Structural insights into the octamerization of glycerol dehydrogenase.

Glycerol dehydrogenase (GDH) catalyzes glycerol oxidation to dihydroxyacetone in a NAD+-dependent manner. As an initiator of the oxidative pathway of glycerol metabolism, a variety of functional and structural studies of GDH have been conducted previously. Structural studies revealed intriguing features of GDH, like the flexible ß-hairpin and its significance. Another commonly reported structural feature is the enzyme's octameric oligomerization, though its structural details and functional significance remained unclear. Here, with a newly reported GDH structure, complexed with both NAD+ and glycerol, we analyzed the octamerization of GDH. Structural analyses revealed that octamerization reduces the structural dynamics of the N-domain, which contributes to more consistently maintaining a distance required for catalysis between the cofactor and substrate. This suggests that octamerization may play a key role in increasing the likelihood of the enzyme reaction by maintaining the ligands in an appropriate configuration for catalysis. These findings expand our understanding of the structure of GDH and its relation to the enzyme's activity.

Fluorometric and Colorimetric Method for SARS-CoV-2 Detection Using Designed Aptamer Display Particles.

SARS-CoV-2, the virus responsible for the COVID-19 pandemic, has spurred the urgent need for practical diagnostics with high sensitivity and selectivity. Although advanced diagnostic tools have emerged to efficiently control pandemics, they still have costly limitations owing to their reliance on antibodies or enzymes and require high-tech equipment. Therefore, there is still a need to develop rapid and low-cost diagnostics with high sensitivity and selectivity. In this study, we generated aptamer display particles (AdP), enabling easy fabrication of a SARS-CoV-2 detection matrix through particle PCR, and applied it to diagnosis using fluorometric and colorimetric assays. We designed two AdPs, C1-AdP and C4-AdP, displayed with SpS1-C1 and SpS1-C4 aptamers, respectively, and showed their high binding ability against SARS-CoV-2 spike protein with a concentration-dependent fluorescence increase. This enabled detection even at low concentrations (0.5 nM). To validate its use as a diagnostic tool for SARS-CoV-2, we designed a sandwich-type assay using two AdPs and high-quality aptamers targeting SARS-CoV-2 pseudoviruses. The fluorometric assay achieved a detection limit of 3.9 × 103 pseudoviruses/mL. The colorimetric assay using an amplification approach exhibited higher sensitivity, with a detection limit of 1 × 101 pseudoviruses/mL, and a broad range of over four orders of magnitude was observed.

Aptamers and Nanobodies as New Bioprobes for SARS-CoV-2 Diagnostic and Therapeutic System Applications.

The global challenges posed by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic have underscored the critical importance of innovative and efficient control systems for addressing future pandemics. The most effective way to control the pandemic is to rapidly suppress the spread of the virus through early detection using a rapid, accurate, and easy-to-use diagnostic platform. In biosensors that use bioprobes, the binding affinity of molecular recognition elements (MREs) is the primary factor determining the dynamic range of the sensing platform. Furthermore, the sensitivity relies mainly on bioprobe quality with sufficient functionality. This comprehensive review investigates aptamers and nanobodies recently developed as advanced MREs for SARS-CoV-2 diagnostic and therapeutic applications. These bioprobes might be integrated into organic bioelectronic materials and devices, with promising enhanced sensitivity and specificity. This review offers valuable insights into advancing biosensing technologies for infectious disease diagnosis and treatment using aptamers and nanobodies as new bioprobes.

Structural and functional insights into the flexible ß-hairpin of glycerol dehydrogenase.

During glycerol metabolism, the initial step of glycerol oxidation is catalysed by glycerol dehydrogenase (GDH), which converts glycerol to dihydroxyacetone in a NAD+ -dependent manner via an ordered Bi-Bi kinetic mechanism. Structural studies conducted with GDH from various species have mainly elucidated structural details of the active site and ligand binding. However, the structure of the full GDH complex with both cofactor and substrate bound is not determined, and thus, the structural basis of the kinetic mechanism of GDH remains unclear. Here, we report the crystal structures of Escherichia coli GDH with a substrate analogue bound in the absence or presence of NAD+ . Structural analyses including molecular dynamics simulations revealed that GDH possesses a flexible ß-hairpin, and that during the ordered progression of the kinetic mechanism, the flexibility of the ß-hairpin is reduced after NAD+ binding. It was also observed that this alterable flexibility of the ß-hairpin contributes to the cofactor binding and possibly to the catalytic efficiency of GDH. These findings suggest the importance of the flexible ß-hairpin to GDH enzymatic activity and shed new light on the kinetic mechanism of GDH.

Biomimetic Silica Particles with Self-Loading BMP-2 Knuckle Epitope Peptide and Its Delivery for Bone Regeneration.

Biomimetic silica deposition is an in-situ immobilization method for bioactive molecules under biocompatible conditions. The osteoinductive P4 peptide derived from the knuckle epitope of bone morphogenetic protein (BMP), which binds to BMP receptor-II (BMPRII), has been newly found to contain silica formation ability. We found that the two lysine residues at the N-terminus of P4 played a vital role in silica deposition. The P4 peptide co-precipitated with silica during P4-mediated silicification, yielding P4/silica hybrid particles (P4@Si) with a high loading efficiency of 87%. P4 was released from P4@Si at a constant rate for over 250 h, representing a zero-order kinetic model. In flow cytometric analysis, P4@Si showed a 1.5-fold increase in the delivery capacity to MC3T3 E1 cells than the free form of P4. Furthermore, P4 was found anchored to hydroxyapatite (HA) through a hexa-glutamate tag, followed by P4-mediated silicification, yielding P4@Si coated HA. This suggested a superior osteoinductive potential compared to silica or P4 alone coated HA in the in vitro study. In conclusion, the co-delivery of the osteoinductive P4 peptide and silica by P4-mediated silica deposition is an efficient method for capturing and delivering its molecules and inducing synergistic osteogenesis.

Structural and biochemical insights into Zn2+-bound EF-hand proteins, EFhd1 and EFhd2.

EF-hand proteins, which contain a Ca2+-binding EF-hand motif, are involved in regulating diverse cellular functions. Ca2+ binding induces conformational changes that modulate the activities of EF-hand proteins. Moreover, these proteins occasionally modify their activities by coordinating metals other than Ca2+, including Mg2+, Pb2+ and Zn2+, within their EF-hands. EFhd1 and EFhd2 are homologous EF-hand proteins with similar structures. Although separately localized within cells, both are actin-binding proteins that modulate F-actin rearrangement through Ca2+-independent actin-binding and Ca2+-dependent actin-bundling activity. Although Ca2+ is known to affect the activities of EFhd1 and EFhd2, it is not known whether their actin-related activities are affected by other metals. Here, the crystal structures of the EFhd1 and EFhd2 core domains coordinating Zn2+ ions within their EF-hands are reported. The presence of Zn2+ within EFhd1 and EFhd2 was confirmed by analyzing anomalous signals and the difference between anomalous signals using data collected at the peak positions as well as low-energy remote positions at the Zn K-edge. EFhd1 and EFhd2 were also found to exhibit Zn2+-independent actin-binding and Zn2+-dependent actin-bundling activity. This suggests the actin-related activities of EFhd1 and EFhd2 could be regulated by Zn2+ as well as Ca2+.

Ultra-sensitive label-free SERS biosensor with high-throughput screened DNA aptamer for universal detection of SARS-CoV-2 variants from clinical samples.

COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused an ongoing global pandemic with economic and social disruption. Moreover, the virus has persistently and rapidly evolved into novel lineages with mutations. The most effective strategy to control the pandemic is suppressing virus spread through early detection of infections. Therefore, developing a rapid, accurate, easy-to-use diagnostic platform against SARS-CoV-2 variants of concern remains necessary. Here, we developed an ultra-sensitive label-free surface-enhanced Raman scattering-based aptasensor as a countermeasure for the universal detection of SARS-CoV-2 variants of concern. In this aptasensor platform, we discovered two DNA aptamers that enable binding to SARS-CoV-2 spike protein via the Particle Display, a high-throughput screening approach. These showed high affinity that exhibited dissociation constants of 1.47 ± 0.30 nM and 1.81 ± 0.39 nM. We designed a combination with the aptamers and silver nanoforest for developing an ultra-sensitive SERS platform and achieved an attomolar (10-18 M) level detection limit with a recombinant trimeric spike protein. Furthermore, using the intrinsic properties of the aptamer signal, we demonstrated a label-free aptasensor approach, enabling use without the Raman tag. Finally, our label-free SERS-combined aptasensor succeeded in detecting SARS-CoV-2 with excellent accuracy, even in clinical samples with variants of concern, including the wild-type, delta, and omicron variants.

Clinical implication of minimal presence of solid or micropapillary subtype in early-stage lung adenocarcinoma.

BACKGROUND: We investigated the clinical features and surgical outcomes of lung adenocarcinoma with minimal solid or micropapillary (S/MP) components, with a focus on stage IA. METHODS: We enrolled 506 patients with lung adenocarcinoma who underwent curative resection in this study. Clinical features and surgical outcomes were compared between the groups with and without the S/MP subtype (S/MP+ and S/MP-, respectively), and between the group with an S/MP proportion of ≤5% (S/MP5) and the S/MP-. RESULTS: The S/MP subtype was present in 247 patients (48.8%); 129 (25.5%) were grouped as the S/MP5 group. The S/MP+ and S/MP5 groups had larger tumors, higher frequency of lymph node metastasis, and more advanced stages of disease than the S/MP- group (P < 0.001, all comparisons). Pleural, lymphatic, and vascular invasions occurred more frequently in the S/MP+ and S/MP5 groups (P < 0.001, all comparisons for S/MP+ vs. S/MP-; P ≤ 0.01, all comparisons for S/MP5 vs. S/MP-). The S/MP+ and S/MP5 groups showed a shorter time to recurrence and cancer-related death than the S/MP- group(P < 0.001, both comparisons). For stage I, the presence or absence of the S/MP subtype defined prognostic subgroups better than the stage IA/IB classification. Notably, in the multivariate analysis, the minimal S/MP component was a significant predictor of recurrence, even in stage IA. CONCLUSIONS: The presence of the minimal S/MP component was a significant predictor of poor prognosis after surgery, even in stage IA patients. Clinical trials to evaluate the advantages of adjuvant chemotherapy for this subset of patients and further investigations to understand underlying biological mechanisms of poor prognosis are needed. KEY POINTS: Significant findings of the study: We demonstrated that only minimal presence of solid or micropapillary component was profoundly associated with aggressive clinicopathological features and poor prognosis after complete resection even in stage IA lung adenocarcinoma. WHAT THIS STUDY ADDS: Our results suggest that minimal presence of these subtypes is a strong prognostic factor which should be taken into account in the risk assessment for adjuvant chemotherapy in lung adenocarcinoma.

TGFBI Expression Predicts the Survival of Patients With Oropharyngeal Squamous Cell Carcinoma.

BACKGROUND/AIM: This study was conducted to investigate transforming growth factor beta-induced protein (TGFBI) expression and analyze the clinical and prognostic significance of TGFBI in oropharyngeal squamous cell carcinoma (OPSCC). PATIENTS AND METHODS: We evaluated TGFBI expression by immunohistochemistry in 94 patients with OPSCC. For comprehensive analysis, TGFBI expression was subdivided into tumor cell score (T), stroma score (S), and the sum of two scores (TS) calculated using H-score. Clinicopathological features and survival outcomes were compared between groups of high expression and low expression of TGFBI in each area. RESULTS: Overall, 12 patients (12.8%) showed high T score, and 41 patients (43.6%) revealed high S score. Although T score showed no significant difference both in overall survival (OS) (p=0.080) and recurrence free survival (RFS) (p=0.272), high S score patients had significantly worse OS (p=0.003) and worse RFS (p=0.043). High TS score also showed significant association with worse OS (p=0.011) and worse RFS (p=0.021). High S score was an independent prognostic factor predicting shorter OS (HR=6.352, 95%CI=1.206-40.050, p=0.029) and RFS (HR=18.843, 95%CI=1.030-344.799, p=0.048) in the multivariate analysis. CONCLUSION: High S score of TGFBI was a significant predictor of poor prognosis in OPSCC. TGFBI could be a useful new predictive and prognostic biomarker in OPSCC.

Potent Small-Molecule Inhibitors Targeting Acetylated Microtubules as Anticancer Agents Against Triple-Negative Breast Cancer.

Microtubules are one of the major targets for anticancer drugs because of their role in cell proliferation and migration. However, as anticancer drugs targeting microtubules have side effects, including the death of normal cells, it is necessary to develop anticancer agents that can target microtubules by specifically acting on cancer cells only. In this study, we identified chemicals that can act as anticancer agents by specifically binding to acetylated microtubules, which are predominant in triple-negative breast cancer (TNBC). The chemical compounds disrupted acetylated microtubule lattices by interfering with microtubule access to alpha-tubulin acetyltransferase 1 (αTAT1), a major acetyltransferase of microtubules, resulting in the increased apoptotic cell death of MDA-MB-231 cells (a TNBC cell line) compared with other cells, such as MCF-10A and MCF-7, which lack microtubule acetylation. Moreover, mouse xenograft experiments showed that treatment with the chemical compounds markedly reduced tumor growth progression. Taken together, the newly identified chemical compounds can be selective for acetylated microtubules and act as potential therapeutic agents against microtubule acetylation enrichment in TNBC.

Comprehensive Analysis and Clinical Implication of PD-L1 Expression Considering HPV Status in Oropharyngeal Squamous Cell Carcinoma.

BACKGROUND/AIM: This study was conducted to comprehensively evaluate programmed cell death ligand 1 (PD-L1) expression, and analyze the clinical and prognostic implications of PD-L1 expression in oropharyngeal squamous cell carcinoma (OPSCC). PATIENTS AND METHODS: We evaluated the expression of PD-L1 using the antibodies SP263 and SP142 in 106 patients with OPSCC, using immunohistochemistry. PD-L1 expression was subdivided into tumor cell score (TC), immune cell score (IC), and combined score (CS). Correlations between each PD-L1 expression and HPV status, clinicopathological features, and survival were analyzed. RESULTS: The expression levels of PD-L1 SP263 and SP142 were significantly correlated. High PD-L1 SP263 TC and CS and SP142 IC and CS were associated with HPV positivity. PD-L1 expression showed no effect on survival in all patients' group. However, in the subgroup analysis, high TC and CS of both PD-L1 SP263 and SP142 were correlated with shorter time to recurrence in the HPV positive group. CONCLUSION: High expression of PD-L1 was associated with HPV positivity in OPSCC. In addition, high expression of PD-L1 might suggest a poorer outcome, especially in the HPV positive subgroup. PD-L1 could be a useful predictive and prognostic biomarker in OPSCC.

Structure of the MICU1-MICU2 heterodimer provides insights into the gatekeeping threshold shift.

Mitochondrial calcium uptake proteins 1 and 2 (MICU1 and MICU2) mediate mitochondrial Ca2+ influx via the mitochondrial calcium uniporter (MCU). Its molecular action for Ca2+ uptake is tightly controlled by the MICU1-MICU2 heterodimer, which comprises Ca2+ sensing proteins which act as gatekeepers at low [Ca2+] or facilitators at high [Ca2+]. However, the mechanism underlying the regulation of the Ca2+ gatekeeping threshold for mitochondrial Ca2+ uptake through the MCU by the MICU1-MICU2 heterodimer remains unclear. In this study, we determined the crystal structure of the apo form of the human MICU1-MICU2 heterodimer that functions as the MCU gatekeeper. MICU1 and MICU2 assemble in the face-to-face heterodimer with salt bridges and me-thio-nine knobs stabilizing the heterodimer in an apo state. Structural analysis suggests how the heterodimer sets a higher Ca2+ threshold than the MICU1 homodimer. The structure of the heterodimer in the apo state provides a framework for understanding the gatekeeping role of the MICU1-MICU2 heterodimer.

Increased Expression of Interleukin-12 in Lesional Skin of Atopic Dermatitis Patients with Psoriasiform Features on Histopathology: An Immunohistochemical Study.

BACKGROUND: Based on clinical and genetic differences, atopic dermatitis (AD) and psoriasis have been classified in two different diseases, but recently, some authors regarded them as in one spectrum. The histological similarities including epidermal hyperplasia between chronic stages of AD and psoriasis supports the presence of two diseases in one spectrum. OBJECTIVE: We investigated clinical and immunohistopathological characteristics of adult Korean patients with AD showing psoriasiform chronic dermatitis on histopathology. METHODS: In total, 59 Korean patients with chronic AD were enrolled. Clinical, laboratory, and histopathological features were compared between AD patients with psoriasiform features and those with non-psoriasiform chronic dermatitis features on histology. In addition, immunohistopathological characteristics were analyzed using antibodies for key regulatory and effector cytokines in psoriasis. RESULTS: Fifteen patients (25.4%) showed a more "psoriasiform" histological appearance. The lesions in patients with psoriasiform features often showed clearer boundaries and noticeable scaling. The interleukin (IL)-23 expression in the psoriasiform chronic dermatitis group was not different from that in the psoriasis group, but the IL-17 expression was less than that in the psoriasis group. In the case of IL-12, multiple dermal inflammatory cells with dendrites were stained in the psoriasiform chronic dermatitis group compared with the 2 other non-psoriasiform subgroups. CONCLUSION: The results suggest that IL-12 secreted from dermal inflammatory cells might be one of the important factors associated with the formation of psoriasiform features in chronic AD. However, further studies are required to better define the specific role of IL-12.

Structural and Biochemical Characterization of EFhd1/Swiprosin-2, an Actin-Binding Protein in Mitochondria.

Ca2+ regulates several cellular functions, including signaling events, energy production, and cell survival. These cellular processes are mediated by Ca2+-binding proteins, such as EF-hand superfamily proteins. Among the EF-hand superfamily proteins, allograft inflammatory factor-1 (AIF-1) and swiprosin-1/EF-hand domain-containing protein 2 (EFhd2) are cytosolic actin-binding proteins. AIF-1 modulates the cytoskeleton and increases the migration of immune cells. EFhd2 is also a cytoskeletal protein implicated in immune cell activation and brain cell functions. EFhd1, a mitochondrial fraternal twin of EFhd2, mediates neuronal and pro-/pre-B cell differentiation and mitoflash activation. Although EFhd1 is important for maintaining mitochondrial morphology and energy synthesis, its mechanism of action remains unclear. Here, we report the crystal structure of the EFhd1 core domain comprising a C-terminus of a proline-rich region, two EF-hand domains, and a ligand mimic helix. Structural comparisons of EFhd1, EFhd2, and AIF-1 revealed similarities in their overall structures. In the structure of the EFhd1 core domain, two Zn2+ ions were observed at the interface of the crystal contact, suggesting the possibility of Zn2+-mediated multimerization. In addition, we found that EFhd1 has Ca2+-independent ß-actin-binding and Ca2+-dependent ß-actin-bundling activities. These findings suggest that EFhd1, an actin-binding and -bundling protein in the mitochondria, may contribute to the Ca2+-dependent regulation of mitochondrial morphology and energy synthesis.

Aquaporin 5 expression correlates with tumor multiplicity and vascular invasion in hepatocellular carcinoma.

Aquaporins (AQPs) are a family of water channel transmembrane proteins that play a key role in transcellular water movement and transport. Recent studies have reported that AQPs are involved in cancer biology and can be a novel biomarker for predicting prognosis. The aim of this study was to identify clinical significance and prognostic impact of AQP5 in surgically resected hepatocellular carcinomas (HCCs). We analyzed the association between the expression of AQP5, Ki-67, and E-cadherin. Immunohistochemical stains for AQP5, KI-67, and E-cadherin were performed on 72 surgically resected HCCs. As a result, 46 patients (63.9%) showed AQP5 expression, 46 patients (63.9%) revealed high expression of Ki-67, and E-cadherin loss was identified in 8 patients (11.1%). No significant relationship among the three markers was found (all P > 0.05). AQP5 expression was associated with tumor multiplicity (P = 0.039), microvascular invasion (P = 0.040), and major vessel invasion (P = 0.044). High expression of Ki-67 was related to high serum AFP level (P = 0.006), tumor grade (P = 0.002), and microvascular invasion (P = 0.040). AQP5 expression tended to be associated with worse overall survival (OS) (P = 0.093) in the univariate analysis, but no significance was found in the multivariate survival analysis. High expression of Ki-67 was associated with shorter recurrence-free survival (RFS) in both univariate (P = 0.012) and multivariate analysis (P = 0.020). In conclusion, AQP5 might be a prognostic marker in HCC based on its association with tumor multiplicity, microvascular invasion, and major vessel invasion; and Ki-67 is an independent prognostic factor in HCC.

Can Quantitative Volumetric Analysis Predict Tumor Recurrence in the Patients with Mucinous Adenocarcinoma of the Lung After Surgical Resection?

RATIONALE AND OBJECTIVES: Mucinous adenocarcinoma (MAC) is a distinct histologic variant subtype of lung adenocarcinomas. However, detailed radiologic findings and prognostic factors are still poorly understood. Thus, this study aimed to investigate the prognostic value of quantitative volumetric analysis of the computed tomography images of patients with MAC after. surgical resection. MATERIALS AND METHODS: Semiautomatic segmentation from computed tomography images of 60 patients with pathologically confirmed MAC was performed and retrospectively reviewed. The main cutoff value in Hounsfield Units (HU) to predict tumor recurrence was defined by receiver-operating curve analysis. Solid volume of mass (SVM) was defined as the volume of HU greater than this cutoff, and solid ratio (Sratio) was defined as SVM divided by total volume. Each parameter was compared to clinicopathologic characteristics and maximum standardized uptake value. Disease-free survival (DFS) was assessed and was compared among patients. Univariate and multivariate Cox regression was performed to predict DFS of MAC. RESULTS: The cutoff value of HU as determined by ROC analysis was 20 HU. SVM and Sratio were positively correlated with the maximum standardized uptake and pathologic invasion size, respectively (p < 0.001). SVM and Sratio were significantly higher in the recurrence group than in the no-recurrence group (p < 0.001). Multivariate Cox proportional hazards regression analysis revealed that the SVM (Hazard Ratio 1.016; 95% Confidence Interval 1.000-1.032; p = 0.048) and Sratio (Hazard Ratio 29.136; 95% Confidence Interval 1.419-598.191; p = 0.029) were independent significant predictors of DFS. CONCLUSION: Quantitative volumetric parameters can predict the prognosis of patients with MAC after surgical resection.

Immunohistochemical Comparison of IL-36 and the IL-23/Th17 Axis of Generalized Pustular Psoriasis and Acute Generalized Exanthematous Pustulosis.

BACKGROUND: Cutaneous pustular disorders include generalized pustular psoriasis (GPP) and acute generalized exanthematous pustulosis (AGEP). OBJECTIVE: To identify differences between GPP and AGEP, here we immunohistochemically evaluated interleukin (IL)-36 and the IL-23/Th17 axis. METHODS: This retrospective comparative immunohistochemical study was completed using 11 biopsies of 11 cases of GPP and 11 biopsies of 11 cases of AGEP. Through staining with the anti-IL-36-alpha (IL-36α), anti-IL-36 receptor antagonist (IL-36Ra), anti-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), anti-IL-23, anti-IL-17, and anti-IL-8 antibodies, main expression location and intensity were visualized in the epidermis and dermis. RESULTS: In both diseases, diffuse IL-36α expression was observed in the epidermis. IL-36Ra expression was observed in the dermal perivascular area as well as in the epidermis. NF-κB expression was observed in the epidermis and perivascular dermal area. Diffuse IL-23 and IL-17 expression was seen in the whole epidermis and the perivascular dermal area. IL-8 was expressed in the subcorneal pustules and parakeratotic area. Contrary to other cytokines, IL-23 expression in the epidermis of patients with GPP was more intense than only that in patients with AGEP. CONCLUSION: Common pathomechanisms might exist in the development of GPP and AGEP based on these immunohistochemical results, but further studies are needed.

The Transition from Thin Basement Membranes to Typical Alport Syndrome Morphology in Children.

Some children with thin basement membranes (TBM) turn out to have Alport syndrome (AS). In our population of 58 children initially diagnosed with TBM, three were eventually diagnosed with AS. As a group, these three were first biopsied at a younger age, and had gross rather than microscopic hematuria. Only one had lamellations initially. Seven others had some degree of basement membrane lamellations at initial biopsy, but none of these have developed other features of AS. We concluded that at least 5% of children initially demonstrating TBM go on to manifest the classical electron-microscopic findings of AS during childhood. Episodes of gross hematuria with TBM can be a significant clue of AS. Genetic and/or immunofluorescent studies for type IV collagen, and continued long-term follow-up should be done in all children with TBM.

Enhanced delivery of liposomes to lung tumor through targeting interleukin-4 receptor on both tumor cells and tumor endothelial cells.

A growing body of evidence suggests that pathological lesions express tissue-specific molecular targets or biomarkers within the tissue. Interleukin-4 receptor (IL-4R) is overexpressed in many types of cancer cells, including lung cancer. Here we investigated the properties of IL-4R-binding peptide-1 (IL4RPep-1), a CRKRLDRNC peptide, and its ability to target the delivery of liposomes to lung tumor. IL4RPep-1 preferentially bound to H226 lung tumor cells which express higher levers of IL-4R compared to H460 lung tumor cells which express less IL-4R. Mutational analysis revealed that C1, R2, and R4 residues of IL4RPep-1 were the key binding determinants. IL4RPep-1-labeled liposomes containing doxorubicin were more efficiently internalized in H226 cells and effectively delivered doxorubicin into the cells compared to unlabeled liposomes. In vivo fluorescence imaging of nude mice subcutaneously xenotransplanted with H226 tumor cells indicated that IL4RPep-1-labeled liposomes accumulate more efficiently in the tumor and inhibit tumor growth more effectively compared to unlabeled liposomes. Interestingly, expression of IL-4R was high in vascular endothelial cells of tumor, while little was detected in vascular endothelial cells of control organs including the liver. IL-4R expression in cultured human vascular endothelial cells was also up-regulated when activated by a pro-inflammatory cytokine tumor necrosis factor-α. Moreover, the up-regulation of IL-4R expression was observed in primary human lung cancer tissues. These results indicate that IL-4R-targeting nanocarriers may be a useful strategy to enhance drug delivery through the recognition of IL-4R in both tumor cells and tumor endothelial cells.