All-stage targeted red blood cell membrane-coated docetaxel nanocrystals for glioma treatment.

Challenges for glioma treatment with nanomedicines include physio-anatomical barriers (the blood-brain barrier and blood-brain tumor barrier), low drug loading capacity, and limited circulation time. Here, a red blood cell membrane-coated docetaxel drug nanocrystal (pV-RBCm-NC(DTX)), modified with pHA-VAP (pV) for all-stage targeting of glioma, was designed. The NC(DTX) core exhibited a high drug loading capacity but low in vivo stability, and the RBCm coating significantly enhanced the stability and prolonged in vivo circulation. Moreover, the Y-shaped targeting ligand pV was modified by a mild avidin-biotin interaction, which endowed RBCm-NC(DTX) with superior barrier-crossing ability and therapeutic efficacy. The integration of nanocrystal technology, cell membrane coating, and the avidin-biotin insertion method into this active targeting biomimetic formulation represents a promising drug delivery strategy for glioma.

All-stage targeted therapy for the brain metastasis from triple-negative breast cancer.

Brain metastasis is a common and serious complication of breast cancer, which is commonly associated with poor survival and prognosis. In particular, the treatment of brain metastasis from triple-negative breast cancer (BM-TNBC) has to face the distinct therapeutic challenges from tumor heterogeneity, circulating tumor cells (CTCs), blood-brain barrier (BBB) and blood-tumor barrier (BTB), which is in unmet clinical needs. Herein, combining with the advantages of synthetic and natural targeting moieties, we develop a "Y-shaped" peptide pVAP-decorated platelet-hybrid liposome drug delivery system to address the all-stage targeted drug delivery for the whole progression of BM-TNBC. Inherited from the activated platelet, the hybrid liposomes still retain the native affinity toward CTCs. Further, the peptide-mediated targeting to breast cancer cells and transport across BBB/BTB are demonstrated in vitro and in vivo. The resultant delivery platform significantly improves the drug accumulation both in orthotopic breast tumors and brain metastatic lesions, and eventually exhibits an outperformance in the inhibition of BM-TNBC compared with the free drug. Overall, this work provides a promising prospect for the comprehensive treatment of BM-TNBC, which could be generalized to other cell types or used in imaging platforms in the future.

Aptamer-Modified Erythrocyte Membrane-Coated pH-Sensitive Nanoparticles for c-Met-Targeted Therapy of Glioblastoma Multiforme.

Biomimetic drug delivery systems, especially red blood cell (RBC) membrane-based nanoparticle drug delivery systems (RNP), have been extensively utilized in tumor drug delivery because of their excellent biocompatibility and prolonged circulation. In this study, we developed an active targeting pH-sensitive RNP loaded with DOX by decorating an aptamer SL1 on RBC membranes (SL1-RNP-DOX) for c-Met-targeted therapy of glioblastoma multiforme (GBM). SL1 could specifically bind to c-Met, which is highly expressed in GBM U87MG cells and facilitate DOX delivery to GBM cells. In vitro studies demonstrated that U87MG cells had a higher uptake of SL1-RNP-DOX (3.25 folds) and a stronger pro-apoptosis effect than unmodified RNP-DOX. In vivo fluorescence imaging and tissue distribution further demonstrated the higher tumor distribution of SL1-RNP-DOX (2.17 folds) compared with RNP-DOX. As a result, SL1-RNP-DOX presented the best anti-GBM effect with a prolonged median survival time (23 days vs. 15.5 days) and the strongest tumor cell apoptosis in vivo among all groups. In conclusion, SL1-RNP-DOX exhibited a promising targeting delivery strategy for GBM therapy.

In vivo self-assembled drug nanocrystals for metastatic breast cancer all-stage targeted therapy.

Chemotherapy is still the mainstay treatment for metastatic triple-negative breast cancers (TNBC) currently in clinical practice. The unmet needs of chemotherapy for metastatic TNBC are mainly from the insufficient drug delivery and unavailable targeting strategy that thwart the whole progression of metastatic TNBC. The in vivo ligands-mediated active targeting efficiency is usually affected by protein corona. While, the protein corona-bridged natural targeting, in turn, provides a new way for specific drug delivery. Herein, we develop a novel metastatic progression-oriented in vivo self-assembled Cabazitaxel nanocrystals (CNC) delivery system (PC/CNC) through the CNC automatically absorbing functional plasma proteins (transferrin, apolipoprotein A-IV and apolipoprotein E) in vivo, aiming to achieve the simultaneously targeted delivery to primary tumors, circulating tumor cells and metastatic lesions. With the unique advantages of superhigh drug-loading and protein corona empowered active targeting properties to tumor cells, HUVECs, active-platelets and blood-brain barrier/blood-tumor barrier, the PC/CNC exhibits a significantly improved therapeutic effect in metastatic TNBC therapy compared with free drug and CNC-loaded liposomes.

Drug Nanocrystals for Active Tumor-Targeted Drug Delivery.

Drug nanocrystals, which are comprised of active pharmaceutical ingredients and only a small amount of essential stabilizers, have the ability to improve the solubility, dissolution and bioavailability of poorly water-soluble drugs; in turn, drug nanocrystal technology can be utilized to develop novel formulations of chemotherapeutic drugs. Compared with passive targeting strategy, active tumor-targeted drug delivery, typically enabled by specific targeting ligands or molecules modified onto the surface of nanomedicines, circumvents the weak and heterogeneous enhanced permeability and retention (EPR) effect in human tumors and overcomes the disadvantages of nonspecific drug distribution, high administration dosage and undesired side effects, thereby contributing to improving the efficacy and safety of conventional nanomedicines for chemotherapy. Continuous efforts have been made in the development of active tumor-targeted drug nanocrystals delivery systems in recent years, most of which are encouraging and also enlightening for further investigation and clinical translation.

All-stage targeted therapy for glioblastoma based on lipid membrane coated cabazitaxel nanocrystals.

Glioblastoma (GBM) is the most aggressive brain tumor with poor prognosis and frequent recurrence. The blood-brain barrier (BBB), blood-brain tumor barrier (BBTB) hinder the entry of therapeutics into the glioma region. Vasculogenic mimicry (VM) formed by invasive glioma cells is also related to recurrence of GBM. VAP is a D-peptide ligand of GRP78 protein overexpressed on BBTB, VM, and glioma cells but not on normal tissues. Besides, p-hydroxybenzoic acid (pHA) can effectively traverse the BBB. Herein we developed an all-stage glioma-targeted cabazitaxel (CBZ) nanocrystal loaded liposome modified with a "Y" shaped targeting ligand composed of pHA and VAP (pV-Lip/cNC). The pure drug nanocrystal core provided high drug loading, while lipid membrane promoted the stability and circulation time. pV-Lip/cNC exhibited excellent glioma homing, barriers crossing, and tumor spheroid penetrating capability in vitro. Treatment of pV-Lip/cNC displayed enhanced CBZ accumulation in glioma and anti-glioma effect with a median survival time (53 days) significantly longer than that of cNC loaded liposomes modified with either single ligand (42 days for VAP and 45 days for pHA) in the murine orthotopic GBM model. These results indicated pV-Lip/cNC could traverse the BBB and BBTB, destruct VM, and finally kill glioma cells to realize all-stage glioma therapy.

Engineered superparamagnetic iron oxide nanoparticles (SPIONs) for dual-modality imaging of intracranial glioblastoma via EGFRvIII targeting.

In this work, a peptide-modified, biodegradable, nontoxic, brain-tumor-targeting nanoprobe based on superparamagnetic iron oxide nanoparticles (SPIONs) (which have been commonly used as T 2-weighted magnetic resonance (MR) contrast agents) was successfully synthesized and applied for accurate molecular MR imaging and sensitive optical imaging. PEPHC1, a short peptide which can specifically bind to epidermal growth factor receptor variant III (EGFRvIII) that is overexpressed in glioblastoma, was conjugated with SPIONs to construct the nanoprobe. Both in vitro and in vivo MR and optical imaging demonstrated that the as-constructed nanoprobe was effective and sensitive for tumor targeting with desirable biosafety. Given its desirable properties such as a 100 nm diameter (capable of penetration of the blood-brain barrier) and bimodal imaging capability, this novel and versatile multimodal nanoprobe could bring a new perspective for elucidating intracranial glioblastoma preoperative diagnosis and the accuracy of tumor resection.

Biomimetic nanoparticles for inflammation targeting.

There have been many recent exciting developments in biomimetic nanoparticles for biomedical applications. Inflammation, a protective response involving immune cells, blood vessels, and molecular mediators directed against harmful stimuli, is closely associated with many human diseases. As a result, biomimetic nanoparticles mimicking immune cells can help achieve molecular imaging and precise drug delivery to these inflammatory sites. This review is focused on inflammation-targeting biomimetic nanoparticles and will provide an in-depth look at the design of these nanoparticles to maximize their benefits for disease diagnosis and treatment.

Cyclopamine treatment disrupts extracellular matrix and alleviates solid stress to improve nanomedicine delivery for pancreatic cancer.

As one of the most intractable tumours, pancreatic ductal adenocarcinoma (PDA) has a dense extracellular matrix (ECM) which could increase solid stress within tumours to compress tumour vessels, reduce tumour perfusion and compromise nanomedicine delivery for PDA. Thus, alleviating solid stress represents a potential therapeutic target for PDA treatment. In this study, cyclopamine, a special inhibitor of the hedgehog signalling pathway which contributes a lot to ECM formation of PDA, was exploited to alleviate solid stress and improve nanomedicine delivery to PDA. Results demonstrated that cyclopamine successfully disrupted ECM and lowered solid stress within PDA, which increased functional tumour vessels and resulted in enhanced tumour perfusion as well as improved tumour nanomedicine delivery in PDA-bearing animal models. Therefore, solid stress within PDA represents a new therapeutic target for PDA treatment.

Biomimetic nanoparticles delivered hedgehog pathway inhibitor to modify tumour microenvironment and improved chemotherapy for pancreatic carcinoma.

The unique tumour microenvironment (TM) of pancreatic ductal adenocarcinoma (PDA) including highly desmoplastic ECM and low tumour perfusion supports a considerable barrier for effective delivery of nanomedicines. Effectively modulating PDA microenvironment to enhance tumour drug delivery represents a pinpoint in the field of PDA treatment. In this study, it was the first time that biomimetic nanoparticles, which were designed in the form of erythrocyte membrane-camouflaged PLGA nanoparticles (MNP), were utilized for PDA microenvironment modulation. Cyclopamine (CYC), an inhibitor of Hedgehog pathway that contributed a lot to desmoplastic ECM of PDA, was selected as the model drug and successfully encapsulated into MNP. Advantages of CYC-loaded MNP (CMNP) included favourable biocompatibility, long circulation time, and powerful TM modulation effect. CMNP could effectively deliver CYC to the tumour site, disrupt tumour ECM, increase functional vessels, and improve tumour perfusion significantly. The combination treatment with CMNP and PTX-loaded MNP (PMNP) successfully improved PTX delivery to tumour, resulting in remarkable tumour growth inhibition in vivo. Therefore, biomimetic nanoparticles provide a new strategy for modulating PDA TM and will have great potential to improve the therapeutic effects of nanomedicines for PDA patients.

BQ123 selectively improved tumor perfusion and enhanced nanomedicine delivery for glioblastomas treatment.

Blood perfusion was always lower in tumor tissues as compared with that in surrounding normal tissues which lead to inadequate nanomedicine delivery to tumors. Inspired by the upregulation of both endothelin-1 (ET1) and its ETA receptor in tumor tissues and the crucial contribution of ET1-ETA receptor signaling to maintain myogenic tone of tumor vessels, we supposed that inhibition of ET1-ETA receptor signaling might selectively improve tumor perfusion and help deliver nanomedicine to tumors. Using human U87 MG glioblastomas with abundant vessels as the tumor model, immunofluorescence staining demonstrated that ETA receptor was overexpressed by in glioblastomas tissues compared with normal brain tissues. A single administration of ETA receptor antagonist BQ123 at the dose of 0.5 mg/kg could effectively improve tumor perfusion which was evidenced by in vivo photoacoustic imaging. Additionally, a single treatment of BQ123 could significantly improve the accumulation of nanoparticles (NPs) around 115 nm in tumors with a more homogeneous distribution pattern by in vivo imaging, ex vivo imaging as well as in vivo distribution experiments. Furthermore, BQ123 successfully increased the therapeutic benefits of paclitaxel-loaded NPs and significantly elongated the survival time of orthotropic glioblastomas-bearing animal models. In summary, the present study provided a new strategy to selectively improve tumor perfusion and therefore benefit nanomedicine delivery for tumor therapy. As ET1-ETA receptor signaling was upregulated in a variety of tumors, this strategy might open a new avenue for tumor treatment.

Celecoxib normalizes the tumor microenvironment and enhances small nanotherapeutics delivery to A549 tumors in nude mice.

Barriers presented by the tumor microenvironment including the abnormal tumor vasculature and interstitial matrix invariably lead to heterogeneous distribution of nanotherapeutics. Inspired by the close association between cyclooxygenase-2 (COX-2) and tumor-associated angiogenesis, as well as tumor matrix formation, we proposed that tumor microenvironment normalization by COX-2 inhibitors might improve the distribution and efficacy of nanotherapeutics for solid tumors. The present study represents the first time that celecoxib, a special COX-2 inhibitor widely used in clinics, was explored to normalize the tumor microenvironment and to improve tumor nanotherapeutics delivery using a human-derived A549 tumor xenograft as the solid tumor model. Immunofluorescence staining of tumor slices demonstrated that oral celecoxib treatment at a dose of 200 mg/kg for two weeks successfully normalized the tumor microenvironment, including tumor-associated fibroblast reduction, fibronectin bundle disruption, tumor vessel normalization, and tumor perfusion improvement. Furthermore, it also significantly enhanced the in vivo accumulation and deep penetration of 22-nm micelles rather than 100-nm nanoparticles in tumor tissues by in vivo imaging and distribution experiments and improved the therapeutic efficacy of paclitaxel-loaded micelles in tumor xenograft-bearing mouse models in the pharmacodynamics experiment. As celecoxib is widely and safely used in clinics, our findings may have great potential in clinics to improve solid tumor treatment.

Captopril improves tumor nanomedicine delivery by increasing tumor blood perfusion and enlarging endothelial gaps in tumor blood vessels.

Poor tumor perfusion and unfavorable vessel permeability compromise nanomedicine drug delivery to tumors. Captopril dilates blood vessels, reducing blood pressure clinically and bradykinin, as the downstream signaling moiety of captopril, is capable of dilating blood vessels and effectively increasing vessel permeability. The hypothesis behind this study was that captopril can dilate tumor blood vessels, improving tumor perfusion and simultaneously enlarge the endothelial gaps of tumor vessels, therefore enhancing nanomedicine drug delivery for tumor therapy. Using the U87 tumor xenograft with abundant blood vessels as the tumor model, tumor perfusion experiments were carried out using laser Doppler imaging and lectin-labeling experiments. A single treatment of captopril at a dose of 100 mg/kg significantly increased the percentage of functional vessels in tumor tissues and improved tumor blood perfusion. Scanning electron microscopy of tumor vessels also indicated that the endothelial gaps of tumor vessels were enlarged after captopril treatment. Immunofluorescence-staining of tumor slices demonstrated that captopril significantly increased bradykinin expression, possibly explaining tumor perfusion improvements and endothelial gap enlargement. Additionally, imaging in vivo, imaging ex vivo and nanoparticle distribution in tumor slices indicated that after a single treatment with captopril, the accumulation of 115-nm nanoparticles in tumors had increased 2.81-fold with a more homogeneous distribution pattern in comparison to non-captopril treated controls. Finally, pharmacodynamics experiments demonstrated that captopril combined with paclitaxel-loaded nanoparticles resulted in the greatest tumor shrinkage and the most extensive necrosis in tumor tissues among all treatment groups. Taken together, the data from the present study suggest a novel strategy for improving tumor perfusion and enlarging blood vessel permeability simultaneously in order to improve nanomedicine delivery for tumor therapy. As captopril has already been extensively used clinically, such a strategy has great therapeutic potential.

On-Demand Drug Release from Dual-Targeting Small Nanoparticles Triggered by High-Intensity Focused Ultrasound Enhanced Glioblastoma-Targeting Therapy.

Glioblastoma is one of the most challenging and intractable tumors with the difficult treatment and poor prognosis. Unsatisfactory traditional systemic chemotherapies for glioblastoma are mainly attributed to the insufficient and nonspecific drug delivery into the brain tumors as well as the incomplete drug release at the tumor sites. Inspired by the facts that angiopep-2 peptide is an acknowledged dual-targeting moiety for brain tumor-targeting delivery and high-intensity focused ultrasound (HIFU) is an ideal trigger for drug release with an ultrahigh energy and millimeter-sized focus ability, in the present study, a novel HIFU-responsive angiopep-2-modified small poly(lactic-co-glycolic acid) (PLGA) hybrid nanoparticle (NP) drug delivery system holding doxorubicin/perfluorooctyl bromide (ANP-D/P) was designed to increase the intratumoral drug accumulation, further trigger on-demand drug release at the glioblastoma sites, and enhance glioblastoma therapy. It was shown that the ANP-D/P was stable and had a small size of 41 nm. The angiopep-2 modification endowed the ANP-D/P with improved blood-brain barrier transportation and specific accumulation in glioblastoma tissues by 17 folds and 13.4 folds compared with unmodified NPs, respectively. Under HIFU irradiation, the ANP-D/P could release 47% of the drug within 2 min and induce the apoptosis of most tumor cells. HIFU-triggered instantaneous drug release at the glioblastoma sites eventually enabled the ANP-D/P to achieve the strongest antiglioblastoma efficacy with the longest median survival time (56 days) of glioblastoma-bearing mice and the minimum vestiges of tumor cells in the pathological slices among all groups. In conclusion, the HIFU-responsive ANP-D/P in this study provided a new way for glioblastoma therapy with a great potential for clinical applications.

Tumor Microenvironment Modulation by Cyclopamine Improved Photothermal Therapy of Biomimetic Gold Nanorods for Pancreatic Ductal Adenocarcinomas.

Due to the rich stroma content and poor blood perfusion, pancreatic ductal adenocarcinoma (PDA) is a tough cancer that can hardly be effectively treated by chemotherapeutic drugs. Tumor microenvironment modulation or advanced design of nanomedicine to achieve better therapeutic benefits for PDA treatment was widely advocated by many reviews. In the present study, a new photothermal therapy strategy of PDA was developed by combination of tumor microenvironment modulation and advanced design of biomimetic gold nanorods. On one hand, biomimetic gold nanorods were developed by coating gold nanorods (GNRs) with erythrocyte membrane (MGNRs). It was shown that MGNRs exhibited significantly higher colloidal stability in vitro, stronger photothermal therapeutic efficacy in vitro, and longer circulation in vivo than GNRs. On the other hand, tumor microenvironment modulation by cyclopamine treatment successfully disrupted the extracellular matrix of PDA and improved tumor blood perfusion. Moreover, cyclopamine treatment significantly increased the accumulation of MGNRs in tumors by 1.8-fold and therefore produced higher photothermal efficiency in vivo than the control group. Finally, cyclopamine treatment combined with photothermal MGNRs achieved the most significant shrinkage of Capan-2 tumor xenografts among all the treatment groups. Therefore, with the integrated advantages of tumor microenvironment regulation and long-circulation biomimetic MGNRs, effective photothermal therapy of PDA was achieved. In general, this new strategy of combining tumor microenvironment modulation and advanced design of biomimetic nanoparticles might have great potential in PDA therapy.

Enhanced photothermal therapy of biomimetic polypyrrole nanoparticles through improving blood flow perfusion.

In this study, we reported a strategy to improve delivery efficiency of a long-circulation biomimetic photothermal nanoagent for enhanced photothermal therapy through selectively dilating tumor vasculature. By using a simply nanocoating technology, a biomimetic layer of natural red blood cell (RBC) membranes was camouflaged on the surface of photothermal polypyrrole nanoparticles (PPy@RBC NPs). The erythrocyte-mimicking PPy NPs inherited the immune evasion ability from natural RBC resulting in superior prolonged blood retention time. Additionally, excellent photothermal and photoacoustic imaging functionalities were all retained attributing to PPy NPs cores. To further improve the photothermal outcome, the endothelin A (ETA) receptor antagonist BQ123 was jointly employed to regulate tumor microenvironment. The BQ123 could induce tumor vascular relaxation and increase blood flow perfusion through modulating an ET-1/ETA transduction pathway and blocking the ETA receptor, whereas the vessel perfusion of normal tissues was not altered. Through our well-designed tactic, the concentration of biomimetic PPy NPs in tumor site was significantly improved when administered systematically. The study documented that the antitumor efficiency of biomimetic PPy NPs combined with specific antagonist BQ123 was particularly prominent and was superior to biomimetic PPy NPs (P < 0.05) and PEGylated PPy NPs with BQ123 (P < 0.01), showing that the greatly enhanced photothermal treatment could be achieved with low-dose administration of photothermal agents. Our findings would provide a promising procedure for other similar enhanced photothermal treatment by blocking ETA receptor to dramatically increase the delivery of biomimetic photothermal nanomaterials.

Red blood cell membrane-camouflaged melanin nanoparticles for enhanced photothermal therapy.

Photothermal therapy (PTT) has represented a promising noninvasive approach for cancer treatment in recent years. However, there still remain challenges in developing non-toxic and biodegradable biomaterials with high photothermal efficiency in vivo. Herein, we explored natural melanin nanoparticles extracted from living cuttlefish as effective photothermal agents and developed red blood cell (RBC) membrane-camouflaged melanin (Melanin@RBC) nanoparticles as a platform for in vivo antitumor PTT. The as-obtained natural melanin nanoparticles demonstrated strong absorption at NIR region, higher photothermal conversion efficiency (∼40%) than synthesized melanin-like polydopamine nanoparticles (∼29%), as well as favorable biocompatibility and biodegradability. It was shown that RBC membrane coating on melanin nanoparticles retained their excellent photothermal property, enhanced their blood retention and effectively improved their accumulation at tumor sites. With the guidance of their inherited photoacoustic imaging capability, optimal accumulation of Melanin@RBC at tumors was achieved around 4 h post intravenous injection. Upon irradiation by an 808-nm laser, the developed Melanin@RBC nanoparticles exhibited significantly higher PTT efficacy than that of bare melanin nanoparticles in A549 tumor-bearing mice. Given that both melanin nanoparticles and RBC membrane are native biomaterials, the developed Melanin@RBC platform could have great potential in clinics for anticancer PTT.

Precise glioblastoma targeting by AS1411 aptamer-functionalized poly (l-γ-glutamylglutamine)-paclitaxel nanoconjugates.

Chemotherapy is still the main adjuvant strategy after surgery in glioblastoma therapy. As the main obstacles of chemotherapeutic drugs for glioblastoma treatment, the blood brain barrier (BBB) and non-specific delivery to non-tumor tissues greatly limit the accumulation of drugs into tumor tissues and simultaneously cause serious toxicity to nearby normal tissues which altogether compromised the chemotherapeutic effect. In the present study, we established an aptamer AS1411-functionalized poly (l-γ-glutamyl-glutamine)-paclitaxel (PGG-PTX) nanoconjugates drug delivery system (AS1411-PGG-PTX), providing an advantageous solution of combining the precisely active targeting and the optimized solubilization of paclitaxel. The receptor nucleolin, highly expressed in glioblastoma U87 MG cells as well as neo-vascular endothelial cells, mediated the binding and endocytosis of AS1411-PGG-PTX nanoconjugates, leading to significantly enhanced uptake of AS1411-PGG-PTX nanoconjugates by tumor cells and three-dimension tumor spheroids, and intensive pro-apoptosis effect of AS1411-PGG-PTX nanoconjugates. In vivo fluorescence imaging and tissue distribution further demonstrated the higher tumor distribution of AS1411-PGG-PTX as compared with PGG-PTX. As a result, the AS1411-PGG-PTX nanoconjugates presented the best anti-glioblastoma effect with prolonged median survival time and most tumor cell apoptosis in vivo as compared with other groups. In conclusion, the AS1411-PGG-PTX nanoconjugates exhibited a promising targeting delivery strategy for glioblastoma therapy.

Effects of echinomycin on endothelin-2 expression and ovulation in immature rats primed with gonadotropins.

Echinomycin is a small-molecule inhibitor of hypoxia- inducible factor-1 DNA-binding activity, which plays a crucial role in ovarian ovulation in mammalians. The present study was designed to test the hypothesis that hypoxia-inducible factor (HIF)-1α-mediated endothelin (ET)-2 expressions contributed to ovarian ovulation in response to human chorionic gonadotropin (hCG) during gonadotropin-induced superuvulation. By real-time RT-PCR analysis, ET-2 mRNA level was found to significantly decrease in the ovaries after echinomycin treatment, while HIF-1α mRNA and protein expression was not obviously changed. Further analysis also showed that these changes of ET-2 mRNA were consistent with HIF-1 activity in the ovaires, which is similar with HIF-1α and ET-2 expression in the granulosa cells with gonadotropin and echinomycin treatments. The results of HIF-1α and ET-2 expression in the granulosa cells transfected with cis-element oligodeoxynucleotide (dsODN) under gonadotropin treatment further indicated HIF-1α directly mediated the transcriptional activation of ET-2 during gonadotropin- induced superuvulation. Taken together, these results demonstrated that HIF-1α-mediated ET-2 transcriptional activation is one of the important mechanisms regulating gonadotropin-induced mammalian ovulatory precess in vivo.