Construction of the Glycolysis Metabolic Pathway Inside an Artificial Cell for the Synthesis of Amino Acid and Its Reversible Deformation.

The bottom-up construction of artificial cells is beneficial for understanding cell working mechanisms. The glycolysis metabolism mimicry inside artificial cells is challenging. Herein, the glycolytic pathway (Entner-Doudoroff pathway in archaea) is reconstituted inside artificial cells. The glycolytic pathway comprising glucose dehydrogenase (GDH), gluconate dehydratase (GAD), and 2-keto-3-deoxygluconate aldolase (KDGA) converts glucose molecules to pyruvate molecules. Inside artificial cells, pyruvate molecules are further converted into alanine with the help of alanine dehydrogenase (AlaDH) to build a metabolic pathway for synthesizing amino acid. On the other hand, the pyruvate molecules from glycolysis stimulate the living mitochondria to produce ATP inside artificial cells, which further trigger actin monomers to polymerize to form actin filaments. With the addition of methylcellulose inside the artificial cell, the actin filaments form adjacent to the inner lipid bilayer, deforming the artificial cell from a spherical shape to a spindle shape. The spindle-shaped artificial cell reverses to a spherical shape by depolymerizing the actin filament upon laser irradiation. The glycolytic pathway and its further extension to produce amino acids (or ATP) inside artificial cells pave the path to build functional artificial cells with more complicated metabolic pathways.

Mimicking Cellular Metabolism in Artificial Cells: Universal Molecule Transport across the Membrane through Vesicle Fusion.

Mass transport across cell membranes is a primary process for cellular metabolism. For this purpose, electrostatically mediated membrane fusion is exploited to transport various small molecules including glucose-6-phosphate, isopropyl ß-D-thiogalactoside, and macromolecules such as DNA plasmids from negatively charged large unilamellar vesicles (LUVs) to positively charged giant unilamellar vesicles (GUVs). After membrane fusion between these oppositely charged vesicles, molecules are transported into GUVs to trigger the NAD+ involved enzyme reaction, bacterial gene expression, and in vitro gene expression of green fluorescent protein from a DNA plasmid. The optimized charged lipid percentages are 10% for both positively charged GUVs and negatively charged LUVs to ensure the fusion process. The experimental results demonstrate a universal way for mass transport into the artificial cells through vesicle fusions, which paves a crucial step for the investigation of complicated cellular metabolism.

Ligustilide-loaded liposome ameliorates mitochondrial impairments and improves cognitive function via the PKA/AKAP1 signaling pathway in a mouse model of Alzheimer's disease.

BACKGROUND: Oxidative stress is an early event in the development of Alzheimer's disease (AD) and maybe a pivotal point of interaction governing AD pathogenesis; oxidative stress contributes to metabolism imbalance, protein misfolding, neuroinflammation and apoptosis. Excess reactive oxygen species (ROS) are a major contributor to oxidative stress. As vital sources of ROS, mitochondria are also the primary targets of ROS attack. Seeking effective avenues to reduce oxidative stress has attracted increasing attention for AD intervention. METHODS: We developed liposome-packaged Ligustilide (LIG) and investigated its effects on mitochondrial function and AD-like pathology in the APPswe/PS1dE9 (APP/PS1) mouse model of AD, and analyzed possible mechanisms. RESULTS: We observed that LIG-loaded liposome (LIG-LPs) treatment reduced oxidative stress and ß-amyloid (Aß) deposition and mitigated cognitive impairment in APP/PS1 mice. LIG management alleviated the destruction of the inner structure in the hippocampal mitochondria and ameliorated the imbalance between mitochondrial fission and fusion in the APP/PS1 mouse brain. We showed that the decline in cAMP-dependent protein kinase A (PKA) and A-kinase anchor protein 1 for PKA (AKAP1) was associated with oxidative stress and AD-like pathology. We confirmed that LIG-mediated antioxidant properties and neuroprotection were involved in upregulating the PKA/AKAP1 signaling in APPswe cells in vitro. CONCLUSION: Liposome packaging for LIG is relatively biosafe and can overcome the instability of LIG. LIG alleviates mitochondrial dysfunctions and cognitive impairment via the PKA/AKAP1 signaling pathway. Our results provide experimental evidence that LIG-LPs may be a promising agent for AD therapy.

High-throughput production of functional prototissues capable of producing NO for vasodilation.

Bottom-up synthesis of prototissues helps us to understand the internal cellular communications in the natural tissues and their functions, as well as to improve or repair the damaged tissues. The existed prototissues are rarely used to improve the function of living tissues. We demonstrate a methodology to produce spatially programmable prototissues based on the magneto-Archimedes effect in a high-throughput manner. More than 2000 prototissues are produced once within 2 h. Two-component and three-component spatial coded prototissues are fabricated by varying the addition giant unilamellar vesicles order/number, and the magnetic field distributions. Two-step and three-step signal communications in the prototissues are realized using cascade enzyme reactions. More importantly, the two-component prototissues capable of producing nitric oxide cause vasodilation of rat blood vessels in the presence of glucose and hydroxyurea. The tension force decreases 2.59 g, meanwhile the blood vessel relaxation is of 31.2%. Our works pave the path to fabricate complicated programmable prototissues, and hold great potential in the biomedical field.

Reversible Deformation of Artificial Cell Colonies Triggered by Actin Polymerization for Muscle Behavior Mimicry.

The use of artificial cells to mimic living tissues is beneficial for understanding the mechanism of interaction among cells. Artificial cells hold immense potential in the field of tissue engineering. Self-powered artificial cells capable of reversible deformation are developed by encapsulating living mitochondria, actins, and methylcellulose. Upon addition of pyruvate molecules, the mitochondria produce adenosine triphosphate (ATP), which acts as an energy source to trigger actin polymerization. The reversible deformation of artificial cells occurs with a spindle shape resulting from the polymerization of actins to form filaments adjacent to the lipid bilayer that subsequently returns to a spherical shape resulting from the depolymerization of actin filaments upon laser irradiation. The linear colonies composed of these artificial cells exhibit collective contraction and relaxation to mimic muscle tissues. At maximum contraction, the long axis of each giant unilamellar vesicle (GUV) is parallel to each other. All the colonies are synchronized in the contraction phase. The deformation of each GUV in the colonies is influenced by its adjacent GUVs. The muscle-like artificial cell colonies described here pave the way to develop sustainably self-powered artificial tissues.

HA-DOPE-Modified Honokiol-Loaded Liposomes Targeted Therapy for Osteosarcoma.

Purpose: Osteosarcoma (OS) is the most common bone cancer with a high risk of metastasis, high growth rate, and poor prognosis. Honokiol (HNK) is a general ingredient of traditional Chinese medicine, with a potential anti-tumor effect. However, HNK is insoluble in water and lacks drug targeting, which limits its clinical application. To improve the OS therapeutic effect of HNK, we used HNK-loaded liposomes modified with hyaluronic acid-phospholipid conjugates (HA-DOPE) to treat OS based on the HA interaction with CD44. Methods: The HNK-loaded liposomes were prepared via thin-film hydration and sonication. HA-DOPE was used to combine the HNK-loaded liposomes (HA-DOPE@Lips/HNK) via sonication and co-extrusion. HA-DOPE@Lips/HNK were characterized with respect to size, zeta potential, polymer dispersity index (PDI), and stability, and transmission electron microscopy was performed. Cellular uptake, cell viability, cell apoptosis, cell cycle, and mitochondrial activity were utilized to evaluate the antitumor effect in vitro. The biodistribution, xenograft tumor growth inhibition, and safety of HA-DOPE@Lips/HNK were evaluated in 143B OS xenograft mice in vivo. Results: The particle size, PDI, and zeta potential of HA-DOPE@Lips/HNK were 146.20±0.26 nm, 0.20±0.01, and -38.45±0.98 mV, respectively. The encapsulation rate and drug loading were 80.14±0.32% and 3.78±0.09%, respectively. HA-DOPE@Lips/HNK could inhibit cell proliferation, cause apoptosis, block the cell cycle and disrupt mitochondrial activity. HA-DOPE@Lips/HNK specially delivered the drug into the tumor and inhibited tumor growth, and showed no obvious toxicity to normal tissues. Conclusion: HA-DOPE@Lips/HNK could deliver HNK into the tumor site and had a good antitumor ability in vitro and in vivo. In addition, HA-DOPE@Lips/HNK increased the antitumor effects of HNK. Thus, it provides a promising nanocarrier to improve drug delivery in OS therapy.

Programmed magnetic manipulation of vesicles into spatially coded prototissue architectures arrays.

In nature, cells self-assemble into spatially coded tissular configurations to execute higher-order biological functions as a collective. This mechanism has stimulated the recent trend in synthetic biology to construct tissue-like assemblies from protocell entities, with the aim to understand the evolution mechanism of multicellular mechanisms, create smart materials or devices, and engineer tissue-like biomedical implant. However, the formation of spatially coded and communicating micro-architectures from large quantity of protocell entities, especially for lipid vesicle-based systems that mostly resemble cells, is still challenging. Herein, we magnetically assemble giant unilamellar vesicles (GUVs) or cells into various microstructures with spatially coded configurations and spatialized cascade biochemical reactions using a stainless steel mesh. GUVs in these tissue-like aggregates exhibit uncustomary osmotic stability that cannot be achieved by individual GUVs suspensions. This work provides a versatile and cost-effective strategy to form robust tissue-mimics and indicates a possible superiority of protocell colonies to individual protocells.