probe.

Investigating the influence of the ambient chemical environment on molecular behaviors in liposomes is crucial for understanding and manipulating cellular vitality as well as the capabilities of lipid drug carriers in various environments. Here, we designed and synthesized a second harmonic generation (SHG) and fluorescence probe molecule called Pyr-Py+-N+ (PPN), which possesses membrane-targeting capability. We employed PPN to investigate the response of lipid vesicles composed of cardiolipin to the presence of exogenous salt. The kinetic behaviors, including the adsorption and embedding of PPN on the surface of small unilamellar vesicles (SUVs) composed of cardiolipin, were analyzed. The response of the SUVs to the addition of NaCl was also monitored. A rapid decrease in vesicle size can be evidenced through the rapid drop in SHG emission originating from PPN located on the vesicle surface.

Nanoparticles and Their Applications in Lipid Signaling.

This chapter provides an overview of the diverse range of applications associated with nanoparticles. The application of nanoparticles in the medical field has garnered considerable attention due to their unique properties and versatile compositions. They have shown promise in the treatment of cancer, fungal and viral infections, and pain management. These systems provide numerous benefits, such as increased drug stability, improved bioavailability, and targeted delivery to specific tissues or cells. The objective of this chapter is to provide a brief analysis of the differences between nanoparticles and lipid particles, focusing particularly on the importance of nanoparticle size and composition in their interactions with lipids. Additionally, the applications of nanoparticles in lipid signaling will be discussed, considering the vital roles lipids play in cellular signaling pathways. Nanoparticles have shown immense potential in the regulation and control of medical pathways. In this case, we will focus on the manufacture of liposomes, a type of nanoparticle composed of lipids. The reason behind the extensive investigation into liposomes as drug delivery vehicles is their remarkable biocompatibility and adaptability. This section will provide insights into the methods and techniques employed for liposome formulation.

Size Matters: Rethinking Hertz Model Interpretation for Cell Mechanics Using AFM.

Cell mechanics are a biophysical indicator of cell state, such as cancer metastasis, leukocyte activation, and cell cycle progression. Atomic force microscopy (AFM) is a widely used technique to measure cell mechanics, where the Young modulus of a cell is usually derived from the Hertz contact model. However, the Hertz model assumes that the cell is an elastic, isotropic, and homogeneous material and that the indentation is small compared to the cell size. These assumptions neglect the effects of the cytoskeleton, cell size and shape, and cell environment on cell deformation. In this study, we investigated the influence of cell size on the estimated Young's modulus using liposomes as cell models. Liposomes were prepared with different sizes and filled with phosphate buffered saline (PBS) or hyaluronic acid (HA) to mimic the cytoplasm. AFM was used to obtain the force indentation curves and fit them to the Hertz model. We found that the larger the liposome, the lower the estimated Young's modulus for both PBS-filled and HA-filled liposomes. This suggests that the Young modulus obtained from the Hertz model is not only a property of the cell material but also depends on the cell dimensions. Therefore, when comparing or interpreting cell mechanics using the Hertz model, it is essential to account for cell size.

Lipid-based nanosystems: the next generation of cancer immune therapy.

Immunotherapy has become an important part of the oncotherapy arsenal. Its applicability in various cancer types is impressive, as well as its use of endogenous mechanisms to achieve desired ends. However, off-target or on-target-off-tumor toxicity, limited activity, lack of control in combination treatments and, especially for solid tumors, low local accumulation, have collectively limited clinical use thereof. These limitations are partially alleviated by delivery systems. Lipid-based nanoparticles (NPs) have emerged as revolutionary carriers due to favorable physicochemical characteristics, with specific applications and strengths particularly useful in immunotherapeutic agent delivery. The aim of this review is to highlight the challenges faced by immunotherapy and how lipid-based NPs have been, and may be further utilized to address such challenges. We discuss recent fundamental and clinical applications of NPs in a range of areas and provide a detailed discussion of the main obstacles in immune checkpoint inhibition therapies, adoptive cellular therapies, and cytokine therapies. We highlight how lipid-based nanosystems could address these through either delivery, direct modulation of the immune system, or targeting of the immunosuppressive tumor microenvironment. We explore advanced and emerging liposomal and lipid nanoparticle (LNP) systems for nucleic acid delivery, intrinsic and extrinsic stimulus-responsive formulations, and biomimetic lipid-based nanosystems in immunotherapy. Finally, we discuss the key challenges relating to the clinical use of lipid-based NP immunotherapies, suggesting future research directions for the near term to realize the potential of these innovative lipid-based nanosystems, as they become the crucial steppingstone towards the necessary enhancement of the efficacy of immunotherapy.

Acidity-activatable dynamic hybrid nanoplatforms derived from extracellular vesicles of M1 macrophages enhance cancer immunotherapy through synergistic triple immunotherapy.

Immunotherapy exhibits considerable promise for sustained tumor reduction. However, current cancer immunotherapy methods elicit limited responses due to the inadequate immunogenicity exhibited by cancer cells. This obstacle may be addressed using nanoplatforms that can activate synergistic therapies (photodynamic therapy and ferroptosis) in response to the acidic pH of the tumor microenvironment. We previously developed an amphiphilic photosensitizer, SR780, which displays satisfactory photodynamic effects. This photosensitizer is inactivated when bound to Fe3+ (SR780Fe) but is activated upon release in mildly acidic conditions. In this study, M1 macrophage-derived extracellular vesicles (EVs) were fused with REV and SR780Fe-loaded liposomes (REV@SR780Fe@Lip) to form REV@SR780Fe@LEV hybrid nanovesicles. Further modification with the RS17 peptide for tumor targeting enabled a combination of photodynamic therapy, ferroptosis, and cGAS-STING pathway activation, resulting in enhanced antitumor efficacy through a synergistic effect. Upon laser irradiation, REV@SR780Fe@LEV-RS17 demonstrated antitumor effects in 4T1 breast cancer models, including the inhibition of lung and liver metastasis, as well as prevention of tumor recurrence.

[Liposome co-delivery of quercetin and doxorubicin in inhibiting retinoblastoma by regulating epithelial-mesenchymal transition process].

Regulating the process of epithelial-mesenchymal transition(EMT) is an essential strategy to inhibit tumor growth and metastasis. This study is based on the EMT process of retinoblastoma and constructs quercetin(QUE) and doxorubicin(DOX) co-loaded liposome(QD Lipo) to investigate the therapeutic effect and mechanisms of combined QUE and DOX treatment on retinoblastoma. Single-factor experiments were conducted to optimize the prescription process of QD Lipo. Eventually, spherical particles with a diameter of(108.87±1.93) nm, a PDI of 0.13±0.02, and a Zeta potential of(-34.83±1.92) mV were obtained. The encapsulation rates of QUE and DOX were 96.20%±4.40% and 91.17%±4.41%, respectively. Y79 human retinoblastoma cells were used as an in vitro cellular model, and confocal microscopy demonstrated that QD Lipo could enhance Y79 uptake efficiency. The CCK-8 assay confirmed that the optimal combination therapy effect of QUE and DOX occurred at a mass ratio of 1∶1 to 1∶2. Flow cytometry showed that QD Lipo enhanced the induction of apoptosis in Y79 cells. Western blot analysis revealed that QD Lipo significantly reduced the expression of EMT pathway-related proteins vimentin and α-SMA. Fluorescence assays detected a significant decrease in ROS levels in Y79 cells after treatment with QD. These results indicated that liposomal co-delivery of QUE and DOX can enhance drug delivery efficiency to retinoblastoma cells, inhibit the EMT process in retinoblastoma by downregulating ROS levels, and enhance the cytotoxicity of DOX against retinoblastoma.

Peptosome: A New Efficient Transfection Tool as an Alternative to Liposome.

Gene therapy is one of the most promising techniques for treating genetic diseases and cancer. The current most important problem in gene therapy is gene delivery. Viral and non-viral vectors like liposomes, used for gene delivery, have many limitations. We have developed new hybrid peptides by combining cell-penetrating peptides (CPPs) with the DNA-binding domain of the human histone H4 protein. These small peptides bind to DNA molecules through their histone domain, leaving the CPP part free and available for binding and penetration into cells, forming complexes that we named "peptosomes". We evaluated the transfection efficiency of several hybrid peptides by delivering a plasmid carrying the green fluorescent protein gene and following its expression by fluorescent microscopy. Among several hybrid peptides, TM3 achieved a gene delivery efficiency of 76%, compared to 52% for Lipofectamine 2000. TM3 peptosomes may become important gene delivery tools with several advantages over current gene delivery agents.

Karanjin-loaded soya lecithin-based ethosomal nanogel for the therapeutic intervention of psoriasis: formulation development, factorial design based-optimization,in vitroandin vivoassessment.

This study aimed to develop and optimize karanjin-loaded ethosomal nanogel formulation and evaluate its efficacy in alleviating symptoms of psoriasis in an animal model induced by imiquimod. These karanjin-loaded ethosomal nanogel, were formulated to enhance drug penetration into the skin and its epidermal retention. Karanjin was taken to formulate ethosomes due to its potential ani-psoriatic activity. Ethosomes were formulated using the cold method using 32full factorial designs to optimize the formulation components. 9 batches were prepared using two independent variablesX1: concentration of ethanol andX2: concentration of phospholipid whereas vesicle size (Y1) and percentage entrapment efficiency (Y2) were selected as dependent variables. All the dependent variables were found to be statistically significant. The optimized ethosomal suspension (B3) exhibited a vesicle size of 334 ± 2.89 nm with an entrapment efficiency of 94.88 ± 1.24% and showed good stability. The morphology of vesicles appeared spherical with smooth surfaces through transmission electron microscopy analysis. X-ray diffraction analysis confirmed that the drug existed in an amorphous state within the ethosomal formulation. The optimized ethosome was incorporated into carbopol 934 to develop nanogel for easy application on the skin. The nanogel underwent characterization for various parameters including spreadability, viscosity, pH, extrudability, and percentage drug content. The ethosomal formulation remarkably enhanced the skin permeation of karanjin and increased epidermal retention of the drug in psoriatic skin compared to marketed preparation and pure drug. A skin retention study showed that ethosomal nanogel formulation has 48.33% epidermal retention in 6 h.In vivo,the anti-psoriatic activity of karanjin ethosomal nanogel demonstrated significant improvement in psoriasis, indicated by a gradual decrease in skin thickness and scaling as reflected in the Psoriasis Severity Index grading. Therefore, the prepared ethosomal nanogel is a potential vehicle for improved topical delivery of karanjin for better treatment of psoriasis.

Improved efficacy of cisplatin delivery by peanut agglutinin­modified liposomes in non­small cell lung cancer.

Globally, non­small cell lung cancer (NSCLC) is a significant threat to human health, and constitutes >80% of lung cancer cases. Cisplatin (CDDP), a commonly used drug in clinical treatment, has been the focus of research aiming to mitigate its potent toxicity through encapsulation within liposomes. However, challenges, such as a reduced drug loading efficiency and nonspecific release, have emerged as obstacles. The present study aimed to improve the encapsulation efficiency of CDDP within liposomes by pre­preparation of CDDP and modifying the liposome surface through the incorporation of peanut agglutinin (PNA) as a ligand [CDDP­loaded PNA­modified liposomes (CDDP­PNA­Lip)]. This strategy was designed to enhance the delivery of CDDP to tumour tissues, thereby reducing associated side effects. The effect of CDDP­PNA­Lip on the proliferation and migration of NSCLC cell lines with high MUC1 expression was elucidated through in vitro studies. Additionally, the capacity of PNA modification to augment the targeted anti­tumour efficacy of liposomes was assessed through xenograft tumour experiments. The results indicated that in an in vitro uptake assay Rhodamine B (RhB)­loaded PNA­modified liposomes were taken up by cells with ~50% higher efficiency compared with free RhB. In addition, CDDP­PNA­Lip resulted in a 2.65­fold enhancement of tumour suppression in vivo compared with free CDDP. These findings suggested that the encapsulation of CDDP within ligand­modified liposomes may significantly improve its tumour­targeting capabilities, providing valuable insights for clinical drug development.

Antibacterial and antibiofilm potentials of vancomycin-loaded niosomal drug delivery system against methicillin-resistant Staphylococcus aureus (MRSA) infections.

The threat of methicillin-resistant Staphylococcus aureus (MRSA) is increasing worldwide, making it significantly necessary to discover a novel way of dealing with related infections. The quick spread of MRSA isolates among infected individuals has heightened public health concerns and significantly limited treatment options. Vancomycin (VAN) can be applied to treat severe MRSA infections, and the indiscriminate administration of this antimicrobial agent has caused several concerns in medical settings. Owing to several advantageous characteristics, a niosomal drug delivery system may increase the potential of loaded antimicrobial agents. This work aims to examine the antibacterial and anti-biofilm properties of VAN-niosome against MRSA clinical isolates with emphasis on cytotoxicity and stability studies. Furthermore, we aim to suggest an effective approach against MRSA infections by investigating the inhibitory effect of formulated niosome on the expression of the biofilm-associated gene (icaR). The thin-film hydration approach was used to prepare the niosome (Tween 60, Span 60, and cholesterol), and field emission scanning electron microscopy (FE-SEM), an in vitro drug release, dynamic light scattering (DLS), and entrapment efficiency (EE%) were used to investigate the physicochemical properties. The physical stability of VAN-niosome, including hydrodynamic size, polydispersity index (PDI), and EE%, was analyzed for a 30-day storage time at 4 °C and 25 °C. In addition, the human foreskin fibroblast (HFF) cell line was used to evaluate the cytotoxic effect of synthesized niosome. Moreover, minimum inhibitory and bactericidal concentrations (MICs/MBCs) were applied to assess the antibacterial properties of niosomal VAN formulation. Also, the antibiofilm potential of VAN-niosome was investigated by microtiter plate (MTP) and real-time PCR methods. The FE-SEM result revealed that synthesized VAN-niosome had a spherical morphology. The hydrodynamic size and PDI of VAN-niosome reported by the DLS method were 201.2 nm and 0.301, respectively. Also, the surface zeta charge of the prepared niosome was - 35.4 mV, and the EE% ranged between 58.9 and 62.5%. Moreover, in vitro release study revealed a sustained-release profile for synthesized niosomal formulation. Our study showed that VAN-niosome had acceptable stability during a 30-day storage time. Additionally, the VAN-niosome had stronger antibacterial and anti-biofilm properties against MRSA clinical isolates compared with free VAN. In conclusion, the result of our study demonstrated that niosomal VAN could be promising as a successful drug delivery system due to sustained drug release, negligible toxicity, and high encapsulation capacity. Also, the antibacterial and anti-biofilm studies showed the high capacity of VAN-niosome against MRSA clinical isolates. Furthermore, the results of real-time PCR exhibited that VAN-niosome could be proposed as a powerful strategy against MRSA biofilm via down-regulation of icaR gene expression.

Liposomal Nanomaterials: A Rising Star in Glioma Treatment.

Glioma is a primary malignant tumor in the central nervous system. In recent years, the treatment of glioma has developed rapidly, but the overall survival of glioma patients has not significantly improved. Due to the presence of the blood-brain barrier and intracranial tumor barrier, many drugs with good effects to cure glioma in vitro cannot be accurately transported to the corresponding lesions. In order to enable anti-tumor drugs to overcome the barriers and target glioma, nanodrug delivery systems have emerged recently. It is gratifying that liposomes, as a multifunctional nanodrug delivery carrier, which can be compatible with hydrophilic and hydrophobic drugs, easily functionalized by various targeted ligands, biodegradable, and hypoimmunogenic in vivo, has become a quality choice to solve the intractable problem of glioma medication. Therefore, we focused on the liposome nanodrug delivery system, and summarized its current research progress in glioma. Hopefully, this review may provide new ideas for the research and development of liposome-based nanomaterials for the clinical treatment of glioma.

Composite branched and linear F-actin maximize myosin-induced membrane shape changes in a biomimetic cell model.

The architecture of the actin cortex determines the generation and transmission of stresses, during key events from cell division to migration. However, its impact on myosin-induced cell shape changes remains unclear. Here, we reconstitute a minimal model of the actomyosin cortex with branched or linear F-actin architecture within giant unilamellar vesicles (GUVs, liposomes). Upon light activation of myosin, neither the branched nor linear F-actin architecture alone induces significant liposome shape changes. The branched F-actin network forms an integrated, membrane-bound "no-slip boundary" -like cortex that attenuates actomyosin contractility. By contrast, the linear F-actin network forms an unintegrated "slip boundary" -like cortex, where actin asters form without inducing membrane deformations. Notably, liposomes undergo significant deformations at an optimized balance of branched and linear F-actin networks. Our findings highlight the pivotal roles of branched F-actin in force transmission and linear F-actin in force generation to yield membrane shape changes.

Codelivery of methotrexate and silibinin by niosome nanoparticles for enhanced chemotherapy of CT26 colon cancer cells.

Colon cancer (CC) is one of the most prevalent cancers in the world, and chemotherapy is widely applied to combat it. However, chemotherapy drugs have severe side effects and emergence of multi drug resistance (MDR) is common. This bottleneck can be overcome by niosome nanocarriers that minimize drug dose/toxicity meanwhile allow co-loading of incompatible drugs for combination therapy. In this research, silibinin (Sil) as a hydrophobic drug was loaded into the lipophilic part, and methotrexate (MTX) into the hydrophilic part of niosome by the thin film hydration (TFH) method to form Nio@MS NPs for CT26 colon cancer therapyin vitro. Our results indicated synthesis of ideal niosome nanoparticles (NPs) with spherical morphology, size of ∼100 nm, and a zeta potential of -10 mV. The IC50value for Nio@MS was determined ∼2.6 µg ml-1, which was significantly lower than MTX-Sil (∼6.86 µg ml-1), Sil (18.46 µg ml-1), and MTX (9.8 µg ml-1). Further, Nio@MS significantly reduced cell adhesion density, promoted apoptosis and increased gene expression level of caspase 3 and BAX while promoted significant downregulation of BCL2. In conclusion, the design and application of niosome to co-administer Sil and MTX can increase the drugs cytotoxicity, reduce their dose and improve anti-cancer potential by combating MDR.

Investigating the impact of 2-OHOA-embedded liposomes on biophysical properties of cancer cell membranes via Laurdan two-photon microscopy imaging.

2-Hydroxyoleic acid (2-OHOA) has gained attention as a membrane lipid therapy (MLT) anti-cancer drug. However, in the viewpoint of anti-cancer drug, 2-OHOA shows poor water solubility and its effectiveness still has space for improvement. Thus, this study aimed to overcome the problems by formulating 2-OHOA into liposome dosage form. Furthermore, in the context of MLT reagents, the influence of 2-OHOA on the biophysical properties of the cytoplasmic membrane remains largely unexplored. To bridge this gap, our study specifically focused the alterations in cancer cell membrane fluidity and lipid packing characteristics before and after treatment. By using a two-photon microscope and the Laurdan fluorescence probe, we noted that liposomes incorporating 2-OHOA induced a more significant reduction in cancer cell membrane fluidity, accompanied by a heightened rate of cellular apoptosis when compared to the non-formulated 2-OHOA. Importantly, the enhanced efficacy of 2-OHOA within the liposomal formulation demonstrated a correlation with its endocytic uptake mechanism. In conclusion, our findings underscore the significant influence of 2-OHOA on the biophysical properties of cancer plasma membranes, emphasizing the potential of liposomes as an optimized delivery system for 2-OHOA in anti-cancer therapy.

Confinement-Induced Biocatalytic Activity Enhancement of Light- and Thermoresponsive Polymer@Enzyme@MOF Composites.

Metal-organic frameworks (MOFs) are favorable hosting materials for fixing enzymes to construct enzyme@MOF composites and to expand the applications of biocatalysts. However, the rigid structure of MOFs without tunable hollow voids and a confinement effect often limits their catalytic activities. Taking advantage of the smart soft polymers to overcome the limitation, herein, a protection protocol to encapsulate the enzyme in zeolitic imidazolate framework-8 (ZIF-8) was developed using a glutathione-sensitive liposome (L) as a soft template. Glucose oxidase (GOx) and horseradish peroxidase (HRP) were first anchored on a light- and thermoresponsive porous poly(styrene-maleic anhydride-N,N-dimethylaminoethyl methacrylate-spiropyran) membrane (PSMDSP) to produce PSMDSP@GOx-HRP, which could provide a confinement effect by switching the UV irradiation or varying the temperature. Afterward, embedding PSMDSP@GOx-HRP in L and encapsulating PSMDSP@GOx-HRP@L into hollow ZIF-8 (HZIF-8) to form PSMDSP@GOx-HRP@HZIF-8 composites were performed, which proceeded during the crystallization of the framework following the removal of L by adding glutathione. Impressively, the biocatalytic activity of the composites was 4.45-fold higher than that of the free enzyme under UV irradiation at 47 °C, which could benefit from the confinement effect of PSMDSP and the conformational freedom of the enzyme in HZIF-8. The proposed composites contributed to the protection of the enzyme against harsh conditions and exhibited superior stability. Furthermore, a colorimetric assay based on the composites for the detection of serum glucose was established with a linearity range of 0.05-5.0 mM, and the calculated LOD value was 0.001 mM in a cascade reaction system. This work provides a universal design idea and a versatile technique to immobilize enzymes on soft polymer membranes that can be encapsulated in porous rigid MOF-hosts. It also holds potential for the development of smart polymer@enzyme@HMOFs biocatalysts with a tunable confinement effect and high catalytic performance.

Phase separation in model lipid membranes investigated with cryogenic electron microscopy.

We describe a method for investigating lateral membrane heterogeneity using cryogenic electron microscopy (cryo-EM) images of liposomes. The method takes advantage of differences in the thickness and molecular density of ordered and disordered phases that are resolvable in phase contrast cryo-EM. Compared to biophysical techniques like FRET or neutron scattering that yield ensemble-averaged information, cryo-EM provides direct visualization of individual vesicles and can therefore reveal variability that would otherwise be obscured by averaging. Moreover, because the contrast mechanism involves inherent properties of the lipid phases themselves, no extrinsic probes are required. We explain and discuss various complementary analyses of spatially resolved thickness and intensity measurements that enable an assessment of the membrane's phase state. The method opens a window to nanodomain structure in synthetic and biological membranes that should lead to an improved understanding of lipid raft phenomena.