Delivery of mRNA Encoding Interleukin-12 and a Stimulator of Interferon Genes Agonist Potentiates Antitumor Efficacy through Reversing T Cell Exhaustion.

T cell exhaustion has emerged as a major hurdle that impedes the clinical translation of stimulator of interferon genes (STING) agonists. It is crucial to explore innovative strategies to rejuvenate exhausted T cells and potentiate the antitumor efficacy. Here, we propose an approach utilizing MSA-2 as a STING agonist, along with nanoparticle-mediated delivery of mRNA encoding interleukin-12 (IL-12) to restore the function of T cells. We developed a lipid nanoparticle (DMT7-IL12 LNP) that encapsulated IL12 mRNA. Our findings convincingly demonstrated that the combination of MSA-2 and DMT7-IL12 LNP can effectively reverse the exhausted T cell phenotype, as evidenced by the enhanced secretion of cytokines, such as tumor necrosis factor alpha, interferon gamma, and Granzyme B, coupled with reduced levels of inhibitory molecules such as T cell immunoglobulin and mucin domain-3 and programmed cell death protein-1 on CD8+ T cells. Furthermore, this approach led to improved survival and tumor regression without causing any systemic toxicity in melanoma and lung metastasis models. These findings suggest that mRNA encoding IL-12 in conjunction with STING agonists has the potential to confer superior clinical outcomes, representing a promising advancement in cancer immunotherapy.

Modulating Plaque Inflammation via Targeted mRNA Nanoparticles for the Treatment of Atherosclerosis.

Atherosclerosis is a common pathology present in many cardiovascular diseases. Although the current therapies (including statins and inhibitors of the serine protease PCSK9) can effectively reduce low-density lipoprotein (LDL) cholesterol levels to guideline-recommended levels, major adverse cardiovascular events still occur frequently. Indeed, the subendothelial retention of lipoproteins in the artery wall triggers multiple events of inflammation in macrophages and is a major contributor to the pathological progression of atherosclerosis. It has been gradually recognized that modulating inflammation is, therefore, an attractive avenue to forestall and treat atherosclerosis and its complications. Unfortunately, challenges with specificity and efficacy in managing plaque inflammation have hindered progress in atherosclerosis treatment. Herein, we report an NP-mediated mRNA therapeutic approach to target atherosclerotic lesional macrophages, modulating inflammation in advanced atherosclerotic lesions for the treatment of atherosclerosis. We demonstrated that the targeted NPs containing IL-10 mRNA colocalized with M2-like macrophages and induced IL-10 production in atherosclerotic plaques following intravenous administration to Western diet (WD)-fed Ldlr-/- mice. Additionally, the lesions showed a significantly alleviated inflammatory response, as evidenced by reduced oxidative stress and macrophage apoptosis, resulting in decreased lipid deposition, diminished necrotic areas, and increased fiber cap thickness. These results demonstrate the successful delivery of mRNA therapeutics to macrophage-enriched plaques in a preclinical model of advanced atherosclerosis, showing that this targeted NP inflammation management approach has great potential for translation into a wide range of clinical applications.

Nanoparticle-Mediated RNA Therapy Attenuates Nonalcoholic Steatohepatitis and Related Fibrosis by Targeting Activated Hepatic Stellate Cells.

Chronic liver injury and inflammation triggered by metabolic abnormalities initiate the activation of hepatic stellate cells (HSCs), driving fibrosis and parenchymal dysfunction, culminating in disorders such as nonalcoholic steatohepatitis (NASH). Unfortunately, there are currently no approved drugs capable of effectively treating NASH due to the challenges in addressing fibrosis and restoring extracellular matrix (ECM) homeostasis. We discovered a significant up-regulation of interleukin-11 (IL-11) in fibrotic livers using two well-established murine models of NASH. To leverage this signaling pathway, we developed a nanoparticle (NP)-assisted RNA interfering approach that specifically targets activated HSCs (aHSCs), blocking IL-11/ERK signaling to regulate HSC transdifferentiation along with fibrotic remodeling. The most potent NP, designated NP-AEAA, showed enhanced accumulation in fibrotic livers with NASH and was primarily enriched in aHSCs. We further investigated the therapeutic efficacy of aHSC-targeting NP-AEAA encapsulating small interfering RNA (siRNA) against IL11 or its cognate receptor IL11ra1 (termed siIL11@NP-AEAA or siIL11ra1@NP-AEAA, respectively) for resolving fibrosis and NASH. Our results demonstrate that both siIL11@NP-AEAA and siIL11ra1@NP-AEAA effectively inhibit HSC activation and resolve fibrosis and inflammation in two well-established murine models of NASH. Notably, siIL11ra1@NP-AEAA exhibits a superior therapeutic effect over siIL11@NP-AEAA, in terms of reducing liver steatosis and fibrosis as well as recovering liver function. These results constitute a targeted nanoparticulate siRNA therapeutic approach against the IL-11 signaling pathway of aHSCs in the fibrotic liver, offering a promising therapeutic intervention for NASH and other diseases.

Effects of a Ganoderma lucidum Proteoglycan on Type 2 Diabetic Rats and the Recovery of Rat Pancreatic Islets.

Type 2 diabetes (T2D) results from both insulin resistance and pancreatic ß-cell dysfunction. A natural proteoglycan extracted from Ganoderma lucidum, namely, FYGL, has been demonstrated to be capable of ameliorating insulin resistance in previous work. In this work, a T2D rat model induced by streptozocin (STZ) and a high-fat diet was used to investigate the effects of FYGL on pancreatic functions, and the transcriptomics of the rat pancreas was used to investigate the biological processes (BP) and signal pathways influenced by FYGL on the gene basis. Furthermore, the results of transcriptomics were verified both by histopathological analyses and protein expression. The studies showed that FYGL positively regulated T2D-related BP and signaling pathways and recovered the pancreatic function, therefore ameliorating hyperglycemia and hyperlipidemia in vivo. Importantly, the recovery of the pancreatic function suggested a crucial strategy to radically treat T2D.

Rational design of engineered H-ferritin nanoparticles with improved siRNA delivery efficacy across an in vitro model of the mouse BBB.

Gene therapy holds tremendous potential for the treatment of incurable brain diseases including Alzheimer's disease (AD), stroke, glioma, and Parkinson's disease. The main challenge is the lack of effective gene delivery systems traversing the blood-brain barrier (BBB), due to the complex microvessels present in the brain which restrict substances from the circulating blood passing through. Recently, increasing efforts have been made to develop promising gene carriers for brain-related disease therapies. One such development is the self-assembled heavy chain ferritin (HFn) nanoparticles (NPs). HFn NPs have a unique hollow spherical structure that can encapsulate nucleic acid drugs (NADs) and specifically bind to cancer cells and BBB endothelial cells (BBB ECs) via interactions with the transferrin receptor 1 (TfR1) overexpressed on their surfaces, which increases uptake through the BBB. However, the gene-loading capacity of HFn is restricted by its limited interior volume and negatively charged inner surface; therefore, these drawbacks have prompted the demand for strategies to remould the structure of HFn. In this work, we analyzed the three-dimensional (3D) structure of HFn using Chimera software (v 1.14) and developed a class of internally cationic HFn variants (HFn+ NPs) through arginine mutation on the lumenal surface of HFn. These HFn+ NPs presented powerful electrostatic forces in their cavities, and exhibited higher gene encapsulation efficacy than naive HFn. The top-performing candidate, HFn2, effectively delivered siRNA to glioma cells after traversing the BBB and achieved the highest silencing efficacy among HFn+ NPs. Overall, our findings demonstrate that HFn+ NPs obtained by this genetic engineering method provide critical insights into the future development of nucleic acid delivery carriers with BBB-crossing ability.

Antioxidation of a proteoglycan from Ganoderma lucidum protects pancreatic ß-cells against oxidative stress-induced apoptosis in vitro and in vivo.

Oxidative stress is one of the major factors in induction of pancreatic ß-cell apoptosis and diabetes. Here, we investigated systematically the roles of a proteoglycan (namely, FYGL) from Ganoderma lucidum in protection and repair of pancreatic ß-cells against oxidative stress-induced injury and apoptosis on molecular, cellular and animal basis. FYGL in vitro had antioxidant activity in terms of scavenging of free radicals and reduction power. FYGL improved cells viability, insulin secretion, redox indicator expressions, and mitochondrial membrane potential in H2O2-induced INS-1 cell via regulating the activations of apoptosis-related mitogen-activated protein kinases (MAPK) and nuclear factor kappa B (NF-κB) pathways as well as the insulin secretion-related pathway. Thrillingly in vivo, FYGL repaired the injured pancreas, reduced the pancreatic ß-cells apoptosis, and improved insulin secretion because of regulating the balance of oxidation-reduction, therefore well managed blood glucose in db/db diabetic mice. These results demonstrated that FYGL is promising to be used as a novel natural remedy for protection of pancreatic ß-cells against oxidative stress in diabetes treatment.

The thermodynamic and kinetic mechanisms of a Ganoderma lucidum proteoglycan inhibiting hIAPP amyloidosis.

Ganoderma lucidum is a valuable medicinal herbal which has been reported to prevent type 2 diabetes (T2D). A natural hyperbranched proteoglycan extracted from Ganoderma lucidum, namely, FYGL, has been demonstrated to inhibit the amyloidosis of human islet amyloid polypeptide (hIAPP) previously by our lab. However, the effective active components and the mechanisms of FYGL in inhibiting hIAPP amyloidosis are unknown. To identify the effective active components, different components from FYGL were isolated: the polysaccharide FYGL-1, the proteoglycans of FYGL-2 and FYGL-3. We further separated and sequenced the protein moieties of FYGL-2 and FYGL-3, namely, FYGL-2-P and FYGL-3-P, respectively, and compared their abilities to inhibit hIAPP amyloidosis, and systematically explored the inhibitory mechanisms by spectroscopy, microscopy and molecular dynamic simulation methods. Results showed that the protein moieties of FYGL played essential roles in inhibiting hIAPP amyloidosis. The strong, specific, and enthalpy-driven interaction by π-π stacking and electrostatic forces between hIAPP and FYGL-3-P dramatically inhibited hIAPP amyloidosis. These results suggested that FYGL-3-P had enormous potential to prevent hIAPP misfolding-induced diabetes and structurally helped researchers to seek or design inhibitors against polypeptide amyloidosis.

Interaction and Inhibition of a Ganoderma lucidum Proteoglycan on PTP1B Activity for Anti-diabetes.

Protein tyrosine phosphatase 1B (PTP1B) is a key negative regulator of insulin and an effective target for the treatment of type 2 diabetes (T2D). A natural hyperbranched proteoglycan extracted from Ganoderma lucidum, namely, Fudan-Yueyang G. Lucidum (FYGL), was demonstrated capable of inhibiting the activity of PTP1B. Here, to identify the effective active components of FYGL, three different components, the polysaccharide FYGL-1, proteoglycans FYGL-2, and FYGL-3, were isolated from FYGL, and then, the protein moiety of FYGL-3 was further separated, namely, FYGL-3-P. Their abilities to enhance the glucose uptake in cells and inhibit the activity of PTP1B were compared. The inhibitory mechanisms were systematically explored by spectroscopic methods and MD simulations. The results showed that FYGL-3 and FYGL-3-P significantly enhanced the insulin-provoked glucose uptake in insulin-resistant HepG2 cells, detected by the glucose oxidase method. Also, the FYGL-3-P protein moiety in FYGL played an essential role in inhibiting the activity of PTP1B. A strong, enthalpy-driven, and multitargeted interaction by electrostatic forces between PTP1B and FYGL-3-P dramatically inhibited the catalytic activity of PTP1B. These results provided deep insights into the molecular mechanisms of FYGL inhibiting the activity of PTP1B and structurally helped researchers seek natural PTP1B inhibitors.

Pancreatic cancer cell apoptosis is induced by a proteoglycan extracted from Ganoderma lucidum.

The Traditional Chinese Medicine, Ganoderma lucidum, has been widely used for its immunity-related and anti-cancer effects. Fudan-Yueyang-Ganoderma lucidum (FYGL) is a proteoglycan, extracted from Ganoderma lucidum, that has shown safe anti-diabetic activity in vivo. The present study demonstrated that FYGL could selectively inhibit the viability of PANC-1 and BxPC-3 pancreatic cancer cells in a dose dependent manner, but not in Mia PaCa-2 pancreatic cancer cells and HepG2 liver cancer cells. In addition, FYGL could inhibit migration and colony formation, and promote apoptosis in PANC-1 cells, but not in Mia PaCa-2 cells. Further investigation into the underlying mechanism revealed that FYGL could inhibit the expression level of the Bcl-2 protein in PANC-1 cells, but not in Mia PaCa-2 cells, leading to an increase in reactive oxygen species (ROS) and a reduction in the mitochondrial membrane potential and cell apoptosis. The increased ROS also promoted the formation of autophagosomes, along with an increase in the microtubule-associated protein light chain 3 II/I ratio. However, FYGL halted autophagy by preventing the autophagosomes from entering the lysosomes. The inhibition of autophagy increased the accumulation of defective mitochondria, as well as the production of ROS. Taken together, the processes of ROS regulation and autophagy inhibition promoted apoptosis of PANC-1 cells through the caspase-3/cleaved caspase-3 cascade. These results indicated that FYGL could be potentially used as an anti-cancer agent in the treatment of pancreatic cancer.

A proteoglycan extract from Ganoderma Lucidum protects pancreatic beta-cells against STZ-induced apoptosis.

The pancreatic ß-cell death or dysfunction induced by oxidative stress plays an important effect on the development and progression of diabetes mellitus. Based on our previous findings, a natural proteoglycan extracted from Ganoderma Lucidum, named FYGL, could treat T2DM in vivo. In this study, we investigated the effects of FYGL on STZ-induced apoptosis of INS-1 cells and its underlying mechanisms. The results showed that FYGL significantly improved the cell viability and alleviated the apoptosis in STZ-treated INS-1 cells. Moreover, FYGL markedly decreased the intracellular ROS accumulation and NO release, and deactivated NF-κB, JNK, and p38 MAPK signaling pathways in STZ-induced INS-1 cells. Furthermore, FYGL improved the insulin secretion through inhibiting the activation of JNK and improving the expression of Pdx-1 in INS-1 cells damaged by STZ. These results indicated that FYGL could protect pancreatic ß-cells against apoptosis and dysfunction, and be used as a promising pharmacological medicine for diabetes management. Abbreviations: T2DM: type 2 diabetes mellitus; FYGL: Fudan-Yueyang G. lucidum; ROS: reactive oxygen species; NO: reactive oxygen species; NF-κB: nuclear factor kappa beta; JNK: c-jun N-terminal kinase; MAPK: mitogen-activated protein kinase; Pdx-1: Pancreatic duodenal homeobox 1.

Amelioration of the Lipogenesis, Oxidative Stress and Apoptosis of Hepatocytes by a Novel Proteoglycan from Ganoderma lucidum.

The steatosis and resultant oxidative stress and apoptosis play the important roles in the progression of nonalcoholic fatty liver disease (NAFLD), therefore, searching for the effective drugs against NAFLD has been a hot topic. In this work, we investigated a hyperbranched proteoglycan, namely FYGL extracted from Ganoderma lucidum, inhibiting the palmitic acid (PA)-induced steatosis in HepG2 hepatocytes. FYGL compose of hydrophilic polysaccharide and lipophilic protein. Both moieties conclude the reductive residues, such as glucose and cystine, making FYGL capable of anti-oxidation. Herein, we demonstrated that FYGL can significantly inhibit the steatosis, i.e., decrease the contents of triglycerides (TG) and total cholesterol (TC) in hepatic cells on the mechanism of increasing the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC), therefore inhibiting the expressions of sterol regulatory element-binding protein 1 (SREBP1) and fatty acid synthase (FASN), furthermore leading to the carnitine palmitoyl transferase-1 (CPT-1) expression increased against steatosis induced by fatty acids oxidation. Meanwhile, FYGL can alleviate reactive oxygen species (ROS) and malondialdehyde (MDA), promote superoxide dismutase (SOD) and total antioxidant capacity (T-AOC). Moreover, FYGL can prevent the cells from apoptosis by regulating the apoptosis-related protein expressions and alleviating oxidative stress. Notably, FYGL could significantly recover the cells activity and inhibit lactate dehydrogenase (LDH) release which were negatively induced by high concentration PA. These results demonstrated that FYGL has the potential functions to prevent the hepatocytes from lipid accumulation, oxidative stress and apoptosis, therefore against NAFLD.

NIR-controlled HSP90 inhibitor release from hollow mesoporous nanocarbon for synergistic tumor photothermal therapy guided by photoacoustic imaging.

Photothermal therapy (PTT) has been widely studied for tumor therapy. However, the clinical transformation of PTT has encountered significant challenges in tumor recurrence, because the uneven hyperthermia in tumor tissues can result in the survival of cancer cells in the lower temperature regions close to blood vessels (as the blood flow can dissipate the localized heat). It is therefore important for clinical treatments to retain the excellent therapeutic efficiency of PTT at relatively low temperatures. In this article, innocuous hollow mesoporous carbon spheres (HMCS) with a high photothermal conversion efficiency were obtained by a one-pot synthesis method. After modification with DSPE-PEG, the HMCS-PEG exhibited a superior stability in biomedia, which is beneficial for further biological applications. Interestingly, combined with hydrophobic gambogic acid (GA) which can downregulate heat shock protein 90 (HSP90), the HMCS-PEG-GA system showed a significant NIR-enhanced tumor therapeutic effect in vitro and in vivo under mild temperature conditions (∼43 °C), and the combination index (CI) value of HMCS-PEG-GA was found to be 0.72. Meanwhile, this nano-system possessed good photothermal imaging and photoacoustic imaging abilities. Guided by the photoacoustic imaging signal, HMCS-PEG-GA showed enormous potential for use in accurate tumor diagnosis and mild-temperature PPT treatment applications, which is very important for clinical transformation of this nano-system.

Complex of EGCG with Cu(II) Suppresses Amyloid Aggregation and Cu(II)-Induced Cytotoxicity of α-Synuclein.

Accumulation of α-synuclein (α-Syn) is a remarkable pathology for Parkinson's disease (PD), therefore clearing it is possibly a promising strategy for treating PD. Aberrant copper (Cu(II)) homeostasis and oxidative stress play critical roles in the abnormal aggregation of α-Syn in the progress of PD. It is reported that the polyphenol (-)-epi-gallocatechin gallate (EGCG) can inhibit α-Syn fibrillation and aggregation, disaggregate α-Syn mature fibrils, as well as protect α-Syn overexpressed-PC12 cells against damage. Also, previous studies have reported that EGCG can chelate many divalent metal ions. What we investigate here is whether EGCG can interfere with the Cu(II) induced fibrillation of α-Syn and protect the cell viability. In this work, on a molecular and cellulaire basis, we demonstrated that EGCG can form a Cu(II)/EGCG complex, leading to the inhibition of Cu(II)-induced conformation transition of α-Syn from random coil to ß-sheet, which is a dominant structure in α-Syn fibrils and aggregates. Moreover, we found that the mixture of Cu(II) and EGCG in a molar ratio from 0.5 to 2 can efficiently inhibit this process. Furthermore, we demonstrated that in the α-Syn transduced-PC12 cells, EGCG can inhibit the overexpression and fibrillation of α-Syn in the cells, and reduce Cu(II)-induced reactive oxygen species (ROS), protecting the cells against Cu(II)-mediated toxicity.