Purification and characterization of putative endothelin converting enzyme in bovine adrenal medulla: evidence for a cathepsin D-like enzyme

A specific and sensitive assay has been established for measurement of endothelin converting activity in a tissue extract. This assay is based on measuring endothelin-1 generated from big endothelin-1 by endothelin converting enzyme (ECE) with radioimmunoassay using an endothelin C-terminal specific antibody. By using this assay, we purified and characterized ECE in bovine adrenomedullary chromaffin granules ECE was purified over 3,000 times by a combination of DEAE, hydrophobic and gel filtration chromatography. A molecular weight of ECE was estimated to be approximately 30,000 by gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that ECE had three major components with estimated molecular weights of 45,000, 30,000 and 15,000 like bovine spleen cathepsin D. ECE had a pH optimum at 3.5 and was inhibited by pepstatin. These results strongly suggest that ECE is a cathepsin D-like aspartic protease.     

Partial purification of phosphoramidon-sensitive endothelin converting enzyme in porcine aortic endothelial cells: high affinity for Ricinus communis agglutinin.

A membrane-bound endothelin converting enzyme (ECE) of porcine aortic endothelial cells (ECs) was solubilized with Lubrol PX with high efficiency and stability. The solubilized ECE was bound to Ricinus communis agglutinin (RCA) but not to peanut agglutinin (PNA) or wheat germ agglutinin (WGA), suggesting that the ECE has a galactosylated structure possessing a high affinity for RCA. The sequential chromatography on RCA-agarose, PNA-agarose and a TSKgel DEAE-5PW column attained 2,100-fold purification for the ECE over the membrane fractions. The purified ECE was sensitively inhibited by phosphoramidon but not by thiorphan. The present RCA and PNA affinity column procedures may be a powerful approach to isolation of ECE of EC origin.     

Upregulation of miR-199a-5p Protects Spinal Cord Against Ischemia/Reperfusion-Induced Injury via Downregulation of ECE1 in Rat

Ischemia–reperfusion (I/R)-induced spinal cord injury can cause apoptotic damage and subsequently act as a blood–spinal cord barrier damage. MicroRNAs (miRNAs) contributed to the process of I/R injury by regulating their target mRNAs. miR-199a-5p is involved in brain and heart I/R injury; however, its function in the spinal cord is not yet completely clarified. In this study, we investigated the role of miR-199a-5p on spinal cord I/R via the endothelin-converting enzyme 1, especially the apoptosis pathway. In the current study, the rat spinal cord I/R injury model was established, and the Basso Beattie Bresnahan scoring, Evans blue staining, HE staining, and TUNEL assay were used to assess the I/R-induced spinal cord injury. The differentially expressed miRNAs were screened using microarray. miR-199a-5p was selected by unsupervised hierarchical clustering analysis. The dual-luciferase reporter assay was used for detecting the regulatory effects of miR-199a-5p on ECE1. In addition, neuron expression was detected by immunostaining assay, while the expressions of p-ERK, ERK, p-JNK, JNK, caspase-9, Bcl-2, and ECE1 were evaluated by Western blot. The results indicated the successful establishment of the I/R-induced spinal cord injury model; the I/R induced the damage to the lower limb motor. Furthermore, 18 differentially expressed miRNAs were detected in the I/R group compared to the sham group, and miR-199a-5p protected the rat spinal cord injury after I/R. Moreover, miR-199a-5p negatively regulated ECE1, and silencing the ECE1 gene also protected the rat spinal cord injury after I/R. miR-199a-5p or silencing of ECE1 also regulated the expressions of caspase-9, Bcl-2, p-JNK, p-ERK, and ECE1 in rat spinal cord injury after I/R. Therefore, we demonstrated that miR-199a-5p might protect the spinal cord against I/R-induced injury by negatively regulating the ECE1, which could aid in developing new therapeutic strategies for I/R-induced spinal cord injury.     

Lymph flow in the thoracic duct of conscious dogs during lymphatic pump treatment, exercise, and expansion of extracellular fluid volume

BACKGROUND: This investigation examined interactions between expansion of the extracellular fluid volume (ECE), osteopathic lymphatic pump treatment (LPT), and exercise on lymph flow in the thoracic duct of eight instrumented, conscious dogs. METHODS AND RESULTS: After recovery from surgery, LPT was performed for 8 min before and after ECE with normal saline, i.v., 4.4+/-0.3% of body weight. Baseline lymph flow was 1.7+/-0.5 mL/min. LPT rapidly increased lymph flow to 5.0+/-1.1 mL/min at 1 min, and lymph flow remained above baseline for 4 min (p<0.05). LPT produced a net increase in lymph flow of 15.4+/-1.1 mL. Following ECE, baseline lymph flow was 4.8+/-0.6 mL/min (p<0.05). LPT increased lymph flow to 9.9+/-1.1 mL/min at 1 min (p<0.05), and lymph flow remained above baseline for 4 min (p<0.05); all flow values after ECE were greater than corresponding values before ECE. However, the net increase in lymph flow produced by 8 min of LPT (18.3+/-3.8 mL) was not significantly greater than that observed before ECE. Moderate treadmill exercise increased lymph flow for 4 min before ECE and for 6 min after ECE. All lymph flows during exercise were greater after ECE than before ECE. The net increase in lymph flow produced by 8 min of exercise was 24.9+/-5.5 mL before ECE and 39.6+/-5.1 mL after ECE (p<0.05). CONCLUSIONS: Expansion of the extracellular fluid volume produced large increases in thoracic duct lymph flow, that were further augmented by lymphatic pump treatment and by moderate treadmill exercise.     

Applicability of green fluorescence protein in the study of endothelin converting enzyme‐1c trafficking

Endothelin‐1 (ET‐1) is one of the most potent peptide vasoconstrictors known. It is produced upon the cleavage of its precursor big endothelin‐1 by endothelin converting enzyme‐1 (ECE‐1). Production of ET‐1 is thought to be dependent upon the expression of ECE‐1 at the cell surface. Therefore, mechanisms inducing the trafficking of ECE‐1 to the cell surface have been the focus of recent research. This research has identified phosphorylation of the cytoplasmic region of ECE‐1 as a main cellular signal inducing its trafficking to the cell surface. Previous studies have used green fluorescent protein (GFP) tagged ECE‐1 to monitor phosphorylation induced trafficking of ECE‐1 to the cell surface. However, it has been speculated that the addition of the GFP tag can itself alter enzyme activity and phosphorylation of ECE‐1, and hence the suitability of GFP or any other protein tag in studying ECE‐1 distribution and trafficking. ECE‐1c is the most widely expressed isoform in endothelial cells. We therefore expressed ECE‐1c with a GFP tag either at the N or C‐terminus of ECE‐1c. Catalytic activity and effect on protein kinase C (PKC) induced phosphorylation was compared between the two chimeras and wild‐type ECE‐1c. Our results indicate that positioning of the GFP tag on the C‐terminus abrogates activity without effecting PKC‐induced phosphorylation. However, GFP tag on the N‐terminus has the opposite effect. Results of this study shed light on the applicability of GFP or perhaps other protein tags in studying ECE‐1c distribution and trafficking.     

Hemodynamic-dependent patterning of endothelin converting enzyme 1 expression and differentiation of impulse-conducting Purkinje fibers in the embryonic heart

Impulse-conducting Purkinje fibers differentiate from myocytes during embryogenesis. The conversion of contractile myocytes into conduction cells is induced by the stretch/pressure-induced factor, endothelin (ET). Active ET is produced via proteolytic processing from its precursor by ET-converting enzyme 1 (ECE1) and triggers signaling by binding to its receptors. In the embryonic chick heart, ET receptors are expressed by all myocytes, but ECE1 is predominantly expressed in endothelial cells of coronary arteries and endocardium along which Purkinje fiber recruitment from myocytes takes place. Furthermore, co-expression of exogenous ECE1 and ET-precursor in the embryonic heart is sufficient to ectopically convert cardiomyocytes into Purkinje fibers. Thus, localized expression of ECE1 defines the site of Purkinje fiber recruitment in embryonic myocardium. However, it is not known how ECE1 expression is regulated in the embryonic heart. The unique expression pattern of ECE1 in the embryonic heart suggests that blood flow-induced stress/stretch may play a role in patterning ECE1 expression and subsequent induction of Purkinje fiber differentiation. We show that gadolinium, an antagonist for stretch-activated cation channels, downregulates the expression of ECE1 and a conduction cell marker, Cx40, in ventricular chambers, concurrently with delayed maturation of a ventricular conduction pathway. Conversely, pressure-overload in the ventricle by conotruncal banding results in a significant expansion of endocardial ECE1 expression and Cx40-positive putative Purkinje fibers. Coincident with this, an excitation pattern typical of the mature heart is precociously established. These in vivo data suggest that biomechanical forces acting on, and created by, the cardiovascular system during embryogenesis play a crucial role in Purkinje fiber induction and patterning.     

Opioid Receptor Function Is Regulated by Post-endocytic Peptide Processing

Most neuroendocrine peptides are generated in the secretory compartment by proteolysis of the precursors at classical cleavage sites consisting of basic residues by well studied endopeptidases belonging to the subtilisin superfamily. In contrast, a subset of bioactive peptides is generated by processing at non-classical cleavage sites that do not contain basic residues. Neither the peptidases responsible for non-classical cleavages nor the compartment involved in such processing has been well established. Members of the endothelin-converting enzyme (ECE) family are considered good candidate enzymes because they exhibit functional properties that are consistent with such a role. In this study we have explored a role for ECE2 in endocytic processing of δ opioid peptides and its effect on modulating δ opioid receptor function by using selective inhibitors of ECE2 that we had identified previously by homology modeling and virtual screening of a library of small molecules. We found that agonist treatment led to intracellular co-localization of ECE2 with δ opioid receptors. Furthermore, selective inhibitors of ECE2 and reagents that increase the pH of the acidic compartment impaired receptor recycling by protecting the endocytosed peptide from degradation. This, in turn, led to a substantial decrease in surface receptor signaling. Finally, we showed that treatment of primary neurons with the ECE2 inhibitor during recycling led to increased intracellular co-localization of the receptors and ECE2, which in turn led to decreased receptor recycling and signaling by the surface receptors. Together, these results support a role for differential modulation of opioid receptor signaling by post-endocytic processing of peptide agonists by ECE2.     

A potent feed preservative candidate produced by Calcarisporium sp., an endophyte residing in stargrass (Cynodon dactylon)

AIMS: The cultures of an endophytic fungus Calcarisporium sp. were screened for inhibitors on the growth of feed-associated moulds and on the aflatoxin biosynthesis to find a safe and effective feed preservative. METHODS AND RESULTS: Eight test fungi were isolated from the spoiled poultry feed. The endophytic fungus Calcarisporium sp. was separated from the Chinese coastal grass Cynodon dactylon. The antifungal action concerning the endophytic culture extract (ECE) was performed with propionic acid (PPA) as the corresponding reference. The ECE had a similar antifungal efficacy to PPA in a concentration-dependent manner. The susceptibility order of the ECE to the test fungi was found to be Fusarium sp. > Aspergillus spp. > Penicillium spp. Furthermore, the application of the ECE in pelleted-layer duck feed as a preservative was carried out at a humidity of 10, 15 and 20%. It has been discerned that mould growth and aflatoxin biosynthesis could be co-inhibited almost completely by ECE at concentrations higher than 1.0% (w/w). The LD50 of the ECE on mice was shown to be higher than 28 g kg-1. CONCLUSIONS: The ECE can be selected as an inhibitor to preserve poultry feed on inhibiting the growth of mould and aflatoxin biosynthesis during feed storage. SIGNIFICANCE AND IMPACT OF THE STUDY: The ECE may be an effective and biosafe antifungal ingredient for poultry feed and holds a potential market prospect in feed industry.     

The heterogeneity of fibrosis and angiogenesis in endometriosis revealed by single-cell RNA-sequencing

Fibrosis, angiogenesis and chronic inflammation are the inherent characteristics of endometriosis (EMS). The cellular heterogeneity of ectopic and non-ectopic endometrium by single-cell RNA-sequencing (scRNA-seq)at secretory phase without the disturbance of hormone drugs hasn't been explored so far. In this study, scRNA-seq was adopted to explore the properties of ectopic endometrium (ECE), eutopic endometrium (EUE) and normal endometrium (NOE) at secretory phase. We found that (i) The proportion of myofibroblasts, pericytes, endothelial cells and macrophages in ECE overwhelms that of non-ectopic tissues (EUE and NOE), and Myofibro.C2 was the predominant myofibroblast sub-cluster in ECE. (ii) Myofibroblasts were mainly fibroblast-to-myofibroblast transdifferentiation (FMT) and pericytes were endothelial cell-dependent differentiation in ECE. (iii) Both myofibroblasts and pericytes had a low differentiation potential. (iv) The increased inflammation score, deceased NK cells, T cell exhaustion score and antigen-presenting capacity in ECE confirmed the inflammatory properties and immunodeficiency of ECE. These findings suggested that myofibroblasts, pericytes and macrophages may be the potential targets for anti-fibrotic, anti-angiogenic and anti-inflammatory therapy of EMS.     

Nonpeptidic endothelin-converting enzyme inhibitors and their potential therapeutic applications

Endothelins (ETs) are potent vasoconstrictors, promitogens, and inflammatory mediators. They have been implicated in the pathogenesis of various cardiovascular, renal, pulmonary, and central nervous system diseases. Since the final step of the biosynthesis of ETs is catalyzed by a family of endothelin-converting enzymes (ECEs), inhibitors of these enzymes may represent novel therapeutic agents. Currently, seven isoforms of these metalloproteases have been identified; they all share a significant amino acid sequence identity with neutral endopeptidase 24.11 (NEP), another metalloprotease. Therefore, it is not surprising that the majority of ECE inhibitors also possess potent NEP inhibitory activity. To date, three classes of ECE inhibitors have been synthesized: dual ECE/NEP inhibitors, triple ECE/NEP/ACE inhibitors, and selective ECE inhibitors. Potential clinical applications of these compounds in hypertension, chronic heart failure, restenosis, renal failure, and cerebral vasospasm deduced from studies with relevant animal models are reviewed.     

Endothelin-converting enzymes.

The putative endothelin-converting enzyme (ECE) has been the focus of intense research, both within academia and the pharmaceutical industry. Interest in ECE stems mainly from the hypothesis that development of inhibitors of ECE will provide an effective means of preventing production of endothelin in circumstances where it may play a pathogenic role. Both an aspartic and a metalloprotease have been identified that have characteristics of this putative enzyme. Evidence suggests that the metalloprotease, which is inhibited by phosphoramidon, may be the physiologically relevant converting enzyme. However, it remains to be demonstrated conclusively that any inhibitor of an ECE activity directly alters endogenous endothelin production and/or the pathogenesis of a disease condition in which endothelin is thought to play a primary role.     

Characterization of phosphoramidon-sensitive metalloproteinases with endothelin-converting enzyme activity in porcine lung membrane

Endothelin-1 (ET-1), a 21 amino-acid potent vasoconstrictor peptide, is produced from the biologically inactive intermediate big ET-1 via an endoproteolytic cleavage between Trp-21 and Val-22 by endothelin converting enzyme (ECE). cDNA sequence analysis predicts that the two other members of the endothelin family, ET-2 and ET-3, are also generated from the corresponding intermediates called big ET-2 and big ET-3, respectively. The metalloproteinase inhibitor phosphoramidon inhibited the conversion of big ET-1 into mature ET-1 both in vivo and in cultured endothelial cells, suggesting that ECE may be a neutral metalloproteinase. In this study, we solubilized and partially purified ECE from the membrane fraction of porcine lung. Using gel filtration chromatography, we separated two distinct ECE activities, designated M1 (apparent molecular mass approx. 300 kDa) and M2 (approx. 65 kDa). Optimum pH for the cleavage of big ET-1 by M1 and M2 was 7.0 and 7.5, respectively. M1 efficiently converted human big ET-1(1–38) to ET-1, but not human big ET-2(1–37) or human big ET-3(1–41)-amide. In contrast, M2 converted both big ET-1 and big ET-2, but not big ET-3. M1 was inhibited by phosphoramidon (IC₅₀ approx. 1 μM) but not by thiorphan or bacitracin. In contrast, M2 was inhibited by much lower concentrations of phosphoramidon (IC₅₀ approx. 0.3 nM), as well as by thiorphan and bacitracin. ECE activity in M1 was able to bind to a concanavalin A-agarose column and was eluted by α-methyl-d-glucoside, indicating that the ECE is glycosylated. From these results, M1 and M2 from the porcine lung membrane are similar to the candidate of ECE in endothelial cells and neutral endopeptidase in kidney (EC 3.4.24.11), respectively. Taken in conjunction with the previous finding that neither thiorphan nor bacitracin affected the conversion of endogenously synthesized big ET-1 in cultured endothelial cells, we conclude that physiologically relevant ECE found in the endothelial cells is more similar to M1 than to M2.     

Characterization of IAA Production by the Mangrove Cyanobacterium Phormidium sp. MI405019 and Its Influence on Tobacco Seed Germination and Organogenesis

The production of indole-3-acetic acid (IAA) by a mangrove root-associated cyanobacterium, Phormidium sp. MI405019, was demonstrated in this study. The extracellular extract (ECE) of this cyanobacterial culture filtrate was tested on tobacco seed germination and callus differentiation. In ECE treatment, seed germination was increased by 40 % when compared to that of control. In addition, ECE also induced multiple roots from tobacco callus. Further, the factors such as concentration of l-tryptophan and NaCl (salinity) on IAA production were studied. IAA in the medium was initially increased up to a certain period and subsequently decreased in all salinity ranges tested. The amount of IAA production was decreased after 48 h in the culture grown in media amended with 2 % NaCl and without NaCl. However, the IAA concentration was increased up to 96 h in media containing 4 % NaCl. IAA produced by Phormidium sp. MI405019 was extracted from the culture filtrate and its identity was confirmed by thin-layer chromatography and ultra-fast liquid chromatography. To the best of our knowledge, this is the first report of IAA production by cyanobacteria isolated from a mangrove ecosystem. Based on the results in this study, the utilization of Phormidium sp. MI405019 in the aspects of mangrove growth promotion was discussed.     

Superoxide regulation of endothelin-converting enzyme

Reactive oxygen species (ROS) act as signaling molecules in the cardiovascular system, regulating cellular proliferation and migration. However, an excess of ROS can damage cells and alter endothelial cell function. We hypothesized that endogenous mechanisms protect the vasculature from excess levels of ROS. We now show that superoxide can inhibit endothelin-converting enzyme activity (ECE) and decrease endothelin-1 synthesis. Superoxide inhibits ECE but hydrogen peroxide and nitric oxide do not. Superoxide inhibits ECE by ejecting zinc from the enzyme, and the addition of exogenous zinc restores enzymatic activity. Superoxide may inhibit other zinc metalloproteinases by a similar mechanism and may thus play an important role in regulating the biology of blood vessels.     

The Endothelin System and Endothelin-Converting Enzyme in the Brain: Molecular and Cellular Studies

The biologically active vasoactive peptides, the endothelins (ETs), are generated from inactive intermediates, the big endothelins, by a unique processing event catalysed by the zinc metalloprotease, endothelin converting enzyme (ECE). In this overview we examine the actions of endothelins in the brain, and focus on the structure and cellular locations of ECE. The heterogeneous distribution in the brain of ET-1, ET-2, and ET-3 is discussed in relation to their hemodynamic, mitogenic and proliferative properties as well as their possible roles as neurotransmitters. The cellular and subcellular localization of ECE in neuronal and in glial cells is compared with that of other brain membrane metalloproteases, neutral endopeptidase-24.11 (neprilysin), angiotensin converting enzyme and aminopeptidase N, which all function in neuropeptide processing and metabolism. Unlike these ectoenzymes, ECE exhibits a dual localisation in the cell, being present on the plasma membrane and also, in some instances, being concentrated in a perinuclear region. This differential localization may reflect distinct targeting of different ECE isoforms, ECE-l, ECE-1, and ECE-2.     

印度块菌提取物抗氧化活性的研究

对印度块菌Tuber indicum子实体的提取物,包括55%乙醇粗提物(ECE)、石油醚提取物(PEF)和乙酸乙酯提取物(EAF)的清除DPPH自由基和羟基自由基能力、铁离子鳌合能力以及各提取物的总酚含量等进行了研究和测定,结果显示,3种提取物的清除自由基能力和铁离子鳌合能力具有显著差异(P0.05);ECE对DPPH自由基的清除活性最高,其EC50值为1.61g/L;EAF对羟基自由基及铁离子表现出较强的清除或螯合的能力,其EC50值分别为3.31g/L和0.70g/L;EAF的总酚含量(2.964mg GAE/g提取物)最高,其次是ECE,总酚含量为(2.618mg GAE/g提取物);PEF的清除自由基和铁离子鳌合能力较差,其总酚含量也最低(1.124mg GAE/g提取物);总酚含量与印度块菌提取物清除自由基以及鳌合铁离子的能力密切相关。     

ECE1 polymorphisms may contribute to the susceptibility of sporadic congenital heart disease in a Chinese population

Endothelin-converting enzyme-1 (ECE1) plays a key role in the development of a subset of neural crest lineages such as cardiogenesis. Genetic variants of ECE1 C338A (rs213045) and T839G (rs213046) have been shown to alter ECE1 expression. This observation led us to hypothesize that two polymorphisms might influence the susceptibility of sporadic congenital heart disease (CHD). We conducted a case-control study comprised of 945 CHD cases and 972 non-CHD controls in a Chinese population. We tested our hypothesis by genotyping ECE1 C338A and T839G and assessed their association with the risk of CHD. Compared with the 338 CC and the 839 TT genotypes, the ECE1 338 AA/AC and 839 TG/GG genotypes significantly increased the risk of CHD (adjusted odds ratio [OR]=1.38, 95% confidence interval [CI]=1.14-1.68; and adjusted OR=1.30, 95% CI=1.07-1.58, respectively). A combined analysis was performed that showed that the presence of 2-4 risk alleles (the ECE1 338A and 839G allele) increased the risk of CHD by 2.07-fold compared with 0-1 risk alleles. Furthermore, we found that the association between 2-4 risk alleles and CHD risk was stronger in females (adjusted OR=1.77, 95% CI=1.31-2.40) than males (adjusted OR=1.33, 95% CI=1.03-1.71), and in the phenotypes of Tetralogy of Fallot (adjusted OR=1.84, 95% CI=1.10-3.06) and perimembranous ventricular septal defect (pmVSD) (adjusted OR=1.74, 95% CI=1.35-2.24). Our results suggest that ECE1 polymorphisms may contribute to the susceptibility of sporadic CHD in a Chinese population.     

Cyclic expression of endothelin-converting enzyme-1 mediates the functional regulation of seminiferous tubule contraction.

The potent smooth muscle agonist endothelin-1 (ET-1) is involved in the local control of seminiferous tubule contractility, which results in the forward propulsion of tubular fluid and spermatozoa, through its action on peritubular myoid cells. ET-1, known to be produced in the seminiferous epithelium by Sertoli cells, is derived from the inactive intermediate big endothelin-1 (big ET-1) through a specific cleavage operated by the endothelin-converting enzyme (ECE), a membrane-bound metalloprotease with ectoenzymatic activity. The data presented suggest that the timing of seminiferous tubule contractility is controlled locally by the cyclic interplay between different cell types. We have studied the expression of ECE by Sertoli cells and used myoid cell cultures and seminiferous tubule explants to monitor the biological activity of the enzymatic reaction product. Northern blot analysis showed that ECE-1 (and not ECE-2) is specifically expressed in Sertoli cells; competitive enzyme immunoassay of ET production showed that Sertoli cell monolayers are capable of cleaving big ET-1, an activity inhibited by the ECE inhibitor phosphoramidon. Microfluorimetric analysis of intracellular calcium mobilization in single cells showed that myoid cells do not respond to big endothelin, nor to Sertoli cell plain medium, but to the medium conditioned by Sertoli cells in the presence of big ET-1, resulting in cell contraction and desensitization to further ET-1 stimulation; in situ hybridization analysis shows regional differences in ECE expression, suggesting that pulsatile production of endothelin by Sertoli cells (at specific "stages" of the seminiferous epithelium) may regulate the cyclicity of tubular contraction; when viewed in a scanning electron microscope, segments of seminiferous tubules containing the specific stages characterized by high expression of ECE were observed to contract in response to big ET-1, whereas stages with low ECE expression remained virtually unaffected. These data indicate that endothelin-mediated spatiotemporal control of rhythmic tubular contractility might be operated by Sertoli cells through the cyclic expression of ECE-1, which is, in turn, dependent upon the timing of spermatogenesis.     

Study In Vivo Intraocular Biocompatibility of In Situ Gelation Hydrogels: Poly(2-Ethyl Oxazoline)-Block-Poly(ε-Caprolactone)-Block-Poly(2-Ethyl Oxazoline) Copolymer,Matrigel and Pluronic F127

The long term in vivo biocompatibility is an essential feature for the design and development of sustained drug release carriers. In the recent intraocular drug delivery studies, hydrogels were suggested as sustained release carriers. The biocompatibility test for these hydrogels, however, was commonly performed only through in vitro cell culture examination, which is insufficient before the clinical applications. We compared three thermosensitive hydrogels that have been suggested as the carriers for drugs by their gel-solution phase-change properties. A new block terpolymer (PEOz-PCL-PEOz, ECE) and two commercial products (Matrigel® and Pluronic F127) were studied. The results demonstrated that the ocular media remained translucent for ECE and Pluronic F127 in the first 2 weeks, but cataract formation for Matrigel occurred in 2 weeks and for Pluronic F127 in 1 month, while turbid media was observed for both Matrigel and Pluronic F127 in 2 months. The electrophysiology examinations showed significant neuroretinal toxicity of Matrigel and Pluronic F127 but good biocompatibility of ECE. The neuroretinal toxicity of Matrigel and Pluronic F127 and superior biocompatibility of ECE hydrogel suggests ECE as more appropriate biomaterial for use in research and potentially in intraocular application.