Expression, purification and functional identification of the modified hEGF protein.

Human epidermal growth factor (hEGF) plays an important role in the growth and division of epithelial cells and has good application prospects in skin-related injuries and diseases. Weak skin penetration and rapid clearance of hEGF in skin via the mononuclear phagocyte system have restricted the application of hEGF. To overcome these shortcomings, the recombinant gene TAT-hEGF-CD47 was constructed in our experiments, and the fusion protein TAT-hEGF-CD47 was expressed, purified and renatured. The cell proliferation-promoting function, skin penetration and concentration of TAT-hEGF-CD47 in skin after its application were determined. The results showed that TAT-hEGF-CD47 effectively promoted human skin fibroblast and skin epithelial cell proliferation, and the proliferation-promoting effect was positively correlated with the TAT-hEGF-CD47 concentration. After administration to the skin, TAT-hEGF-CD47 effectively penetrated the epidermal layer of the skin because of the TAT domain and stayed in the skin for a long time because the CD47 fragment slowed its clearance via the mononuclear phagocytic system. In conclusion, TAT-hEGF-CD47 exhibits high cell proliferation-promoting activity, high skin penetration efficiency and long retention time in skin and has laid the foundation for its wide application in skin repair, ulcer, diabetes and even cancer treatments.

Dermal toxicity, dermal irritation, and delayed contact sensitization evaluation of oil body linked oleosin-hEGF microgel emulsion via transdermal drug delivery for wound healing.

Objective: The expression of therapeutic proteins in plant oil body bioreactors has attracted much attention. But its safety is not yet clear. This article determines the risk of safety after using the drug. Methods: The oil body-linked oleosin-hEGF microgel emulsion (OBEME) was prepared by mixing the xanthan gum with suitable concentrations in an appropriate proportion. Skin irritation and sensitization reaction were investigated in rats and guinea pigs using OBEME as test article.Results: The OBEME did not produce dermal erythema/eschar or oedema responses. The dermal subacute and subchronic toxicity of OBEME were evaluated in accordance with OECD guidelines. Compared with the control group, the basic physical signs, such as weight, feed, drinking, excretion, and behaviour of experimental animals, were not abnormal. In addition, no abnormality was found in haematological parameters, biochemical indexes, relative organ weight, and histopathological observation of organs, and there was no significant difference compared with normal saline treatment group. Therefore, we conclude that OBEME has no toxic effects and is safe and reliable to be used for topical application.

High expression level of human epidermal growth factor (hEGF) using a well-designed fusion protein-tagged construct in E. coli.

AIM: The study was aimed at design a good fusion construct that would successfully express the recombinant proteins and produce peptides in Escherichia coli. Two different constructs including human epidermal growth factor (hEGF) gene were designed to obtain an efficient expression level of hEGF. The hEGF sequence was inserted in pET32a vector containing thioredoxin (Trx) sequence and modified pET15b vector containing intein and elastin-like polypeptide (ELP). METHODS: The vectors were transformed into E. coli TOP10F' for multiplication and further into E. coli BL21 (DE3) to express protein. The hEGF expression was induced by isopropyl ß-D-1-thiogalactopyranoside (IPTG) while the expression levels were evaluated by SDS-PAGE and western blotting and compared by ImageJ analysis, BCA and Elisa assays. RESULTS: The expression level after 2 hours of IPTG induction was significantly higher than after other induction times. ImageJ, BCA and Elisa analyses demonstrated that the Trx presence enhanced protein expression significantly when compared to ELP-intein-based construct. CONCLUSION: The pET32a-Trx-hEGF construct had a higher expression than pET15b-ELP-intein-hEGF. Overall, considering Trx, the fusion protein in construct design can make it suitable to significantly express hEGF compared to ELP-intein while its combination with ELP-intein may improve the expression of the ELP-intein construct (Tab. 2, Fig. 7, Ref. 34).

Towards Radiolabeled EGFR-Specific Peptides: Alternatives to GE11.

The human epidermal growth factor receptor (EGFR) is closely related to several cancer-promoting processes and overexpressed on a variety of tumor types, rendering it an important target structure for the imaging and therapy of several malignancies. To date, approaches to develop peptidic radioligands able to specifically address and visualize EGFR-positive tumors have been of limited success. Most of the attempts were based on the lead GE11, as this peptide was previously described to be a highly potent EGFR-specific agent. However, since it has recently been shown that GE11 exhibits an insufficient affinity to the EGFR in monomeric form to be suitable as a basis for the development of tracers based on it, in the present work we investigated which other peptides might be suitable as lead structures for the development of EGFR-specific peptidic radiotracers. For this purpose, we developed 68Ga-labeled radioligands based on the peptides D4, P1, P2, CPP, QRH, EGBP and Pep11, having been described before as EGFR-specific. In addition, we also tested three truncated versions of the endogenous EGFR ligand hEGF (human epidermal growth factor) with respect to their ability to specifically target the EGFR with high affinity. Therefore, chelator-modified labeling precursors of the mentioned peptides were synthesized, radiolabeled with 68Ga and the obtained radioligands were evaluated for their hydrophilicity/lipophilicity, stability against degradation by human serum peptidases, in vitro tumor cell uptake, and receptor affinity in competitive displacement experiments on EGFR-positive A431 cells. Although all NODA-GA-modified (NODA-GA: (1,4,7-triazacyclononane-4,7-diyl)diacetic acid-1-glutaric acid) labeling precursors could be obtained more or less efficient in yields between 5 and 74%, the 68Ga-radiolabeling proved to be unsuccessful for two of the three truncated versions of hEGF ([68Ga]Ga-8 and [68Ga]Ga-9), producing several side-products. For the other agents [68Ga]Ga-1-[68Ga]Ga-7, [68Ga]Ga-10 and [68Ga]Ga-11, high radiochemical yields and purities of ≥98% and molar activities of up to 114 GBq/µmol were obtained. In the assay investigating the radiopeptide susceptibilities against serum peptidase degradation, the EGBP-based agent demonstrated a limited stability with a half-life of only 66.4 ± 3.0 min, whereas the other tracers showed considerably higher stabilities of up to an 8000 min half-life. Finally, all radiotracer candidates were evaluated in terms of tumor cell internalization and receptor binding potential on EGFR-positive A431 cell. In these experiments, all developed agents failed to show an EGFR-specific tumor cell uptake or a relevant EGFR-affinity. By contrast, the positive controls tested under identical conditions, [125I]I-hEGF and hEGF demonstrated the expected high EGFR-specific tumor cell uptake (33.6% after 1 h, being reduced to 1.9% under blocking conditions) and affinity (IC50 value of 15.2 ± 3.3 nM). Thus, these results indicate that none of the previously described peptidic agents developed for EGFR targeting appears to be a reasonable choice as a lead structure for the development of radiopeptides for targeting of EGFR-positive tumors. Likewise, the tested truncated variants of the endogenous hEGF do not seem to be promising alternatives for this purpose.

Continuous Production of Human Epidermal Growth Factor Using Escherichia coli Biofilm.

Increasing demand for recombinant proteins necessitates efficient protein production processes. In this study, a continuous process for human epidermal growth factor (hEGF) secretion by Escherichia coli was developed by taking advantage of biofilm formation. Genes bcsB, fimH, and csgAcsgB that have proved to facilitate biofilm formation and some genes moaE, yceA, ychJ, and gshB potentially involved in biofilm formation were examined for their effects on hEGF secretion as well as biofilm formation. Finally, biofilm-based fermentation processes were established, which demonstrated the feasibility of continuous production of hEGF with improved efficiency. The best result was obtained from ychJ-disruption that showed a 28% increase in hEGF secretion over the BL21(DE3) wild strain, from 24 to 32 mg/L. Overexpression of bcsB also showed great potential in continuous immobilized fermentation. Overall, the biofilm engineering here represents an effective strategy to improve hEGF production and can be adapted to produce more recombinant proteins in future.

Refolding of bioactive human epidermal growth factor from E. coli BL21(DE3) inclusion bodies & evaluations on its in vitro & in vivo bioactivity.

Human epidermal growth factor (hEGF) is a mitogenic protein widely used in pharmaceutical and cosmetic industries, thus recombinant DNA technology has been applied to meet the high demand for hEGF. The overexpression of recombinant protein in E. coli often leads to the formation of inclusion bodies (IBs). Mild solubilisation preserves the native secondary protein structure in IBs, thereby the high recovery of active protein from IBs. The redox system also plays a pivotal role in the formation of disulphide bonds during refolding of disulphide bond-containing protein. This study aimed to recover hEGF from bacterial IBs through freeze-thawing solubilisation and glutathione-based oxidative refolding. CBD-Ssp DnaB-hEGF fusion protein was expressed as IBs in E. coli, washed with Triton X-100 and urea to remove most protein contaminants, then the solubilised fusion protein was obtained by freeze-thawing with the addition of 2 M urea. The solubilised protein was subsequently refolded by intein cleavage via a glutathione-based redox system. The refolded hEGF demonstrated heat-resistant properties, interacted with specific antibodies on ELISA, stimulated keratinocyte proliferation and possessed significant in vivo wound healing properties on the 8th day, confirming that hEGF was correctly folded. In summary, the protocol described is suitable for the recovery of refolded hEGF from bacterial IBs by mild solubilisation and oxidative refolding.

Assessment of Antimicrobial Agents, Analgesics, and Epidermal Growth Factors-Embedded Anti-Adhesive Poly(Lactic-Co-Glycolic Acid) Nanofibrous Membranes: In vitro and in vivo Studies.

BACKGROUND: Postoperative tissue adhesion is a major concern for most surgeons and is a nearly unpreventable complication after abdominal or pelvic surgeries. This study explored the use of sandwich-structured antimicrobial agents, analgesics, and human epidermal growth factor (hEGF)-incorporated anti-adhesive poly(lactic-co-glycolic acid) nanofibrous membranes for surgical wounds. MATERIALS AND METHODS: Electrospinning and co-axial electrospinning techniques were utilized in fabricating the membranes. After spinning, the properties of the prepared membranes were assessed. Additionally, high-performance liquid chromatography and enzyme-linked immunosorbent assays were utilized in assessing the in vitro and in vivo liberation profiles of the pharmaceuticals and the hEGF from the membranes. RESULTS: The measured data suggest that the degradable anti-adhesive membranes discharged high levels of vancomycin/ceftazidime, ketorolac, and hEGF in vitro for more than 30, 24, and 27 days, respectively. The in vivo assessment in a rat laparotomy model indicated no adhesion in the peritoneal cavity at 14 days post-operation, demonstrating the anti-adhesive capability of the sandwich-structured nanofibrous membranes. The nanofibers also released effective levels of vancomycin, ceftazidime, and ketorolac for more than 28 days in vivo. Histological examination revealed no adverse effects. CONCLUSION: The outcomes of this study implied that the anti-adhesive nanofibers with sustained release of antimicrobial agents, analgesics, and growth factors might offer postoperative pain relief and infection control, as well as promote postoperative healing of surgical wounds.

Activity and Effectiveness of Recombinant hEGF Excreted by Escherichia coli BL21 on Wound Healing in Induced Diabetic Mice.

CONTEXT: Human epidermal growth factor (hEGF) has biological activities and can be used in medicines and cosmetics. A high level of effectiveness of hEGF can be obtained when three disulfide bonds fold perfectly. Extracellular secretion from E. coli BL21 using the PelB signal peptide is a new way to obtain hEGF with a structure that folds appropriately. OBJECT: This study aimed to determine the activity and effectiveness of recombinant hEGF excreted by E. coli BL21 on wound healing in induced diabetic mice. METHODS: Cell proliferation and migration tests were performed on NIH3T3 cells, followed by wound healing tests in induced diabetic mice, along with histological and endotoxin test at various hEGF concentrations (25, 50, and 75 µg/mL). RESULTS: Based on the results, hEGF at a level of 50 µg/mL showed optimal proliferation and migration activities. Wound healing in induced diabetic mice showed faster-wound closure within 12 days at hEGF 50 and 75 µg/mL with a percentage wound closure of 95% and 98.5%, respectively, which was significant versus control. In the histology test, the number of fibroblasts showed an increase and was significant at hEGF 75 µg/mL compared to the control group. The single test vial (STV) showed that hEGF solution was free of endotoxin. CONCLUSION: Recombinant hEGF produced by extracellular secretion using E. coli BL21 has optimal diabetic wound healing activity through increased fibroblast proliferation.

Anesthetics and human epidermal growth factor incorporated into anti-adhesive nanofibers provide sustained pain relief and promote healing of surgical wounds.

Background: This study exploited sheath-core-structured lidocaine/human EGF (hEGF)-loaded anti-adhesive poly[(d,l)-lactide-co-glycolide] (PLGA) nanofibrous films for surgical wounds via a co-axial electrospinning technique. Materials and methods: After spinning, the properties of the co-axially spun membranes were characterized by scanning electron microscopy, laser-scanning confocal microscopy, Fourier Transform Infrared spectrometry, water contact angle measurements, and tensile tests. Furthermore, a HPLC analysis and an ELISA evaluated the in vitro and in vivo release curves of lidocaine and hEGF from the films. Results: PLGA anti-adhesion nanofibers eluted high levels of lidocaine and hEGF for over 32 and 27 days, respectively, in vitro. The in vivo evaluation of post-surgery recovery in a rat model demonstrated that no adhesion was noticed in tissues at 2 weeks after surgery illustrating the anti-adhesive performance of the sheath-core-structured nanofibers. Nanofibrous films effectively released lidocaine and hEGF for >2 weeks in vivo. In addition, rats implanted with the lidocaine/hEGF nanofibrous membranes exhibited greater activities than the control demonstrating the pain relief efficacy of the films. Conclusion: The empirical outcomes suggested that the anti-adhesive nanofibrous films with extended release of lidocaine and hEGF offer post-operative pain relief and wound healing.

[Purification of recombinant fusion polypeptide hEGF-AWRK6 and effect on wound healing and infection of burn model mice].

To test the therapeutic effect of recombinant fusion polypeptide hEGF-AWRK6 (EK) on burn infection of model mice. EK6 was expressed and purified with Escherichia coli expression system, and the Ⅱ degree burns and Pseudomonas aeruginosa infection model mouse were established. Experiment group was treated with EK (30 mg/L), and the control group was treated with PBS, gentamicin (30 mg/L), burn ointment (10 mg/L). The wound healing rate and colony count were calculated. Wound and surrounding skin were taken for HE staining and collagen western-blot analysis, and the wound pathological changes were observed after 10 days of drug delivery. The results showed that fusion peptide EK was successfully expressed and purified with significant antibacterial activities against Pseudomonas aeruginosa. Compared to the control group, the colony count (CFU) of the wound surface in EK mouse had a remarkable decrease (P<0.01) and healing rate had a significant increase in group EK6 (P<0.01). Pathological analysis result showed that compared to the control group, wound dermal cells in group EK arranged regularly, had more hair growth and a faster epithelization. These results indicated that the fusion peptide EK would be a good candidate for the drug development for the treatment of burning wounds.

Deformable liposomes for skin therapy with human epidermal growth factor: The effect of liposomal surface charge.

The topical administration of exogenous human epidermal growth factor (hEGF) is a promising approach for improved chronic wound therapy. To develop therapeutically superior hEGF formulation, we prepared hEGF-containing neutral (NDLs), cationic (CDLs) and anionic (ADLs) deformable liposomes (DLs), respectively, since it is expected that the liposomal surface charge can affect both the liposomal physicochemical properties, their skin penetration potential and therapeutic efficacy of liposome-associated drug. All prepared liposomes were of similar size (300-350 nm) with high hEGF load (~80% entrapment efficacy). Among the studied DLs, ADLs were found to be most promising for sustained release of hEGF, as assessed in vitro using the polyamide membrane. Ex vivo studies revealed that all DLs were excellent systems for skin therapy with hEGF and no penetration of hEGF through the full thickness human skin was detected. ADLs provided a depot exhibiting the highest hEGF retention onto the human skin surface. ADLs also revealed enhanced mitogenic activities in human fibroblasts compared to both NDLs and CDLs after 48 hrs treatment. Moreover, hEGF-containing ADLs significantly enhanced mitogenic activity in fibroblast as compared to activity of hEGF solution (positive control). Similar trends were observed in human keratinocytes after 24 hrs of treatment. We proved that the liposomal surface charge affects the therapeutic potential of hEGF-containing liposomes. hEGF-containing ADLs can be a promising nanosystem-based formulation for localized therapy of chronic wounds.

Gefitinib enhances radiotherapeutic effects of 131I-hEGF targeted to EGFR by increasing tumor uptake of hEGF in tumor xenografts.

Gefitinib is an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor which has been proven effective for cancer treatment. In this study, we sought to determine whether gefitinib could increase the in vivo tumor uptake of human 131I-EGF (131I-hEGF), thereby enhancing the potential of hEGF as a vehicle for EGFR-targeted radionuclide therapy. Western blot analysis was conducted to detect the effects of gefitinib on EGFR expression in human head and neck squamous carcinoma cell line UM-SCC-22B. Nude mice bearing UM-SCC-22B tumor xenografts were pretreated via i.p. injection of gefitinib or DMSO (vehicle control), followed by i.v. injection of 125I-hEGF; the animals were then subjected to ex vivo biodistribution or injection of 131I-hEGF for planar γ-imaging using SPECT, respectively. Targeted radionuclide therapy using 131I-hEGF combined with gefitinib as a vehicle targeting EGFR was also performed in UM-SCC-22B tumor xenografts. The EGFR level was unchangeable in cells pretreated with gefitinib, but after gefitinib pretreatment, the uptake of 125I-hEGF in 22B tumor xenografts increased substantially while the uptake of 125I-hEGF in normal organs was effectively unchanged. 131I-hEGF as a vehicle for EGFR-targeting therapy combined with gefitinib therefore showed strong therapeutic effects against 22B tumor xenografts tolerant to gefitinib. The uptake of hEGF to EGFR-positive tumors was enhanced significantly after gefitinib pretreatment, suggesting that 131I-hEGF is a potential vehicle for EGFR-targeting radionuclide therapy when combined with gefitinib.

Production of human epidermal growth factor using adenoviral based system.

Epidermal growth factor (EGF), a growth factor involved in cell growth and differentiation, is a small polypeptide with molecular weight of approximately 6 kDa known to be present in a number of different mammalian species. Experimental studies in animals and humans have demonstrated that the topical application of EGF accelerates the rate of epidermal regeneration of partial-thickness wounds and second-degree burns. Due to its commercial applications, Human EGF (hEGF) has been cloned in several forms. In the present study, adenoviral based expression system was used to produce biologically active recombinant hEGF. The presence of secreted recombinant hEGF was confirmed by a dot blot and its expression level was determined by enzyme-linked immuno-sorbent assay. Moreover, biological activity of secreted hEGF was evaluated by a proliferation assay performed on A549 cells. For production of hEGF in a secretory form, a chimeric gene coding for the hEGF fused to the signal peptide was expressed using adenoviral based method. This method enables the production of hEGF at the site of interest and moreover it could be used for cell proliferation and differentiation assays in tissue engineering research experiments instead of using commercially available EGF.

Nuclear localized Akt enhances breast cancer stem-like cells through counter-regulation of p21(Waf1/Cip1) and p27(kip1).

UNLABELLED: Cancer stem-like cells (CSCs) are a rare subpopulation of cancer cells capable of propagating the disease and causing cancer recurrence. In this study, we found that the cellular localization of PKB/Akt kinase affects the maintenance of CSCs. When Akt tagged with nuclear localization signal (Akt-NLS) was overexpressed in SKBR3 and MDA-MB468 cells, these cells showed a 10-15% increase in the number of cells with CSCs enhanced ALDH activity and demonstrated a CD44(+High)/CD24(-Low) phenotype. This effect was completely reversed in the presence of Akt-specific inhibitor, triciribine. Furthermore, cells overexpressing Akt or Akt-NLS were less likely to be in G0/G1 phase of the cell cycle by inactivating p21(Waf1/Cip1) and exhibited increased clonogenicity and proliferation as assayed by colony-forming assay (mammosphere formation). Thus, our data emphasize the importance the intracellular localization of Akt has on stemness in human breast cancer cells. It also indicates a new robust way for improving the enrichment and culture of CSCs for experimental purposes. Hence, it allows for the development of simpler protocols to study stemness, clonogenic potency, and screening of new chemotherapeutic agents that preferentially target cancer stem cells. SUMMARY: The presented data, (i) shows new, stemness-promoting role of nuclear Akt/PKB kinase, (ii) it underlines the effects of nuclear Akt on cell cycle regulation, and finally (iii) it suggests new ways to study cancer stem-like cells.

Phase I trial to evaluate the tumor and normal tissue uptake, radiation dosimetry and safety of (111)In-DTPA-human epidermal growth factor in patients with metastatic EGFR-positive breast cancer.

The safety, pharmacokinetics, biodistribution and radiation dosimetry of (111)In-DTPA-hEGF, an Auger electron-emitting radiopharmaceutical, were evaluated in a first-in-human trial. Dose escalation was performed in patients with EGFR-positive metastatic breast cancer who had received ≥2 prior courses of systemic treatment. (111)In-DTPA-hEGF (0.25 mg) was administered once intravenously (i.v.). Blood was collected for biochemistry/hematology testing and pharmacokinetic and immunogenicity analyses at selected times post injection (p.i.). Whole body planar images were acquired at 1, 4-6, 24 and 72 h p.i. and SPECT images at 24 and/or 72 h p.i. Macrodosimetry (MIRD) for the whole body and organs was estimated using OLINDA. Correlative radiological imaging was obtained at baseline, 1 and 3 months and then 6 monthly. Toxicity was scored using Common Terminology Criteria for Adverse Events (CTCAE)v2.0. Sixteen patients, median age 47 yr (range, 35-59), received (111)In-DTPA-hEGF as follows: 357-434 MBq (7), 754-805 MBq (3), 1,241-1,527 MBq (3) and 2,030-2,290 MBq (3). Fifteen were evaluable for toxicity. The commonest adverse events (AE) were flushing, chills, nausea, and vomiting occurring during or immediately p.i. One patient experienced Grade 3 thrombocytopenia (attributed to bone marrow infiltration by cancer). There were no other Grade 3 or 4 AEs. Maximum tolerated dose was not reached. Clear accumulation of radiopharmaceutical in at least one known site of disease was observed in 47% of patients. (111)In-DTPA-hEGF was cleared biexponentially from the blood with α-phase T½ of 0.16 ± 0.03 h and ß-phase T½ of 9.41 ± 1.93 h. (111)In-DTPA-hEGF was not immunogenic. The mean radiation dose estimates in mGy/MBq for whole body, liver, kidneys, spleen and thyroid were 0.08, 0.86, 0.74, 0.37 and 0.30, respectively. No objective antitumor responses were observed at the doses studied. In summary, administered amounts of up to 2,290 MBq (0.25 mg) of (111)In-DTPA-hEGF were well tolerated as a single i.v. injection.

Purification of recombinant fusion polypeptide hEGF-AWRK6 and effect on wound healing and infection of burn model mice / 生物工程学报

To test the therapeutic effect of recombinant fusion polypeptide hEGF-AWRK6 (EK) on burn infection of model mice. EK6 was expressed and purified with Escherichia coli expression system, and the Ⅱ degree burns and Pseudomonas aeruginosa infection model mouse were established. Experiment group was treated with EK (30 mg/L), and the control group was treated with PBS, gentamicin (30 mg/L), burn ointment (10 mg/L). The wound healing rate and colony count were calculated. Wound and surrounding skin were taken for HE staining and collagen western-blot analysis, and the wound pathological changes were observed after 10 days of drug delivery. The results showed that fusion peptide EK was successfully expressed and purified with significant antibacterial activities against Pseudomonas aeruginosa. Compared to the control group, the colony count (CFU) of the wound surface in EK mouse had a remarkable decrease (P<0.01) and healing rate had a significant increase in group EK6 (P<0.01). Pathological analysis result showed that compared to the control group, wound dermal cells in group EK arranged regularly, had more hair growth and a faster epithelization. These results indicated that the fusion peptide EK would be a good candidate for the drug development for the treatment of burning wounds.

Ability of plasmid DNA complexed with histidinylated lPEI and lPEI to cross in vitro lung and muscle vascular endothelial barriers.

DNA complexes made with cationic polymers (polyplexes) developed as nonviral vectors for gene therapy must be enabled to cross through vascular endothelium to transfect underlying tissues upon their administration in the blood circulation. Here, we evaluated the transendothelial passage (TEP) of DNA complexes made with histidinylated linear polyethylenimine (His-lPEI) or linear polyethylenimine (lPEI). In vitro studies were performed by using established transwell lung and skeletal muscle vascular endothelial barriers. The models were composed of a monolayer of human lung microvascular endothelial (HMVEC-L) cells and mouse cardiac endothelial (MCEC) cells formed on a PET insert and immortalized human tracheal epithelial (ΣCFTE29o-) cells and mouse myoblasts (C2C12) as target cells cultured in the lower chamber, respectively. When the vascular endothelium monolayer was established and characterized, the transfection efficiency of target (ΣCFTE29o- and C2C12) cells with plasmid DNA encoding luciferase was used to evaluate TEP of polyplexes. The luciferase activities with His-lPEI and lPEI polyplexes compared to those obtained in the absence of endothelial cell monolayer were 6.5% and 4.3% into ΣCFTE29o- cells, and 18.5% and 0.23% into C2C12 cells, respectively. The estimated rate for His-lPEI polyplexes was 0.135 µg/cm(2).h and 0.385 µg/cm(2).h through the HMVEC-L and MCEC monolayers, respectively. These results indicate that His-lPEI polyplexes can pass through the lung and skeletal muscle vascular endothelium and can transfect underlying cells.

Production and biomedical applications of virus-like particles derived from polyomaviruses.

Virus-like particles (VLPs), aggregates of capsid proteins devoid of viral genetic material, show great promise in the fields of vaccine development and gene therapy. These particles spontaneously self-assemble after heterologous expression of viral structural proteins. This review will focus on the use of virus-like particles derived from polyomavirus capsid proteins. Since their first recombinant production 27 years ago these particles have been investigated for a myriad of biomedical applications. These virus-like particles are safe, easy to produce, can be loaded with a broad range of diverse cargoes and can be tailored for specific delivery or epitope presentation. We will highlight the structural characteristics of polyomavirus-derived VLPs and give an overview of their applications in diagnostics, vaccine development and gene delivery.

Optimal Concentration of hEGF for Epithelial Healing in Experimental Corneal Alkali Wounds

PURPOSE: An optimal concentration of hEGF to enhance epithelial healing was determined in the rabbit eyes after alkali wounds. METHODS: Alkali-injured corneas (=5.5 mm, 1N NaOH, 60s) were treated with 0 (control), 2, 5, 10, and 50microgram/ml hEGF 3 times per day. The measurement of epithelial healing rate, transmission electron microscopy and immunohistochemical observation were performed after 7 days treatment. RESULTS: No significant difference was observed in the rates of epithelial healing between control and each hEGF-treated group. Among the tested concentrations, 5microgram/ml hEGF induced the most active proliferation of basal cells and 50microgram/ml hEGF remarkably produced a vascular ingrowth to the central wound area. The number of epithelial layers was increased by hEGF in a concentration-dependent manner. CONCLUSIONS: A low concentration of hEGF may selectively enhance epithelial healing without inducing corneal neovascularization. The optimal concentration of hEGF for the stimulation of basal cell proliferation appears to be 5 microgram/ml in rabbit corneal alkali wounds.

Effect of Topically Applied Human Epidermal Growth Factor on Stromal Keratocyte Activity in Experimental Corneal Alkali Wounds

PURPOSE: The Influence of topically applied human epidermal growth factor(hEGF) on stromal keratocyte was evaluated in the experimental corneal alkali wounds. METHODS: Corneal alkali wounds were induced by applying a 5.5 mm round filter paper soaked in 1N NaOH onto the center of rabbit cornea for 60 seconds. The eyes were treated topically with 5 micrometer/ml hEGF(DaeWoong Pharma. Co. Ltd. Seoul, Korea) or PBS, 3 times per day. After 3- and 28-days treatment, the corneas were processed for transmission electron microscopy(TEM). RESULTS: After 3-days treatment, the basement membrane integrity was maintained and the stromal keratocyte remained damaged in both groups. However, after 28-days treatment, the basement membrane was disrupted and the keratocytes were activated in both groups. CONCLUSIONS: The findings of present study indicated that the optimal concentration of hEGF which is known to stimulate the epithelial healing was failed to affect the keratocyte activities as long as the basement membrane integrity was maintained.