The deubiquitinating enzyme 13 retards non-alcoholic steatohepatitis via blocking inactive rhomboid protein 2-dependent pathway.

Nowadays potential preclinical drugs for the treatment of nonalcoholic steatohepatitis (NASH) have failed to achieve expected therapeutic efficacy because the pathogenic mechanisms are underestimated. Inactive rhomboid protein 2 (IRHOM2), a promising target for treatment of inflammation-related diseases, contributes to deregulated hepatocyte metabolism-associated nonalcoholic steatohepatitis (NASH) progression. However, the molecular mechanism underlying Irhom2 regulation is still not completely understood. In this work, we identify the ubiquitin-specific protease 13 (USP13) as a critical and novel endogenous blocker of IRHOM2, and we also indicate that USP13 is an IRHOM2-interacting protein that catalyzes deubiquitination of Irhom2 in hepatocytes. Hepatocyte-specific loss of the Usp13 disrupts liver metabolic homeostasis, followed by glycometabolic disorder, lipid deposition, increased inflammation, and markedly promotes NASH development. Conversely, transgenic mice with Usp13 overexpression, lentivirus (LV)- or adeno-associated virus (AAV)-driven Usp13 gene therapeutics mitigates NASH in 3 models of rodent. Mechanistically, in response to metabolic stresses, USP13 directly interacts with IRHOM2 and removes its K63-linked ubiquitination induced by ubiquitin-conjugating enzyme E2N (UBC13), a ubiquitin E2 conjugating enzyme, and thus prevents its activation of downstream cascade pathway. USP13 is a potential treatment target for NASH therapy by targeting the Irhom2 signaling pathway.

A novel NADP(H)-dependent 3α-HSDH from the intestinal microbiome of Ursus thibetanus.

3α-HSDHs have a crucial role in the bioconversion of steroids, and have been widely applied in the detection of total bile acid (TBA). In this study, we report a novel NADP(H)-dependent 3α-HSDH (named Sc 3α-HSDH) cloned from the intestinal microbiome of Ursus thibetanus. Sc 3α-HSDH was solubly expressed in E. coli (BL21) as a recombinant glutathione-S-transferase (GST)-tagged protein and freed from its GST-fusion by cleavage using the PreScission protease. Sc 3α-HSDH is a new member of the short-chain dehydrogenases/reductase superfamily (SDRs) with a typical α/ß folding pattern, based on protein three-dimensional models predicted by AlphaFold. The best activity of Sc 3α-HSDH occurred at pH 8.5 and the temperature optima was 55 °C, indicating that Sc 3α-HSDH is not an extremozyme. The catalytic efficiencies (kcat/Km) of Sc 3α-HSDH catalyzing the oxidation reaction with the substrates, glycochenodeoxycholic acid (GCDCA) and glycoursodeoxycholic acid (GUDCA), were 183.617 and 34.458 s-1 mM-1, respectively. In addition, multiple metal ions can enhance the activity of Sc 3α-HSDH when used at concentrations ranging from 2 % to 42 %. The results also suggest that the metagenomic approach is an efficient method for identifying novel enzymes.

iRhom2 Promotes Hepatic Steatosis by Activating MAP3K7-Dependent Pathway.

BACKGROUND AND AIMS: Nonalcoholic fatty liver disease (NAFLD) has been widely recognized as a precursor to metabolic complications. Elevated inflammation levels are predictive of NAFLD-associated metabolic disorder. Inactive rhomboid-like protein 2 (iRhom2) is regarded as a key regulator in inflammation. However, the precise mechanisms by which iRhom2-regulated inflammation promotes NAFLD progression remain to be elucidated. APPROACH AND RESULTS: Here, we report that insulin resistance, hepatic steatosis, and specific macrophage inflammatory activation are significantly alleviated in iRhom2-deficient (knockout [KO]) mice, but aggravated in iRhom2 overexpressing mice. We further show that, mechanistically, in response to a high-fat diet (HFD), iRhom2 KO mice and mice with iRhom2 deficiency in myeloid cells only showed less severe hepatic steatosis and insulin resistance than controls. Inversely, transplantation of bone marrow cells from healthy mice to iRhom2 KO mice expedited the severity of insulin resistance and hepatic dyslipidemia. Of note, in response to HFD, hepatic iRhom2 binds to mitogen-activated protein kinase kinase kinase 7 (MAP3K7) to facilitate MAP3K7 phosphorylation and nuclear factor kappa B cascade activation, thereby promoting the activation of c-Jun N-terminal kinase/insulin receptor substrate 1 signaling, but disturbing AKT/glycogen synthase kinase 3ß-associated insulin signaling. The iRhom2/MAP3K7 axis is essential for iRhom2-regulated liver steatosis. CONCLUSIONS: iRhom2 may represent a therapeutic target for the treatment of HFD-induced hepatic steatosis and insulin resistance.

Functional loss of inactive rhomboid-like protein 2 mitigates obesity by suppressing pro-inflammatory macrophage activation-triggered adipose inflammation.

OBJECTIVE: Chronic inflammation of adipose tissues contributes to obesity-triggered insulin resistance. Unfortunately, the potential molecular mechanisms regarding obesity-associated systemic inflammation and metabolic disorder remain complicated. Here, we report that inactive rhomboid-like protein 2 (iRhom2) was increased in overweight mice with adipose inflammation. METHODS: Mice with deletion of iRhom2 on a C57BL/6J background, mice without deletion of this gene (controls), and mice with deficiency of iRhom2 only in myeloid cells were fed a standard chow diet (SCD) or a high-fat diet (HFD; 60% fat calories). Then the adipose tissues or bone marrow cells were isolated for the further detection. RESULTS: After 16 weeks on a high-fat diet (HFD), obesity, chronic inflammation in adipose tissues, and insulin resistance were markedly mitigated in iRhom2 knockout (iRhom2 KO) mice, whereas these parameters were exaggerated in iRhom2 overactivated mice. The adverse influences of iRhom2 on adipose inflammation and associated pathologies were determined in db/db mice. We further demonstrated that, in response to an HFD, iRhom2 KO mice and mice with deletion only in the myeloid cells showed less severe adipose inflammation and insulin resistance than control groups. Conversely, transplantation of bone marrow cells from normal mice to iRhom2 KO mice unleashed severe systemic inflammation and metabolic dysfunction after HFD ingestion. CONCLUSION: We identified iRhom2 as a key regulator that promotes obesity-associated metabolic disorders. Loss of iRhom2 from macrophages in adipose tissues may indirectly restrain inflammation and insulin resistance via blocking crosslinks between macrophages and adipocytes. Hence, iRhom2 may be a therapeutic target for obesity-induced metabolic dysfunction.

Endoplasmic reticulum stress-induced iRhom2 up-regulation promotes macrophage-regulated cardiac inflammation and lipid deposition in high fat diet (HFD)-challenged mice: Intervention of fisetin and metformin.

Endoplasmic reticulum stress (ERS) has been implicated in obesity-associated cardiac remodeling and dysfunction. Inactive rhomboid protein 2 (iRhom2), also known as Rhbdf2, is an inactive member of the rhomboid intramembrane proteinase family, playing an essential role in regulating inflammation. Nevertheless, the role of ERS-meditated iRhom2 pathway in metabolic stress-induced cardiomyopathy remains unknown. In the study, we showed that 4-PBA, as an essential ERS inhibitor, significantly alleviated high fat diet (HFD)-induced metabolic disorder and cardiac dysfunction in mice. Additionally, lipid deposition in heart tissues was prevented by 4-PBA in HFD-challenged mice. Moreover, 4-PBA blunted the expression of iRhom2, TACE, TNFR2 and phosphorylated NF-κB to prevent HFD-induced expression of inflammatory factors. Further, 4-PBA restrained HFD-triggered oxidative stress by promoting Nrf-2 signaling. Importantly, 4-PBA markedly suppressed cardiac ERS in HFD mice. The anti-inflammation, anti-ERS and anti-oxidant effects of 4-PBA were verified in palmitate (PAL)-incubated macrophages and cardiomyocytes. In addition, promoting ERS could obviously enhance iRhom2 signaling in vitro. Intriguingly, our data demonstrated that PAL-induced iRhom2 up-regulation apparently promoted macrophage to generate inflammatory factors that could promote cardiomyocyte inflammation and lipid accumulation. Finally, interventions by adding fisetin or metformin significantly abrogated metabolic stress-induced cardiomyopathy through the mechanisms mentioned above. In conclusion, this study provided a novel mechanism for metabolic stress-induced cardiomyopathy pathogenesis. Therapeutic strategy to restrain ROS/ERS/iRhom2 signaling pathway could be developed to prevent myocardial inflammation and lipid deposition, consequently alleviating obesity-induced cardiomyopathy.

Fisetin supplementation prevents high fat diet-induced diabetic nephropathy by repressing insulin resistance and RIP3-regulated inflammation.

Obesity-related renal disease is related to caloric excess promoting deleterious cellular responses. However, a full understanding of the molecular mechanisms involved in progressive kidney disease, as well as a therapeutic strategy, is still absent. Fisetin (FIS), as a natural flavonoid, possesses various bioactivities in a number of disease models. However, its role in obesity-associated kidney injury is still unclear and requires elucidation. In our study, an obesity animal model was established using C57BL/6 mice fed with a normal chow diet (NCD) or high fat diet (HFD) for 16 weeks with or without FIS administration (20, 40 or 80 mg kg-1). Our results indicated that chronic HFD feeding led to a significant body weight gain in mice compared to the normal control group, accompanied by a marked insulin resistance and glucose intolerance, whereas FIS treatment exerted prominently protective effects. In addition, FIS significantly attenuated HFD-induced histological alterations in renal tissue samples. Moreover, FIS treatment down-regulated expression of kidney injury molecule-1 (KIM-1), and up-regulated nephrin and podocin expression levels in the kidneys of HFD-fed mice, improving their renal dysfunction. After HFD feeding, mice treated with FIS exhibited a decrease in phosphorylated IRS1Ser307, and an increase in phosphorylated glycogen synthase kinase 1 (IRS1Tyr608), AKT, forkhead box protein O1 (FOXO1) and glycogen synthase kinase (GSK)-3ß. Furthermore, FIS administration markedly restrained the inflammatory response in the kidneys of HFD-challenged mice, as evidenced by the reduced pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), IL-1ß and IL-18, which was attributed to the blockage of nuclear factor κB (NF-κB) signaling. Importantly, FIS-treated obese mice exerted a remarkable decrease in RIP3 expressions in the kidneys compared to obese mice in the absence of FIS, along with an evident reduction in the NOD-like receptor protein 3 (NLRP3), an apoptosis-associated speck-like protein containing a Caspase recruitment domain (ASC) and Caspase-1. The protective effects of FIS against HFD-induced renal injury were verified in vitro using palmitate (PAL)-treated HK2 cells, an immortalized proximal tubule epithelial cell line from the adult human kidney. In summary, our results supported the notion that FIS functions as a promising agent to improve insulin resistance and inflammatory response against metabolic stress-induced renal injury.

Activated TNF-α/RIPK3 signaling is involved in prolonged high fat diet-stimulated hepatic inflammation and lipid accumulation: inhibition by dietary fisetin intervention.

Increasing evidence indicates that high-fat diet (HFD) is a predisposing factor for metabolic syndrome-associated systemic inflammation and nonalcoholic fatty liver disease (NAFLD). Tumor necrosis factor-α/receptor-interacting protein kinase 3 (TNF-α/RIPK3) axis has recently become regarded as an important regulator and contributor in many inflammation-related diseases. Fisetin (Fn) is a bioactive flavonoid polyphenol with anti-inflammatory and anti-tumor activity. Lately it has gained increasing attention due to its potential advantages in prevention of tumors, metabolic disorders, neuroinflammation and chronic liver injury. However, its role in NAFLD is still not fully understood. Thus, we studied whether prolonged HFD causes TNF-α/RIPK3 activation-associated hepatic steatosis, further evaluating the protective effects of Fn in HFD-fed mice. As expected, HFD promotes metabolic syndrome and pro-inflammatory cytokine generation in serum, enhances liver inflammatory infiltration-related lipid accumulation by increase of TNF-α/RIPK3 activation, ultimately resulting in development of nonalcoholic steatohepatitis. In contrast to this, dietary Fn intervention could suppress metabolic disorder and HFD-triggered hepatic function loss, downregulate TNF-α/RIPK3 signaling-associated hepatic inflammation, balance lipid metabolism-related gene expression, and finally inhibit lipid accumulation and steatohepatitis. Hence, Fn restrains HFD-induced hepatic inflammation and lipid deposition by downregulating metabolic disorder and excessive liver inflammation-associated TNF-α/RIPK3 axis activation.

Prolonged PM2.5 exposure elevates risk of oxidative stress-driven nonalcoholic fatty liver disease by triggering increase of dyslipidemia.

An increasing number of studies have shown that air pollution containing particulate matter (PM) ≤ 2.5 µm (PM2.5) plays a significant role in the development of metabolic disorder and other chronic diseases. Inflammation and oxidative stress caused by metabolic syndrome are widely determined to be critical factors in the development of nonalcoholic fatty liver disease (NAFLD) pathogenesis. However, there is no direct evidence of this, and the underlying molecular mechanism is still not fully understood. In this study, we investigated the role of inflammation and oxidative stress caused by prolonged PM2.5 exposure in dyslipidemia-associated chronic hepatic injury, and further determined whether an increase in hepatic inflammation and oxidative stress promoted lipid accumulation in the liver, ultimately increasing the risk of NAFLD. Therefore, we studied changes in indicators of metabolic disorder and in symbolic indices of NAFLD. We confirmed increases in insulin resistance, glucose tolerance, peripheral inflammation and dysarteriotony in PM2.5-induced mice. Oxidative stress and inflammatory response in the liver caused by PM2.5 inhalation contributed to abnormal hepatic function, further promoting lipid accumulation in the liver. Moreover, we observed inhibition of oxidative stress and inflammatory response by pyrrolidine dithiocarbamate (PDTC) and N-acetyl-L-cysteine (NAC) in vitro, suggesting that oxidative stress and inflammatory in liver cells aggravated by PM2.5 contributed to hepatic injury by altering normal lipid metabolism. These results indicate a new goal for preventing and treating air pollution-induced diseases: suppression of oxidative stress and inflammatory response.

Activated iRhom2 drives prolonged PM2.5 exposure-triggered renal injury in Nrf2-defective mice.

Research suggests that particulate matter (PM2.5) is a predisposing factor for metabolic syndrome-related systemic inflammation and oxidative stress injury. TNF-α as a major pro-inflammatory cytokine was confirmed to participate in various diseases. Inactive rhomboid protein 2 (iRhom2) was recently determined as a necessary regulator for shedding of TNF-α in immune cells. Importantly, kidney-resident macrophages are critical to inflammation-associated chronic renal injury. Podocyte injury can be induced by stimulants and give rise to nephritis, but how iRhom2 contributes to PM2.5-induced renal injury is unclear. Thus, we studied whether PM2.5 causes renal injury and characterized iRhom2 with respect to TNF-α release in mice macrophages and renal tissues in long-term PM2.5-exposed mouse models. After long-term PM2.5 exposures, renal injury was confirmed via inflammatory cytokine, chemokine expression, and reduced antioxidant activity. Patients with kidney-related diseases had increased TNF-α, which may contribute to renal injury. We observed up-regulation of serum creatinine, serum urea nitrogen, kidney injury molecule 1, uric acid, TNF-α, MDA, H2O2, and O2- in PM2.5-treated mice, which was greater than that found in Nrf2-/- mice. Meanwhile, increases in metabolic disorder-associated indicators were involved in PM2.5-induced nephritis. In vitro, kidney-resident macrophages were observed to be critical to renal inflammatory infiltration and function loss via regulation of iRhom2/TACE/TNF-α signaling, and suppression of Nrf2-associated anti-oxidant response. PM2.5 exposure led to renal injury partly by inflammation-mediated podocyte injury. Reduced SOD1, SOD2, Nrf2 activation, and increased XO, NF-κB activity, TACE, iNOS, IL-1ß, TNF-α, IL-6, MIP-1α, Emr-1, MCP-1, and Cxcr4, were also noted. Long-term PM2.5 exposure causes chronic renal injury by up-regulation of iRhom2/TACE/TNF-α axis in kidney-resident macrophages. Overexpression of TNF-α derived from macrophages causes podocyte injury and kidney function loss. Thus, PM2.5 toxicities are related to exposure duration and iRhom2 may be a potential therapeutic renal target.

Multicombination Approach Suppresses Listeria monocytogenes-Induced Septicemia-Associated Acute Hepatic Failure: The Role of iRhom2 Signaling.

The mortality rate of acute liver failure significantly increases due to fatal septicemia. Inactive rhomboid protein 2 (iRhom2) is an essential regulator of shedding TNF-α by trafficking with TNF-α converting enzyme (TACE). Fisetin, a flavonoid present in various fruits and plants, possesses anti-oxidative stress and anti-inflammatory activities. Here, multi-combination nanoparticles Fe@Au conjugated with fisetin, iRhom2 small interfering RNA (siRNA), and TNF-α inhibitor (FN) are prepared to examine their effects on fatal septicemia-associated hepatic failure induced by Listeria monocytogenes (LM) in mice and to reveal the underlying mechanisms. After LM infection, upregulation of glutamic-oxalacetic transaminease, glutamic-pyruvic transaminase, alkaline phosphatase, TNF-α, malondialdehyde, H2 O2 , and O2- is observedcompared to FN-treated mice. The iRhom2/TACE/TNF-α signals are enhanced in vivo and in vitro, resulting in oxidative stress, which is especially associated with the activation of kupffer cells and other macrophages. Decrease in Nrf2 activation and increase of inflammation-associated regulators are also noted in vivo and in vitro. Furthermore, overexpression of TNF-α derived from macrophages aggravates hepatic failure. Inversely, the processes above are restored by FN nanoparticles through the regulation of the iRhom2/TACE/TNF-α axis and Nrf2 activation. These findings suggest that FN may be a potential approach to protect against bacterial septicemia-related diseases by targeting iRhom2.

iRhom2 deficiency relieves TNF-α associated hepatic dyslipidemia in long-term PM2.5-exposed mice.

Accumulating researches reported that particulate matter (PM2.5) is a risk factor for developing various diseases, including metabolic syndrome. Recently, inactive rhomboid protein 2 (iRhom2) was considered as a necessary modulator for shedding of tumor necrosis factor-α (TNF-α) in immune cells. TNF-α, a major pro-inflammatory cytokine, was linked to various pathogenesis of diseases, including dyslipidemia. Here, wild type (WT) and iRhom2-knockout (iRhom2-/-) mice were used to investigate the effects of iRhom2 on PM2.5-induced hepatic dyslipidemia. The hepatic histology, inflammatory response, glucose tolerance, serum parameters and gene expressions were analyzed. We found that long-term inhalation of PM2.5 resulted in hepatic steatosis. And a significant up-regulation of iRhom2 in liver tissues was observed, accompanied with elevated TNF-α, TNF-α converting enzyme (TACE), TNFα receptor (TNFR)2 and various inflammatory cytokines expressions. Additionally, PM2.5 treatment caused TG and TC accumulation in serum and liver, probably attributed to changes of genes modulating lipid metabolism. Intriguingly, hepatic injury and dyslipidemia were attenuated by iRhom2-/- in mice with PM2.5 challenge. In vitro, iRhom2-knockdwon reduced TNF-α expressions and its associated inflammatory cytokines in Kupffer cells, implying that liver-resident macrophages played an important role in regulating hepatic inflammation and lipid metabolism in cells treated with PM2.5. The findings indicated that long-term PM2.5 exposure caused hepatic steatosis and dyslipidemia through triggering inflammation, which was, at least partly, dependent on iRhom2/TNF-α pathway in liver-resident macrophages.

Drug release from core-shell PVA/silk fibroin nanoparticles fabricated by one-step electrospraying.

Silk fibroin (SF), a FDA-approved natural protein, is renowned for its great biocompatibility, biodegradability, and mechanical properties. SF-based nanoparticles provide new options for drug delivery with their tunable drug loading and release properties. To take advantage of the features of carrier polymers, we present a one-step electrospraying method that combines SF, polyvinyl alcohol (PVA) and therapeutic drugs without an emulsion process. A distinct core-shell structure was obtained with the PVA core and silk shell after the system was properly set up. The model drug, doxorubicin, was encapsulated in the core with a greater than 90% drug encapsulation efficiency. Controllable drug release profiles were achieved by alternating the PVA/SF ratio. Although the initial burst release of the drug was minimized by the SF coating, a large number of drug molecules remained entrapped by the carrier polymers. To promote and trigger drug release on demand, low intensity focused ultrasound (US) was applied. The US was especially advantageous for accelerating the drug diffusion and release. The apoptotic activity of MDA-MB-231 cells incubated with drug-loaded nanoparticles was found to increase with time. In addition, we also observed PVA/SF nanoparticles that could elicit a drug release in response to pH.

Functional contribution of coenzyme specificity-determining sites of 7α-hydroxysteroid dehydrogenase from Clostridium absonum.

Studies of the molecular determinants of coenzyme specificity help to reveal the structure-function relationship of enzymes, especially with regards to coenzyme specificity-determining sites (CSDSs) that usually mediate complex interactions. NADP(H)-dependent 7α-hydroxysteroid dehydrogenase from Clostridium absonum (CA 7α-HSDH), a member of the short-chain dehydrogenase/reductase superfamily (SDRs), possesses positively charged CSDSs that mainly contain T15, R16, R38, and R194, forming complicated polar interactions with the adenosine ribose C2 phosphate group of NADP(H). The R38 residue is crucial for coenzyme anchoring, but the influence of the other residues on coenzyme utilization is still not clear. Hence, we performed alanine scanning mutagenesis and molecular dynamic (MD) simulations. The results suggest that the natural CSDSs have the greatest NADP(H)-binding affinity, but not the best activity (kcat) toward NADP+. Compared with the wild type and other mutants, the mutant R194A showed the highest catalytic efficiency (kcat/Km), which was more than three-times that of the wild type. MD simulation and kinetics analysis suggested that the importance of the CSDSs of CA 7α-HSDH should be in accordance with the following order R38>T15>R16>R194, and S39 may have a supporting role in NADP(H) anchoring for mutants R16A/T194A and T15A/R16A/T194A.

Dual drug release from core-shell nanoparticles with distinct release profiles.

Multiple drug combination is a promising strategy in biomedical fields, such as cancer chemotherapy and tissue engineering. With the aim of codelivering multiple drugs with different characteristics, immiscible and miscible liquids were utilized to fabricate nanoparticles of polyvinylpyrrolidone/poly(lactic-co-glycolic acid) (PLGA) and poly(ε-caprolactone)/PLGA with distinct core-shell structure by coaxial electrospray. Each kind of nanoparticles can encapsulate the hydrophilic rhodamine B and hydrophobic naproxen in one single step efficiently. Encapsulation efficiency was over 85%. The different release patterns of dual-drug encapsulated were demonstrated when the drug location swapped, attributing to the distinct core-shell structures of nanoparticles and the interaction between drug molecules and carrier polymers. Meanwhile, the release profiles of encapsulated drugs with different loading amount were investigated as well. Dual drug release profiles from nanoparticles were affected by the unique architecture of nanocarriers (porous and core-shell structure), physical properties of polymers, and drugs. In addition, polymer-drug and drug-drug molecular interaction may take an important role in drug release behaviors. The results suggested that the distinct release kinetics of multiple drugs fabricated by coaxial electrospray can be obtained and tuned to fulfill the clinical requirement in combination therapy.

Transport barriers and strategies of antitumor nanocarriers delivery system.

Nanocarriers with unique characteristics have attracted great attentions in tumor treatment, but therapeutic efficacy of drugs is limited by the nonuniform delivery of nanocarriers to tumor by three progresses blood circulation system, tumor vessels and tumor interstitium. The total number of nanocarriers usually was reduced dramatically into tumor, because nanocarriers in blood vessels always can be cleared by RES system or accumulated in kidney and liver. Abnormal vessels are almost uneven allocation on tumor surface and cannot contribute to deliver nanocarriers to all the region of tumors, especially the interior of tumors. In addition, nanocarriers will be retained in perivascular space of tumor under high interstitial fluid pressure (IFP) and heterogeneous structure of tumor matrix. In this article, we reviewed the structure of nanocarriers and the barriers in tumor microenvironment influencing delivery of nanocarriers. With the aim of transport over these barriers to increase the delivery efficiency of nanocarriers, two strategies tumor microenvironment normalization and nanocarriers optimization were proposed and designed.

Selective killing of cancer stem cells by a novel dual-targeting strategy.

Chemo- and radio-resistance of cancer stem cells (CSCs) in solid tumors are the root causes of some cancer treatment failures in clinics. To eradicate tumors, in the present study we proposed a novel strategy against CSCs with a core-shell dual-targeting system, which consists of outer layers and inner parts. Anti-tumor therapeutic agents are loaded in the outer layer targeting cancer cells whereas antibody-drug conjugates (ADCs) with CSC targeting are encapsulated in the inner parts. The novel system we proposed can be concentrated in tumor tissue by the enhanced permeability and retention (EPR) effect. The drugs dispersed in the outer layer are supposed to kill the cancer cells, and then ADCs will be released gradually from the inner part at the tumor site which can target CSCs and help to eliminate cancer cells. In this regard, we may be able to kill the CSCs with these therapeutic drug combinations and improve the therapeutic effect of chemotherapy in cancer treatments.

Nanoscale delivery systems for multiple drug combinations in cancer.

Although drug-delivery systems have been developed to improve drug biodistribution and efficiency in cancer therapy, some limitations still hinder successful drug targeting and delivery. Multiple drugs in combination seems a promising strategy for cancer therapy. It enables drugs to be delivered to multiple targets and exhibits the additive or synergistic effects of drugs. Physiological barriers are known to be the main obstacles of insufficient drug efficacy and delivery in tumors, but they are likely to be potential targets in combination therapy as well. This article discusses some general considerations for optimizing multiply drug delivery, including drug-release profiles and loading strategies.