Preparation and antifungal activity of water-soluble graphene-based itraconazole antifungal eye drops / 中华实验眼科杂志

Objective:To prepare water-soluble graphene-based itraconazole antifungal eye drops and evaluate its antifungal activity against Fusarium solani. Methods:By oxidative modification of graphene and modification of polymer materials, water-soluble graphene oxide-modified polyethylene glycol (GO-PEG) composites were prepared.The composites were characterized by scanning electron microscopy, zeta potential, and Raman spectroscopy.The antifungal drug itraconazole was loaded onto the GO-PEG vector by solvent evaporation method, and itraconazole eye drops were obtained.The drug loading of itraconazole eye drops was measured using a UV and visible spectrophotometer.The antifungal effect in vitro was assessed by the microdilution method and light microscopy. Results:Scanning electron microscopy showed that GO-PEG had a two-dimensional nanosheet structure and many wrinkles.The zeta potential of GO-PEG was -42.40 mV.Raman spectroscopy showed that the ID/ IG of GO-PEG was 1.003.Using the water-soluble GO-PEG vector, a maximum itraconazole concentration of 10 mg/ml was achieved with a 10 000-fold increase in apparent solubility (10 mg/ml vs 0.001 mg/ml). The antifungal results showed that the minimum inhibitory concentration of itraconazole eye drops against Fusarium solani was approximately 1.88 μg/ml, but the GO-PEG vector has no significant antifungal activity against Fusarium solani. Conclusions:GO-PEG achieves effective loading and solubilization of itraconazole, demonstrating an in vitro inhibitory effect on Fusarium solani.

The facilitation of corneal permeability of cyclosporine A loaded on chitosan-graft-cyclodextrin copolymers vector / 中华实验眼科杂志

Objective To investigate the corneal permeability of cyclosprin A (CsA) loaded on polymeric vector after topical application.Methods The grafted copolymer chitosan-graft-cyclodextrin (CS-g-CD) was synthesized,and the physicochemical structures of the polymer were investigated using nuclear magnetic resonance spectroscopy (NMR) and fourier transform infrared spectroscopy (FT-IR).A novel CsA eye drop was prepared using the grafted copolymer as carrier material.The physicochemical properties of eye drop,including drug-loading content,osmotic pressure and viscosity were investigated by high performance liquid chromatography-mass spectrometry (HPLC-MS),osmotic pressure gauge and viscometer,respectively.New Zealand albino rabbits were randomly divided into intact cornea CsA group,epithelium debrided CsA group and epithelium debrided control group.The corneal epithelia of the left eyes was debrided in the cornea epithelium debrided group.Cornea irritation test was performed on New Zealand albino rabbits.The aqueous humor was taken and the corneas were collected at 0.5 hour and 1 hour after instilled.The concentration of CsA was measured by HPLC-MS.Cy5 labeled vector loaded with Coumarin 6 served as model copolymers system,the penetration capabilities of the double fluorescent labeling copolymers system were monitored in vivo using two-photon scanning fluorescence microscopy on murine corneas after topical application.The use and care of the animals complied with Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.Results The polymer of CS-g-CD was successfully synthesized and confirmed using NMR and FT-IR.The drug loading of CsA in eye drop solution was 0.06 ％;the osmotic pressure was 305 mOsmol/kg and the viscosity was 36.5 cP.The CsA drug delivery system had a reversible temperature-sensitive drug release behavior and had no obvious irritation on the eyes of New Zealand rabbits.One hour after treatment,the concentration of CsA in the cornea and aqueous humor of epithelium debrided CsA group was (5.88 ± 1.46) μg/g and (149.19 ± 3.93) ng/ml,respectively,which was significantly higher than (3.98 ±0.95) μg/g and (30.25± 11.43) ng/ml in epithelium debrided control group (both at P＜0.05);the concentration of CsA in the aqueous humor of intact cornea CsA group was (7.23 ± 1.31)ng/ml,which was significantly lower than that in epithelium debrided CsA group (P＜0.05).Polymer vectors were mainly retained in the corneal epithelium,and coumarin 6 gradually diffused into the deep corneal stroma with time.Conclusions The grafted copolymer can load CsA,and the eye drop can effectively overcome the corneal barrier and increase the corneal permeability of CsA.

The facilitation of corneal permeability of cyclosporine A loaded on chitosan-graft-cyclodextrin copolymers vector / 中华实验眼科杂志

Objective To investigate the corneal permeability of cyclosprin A (CsA) loaded on polymeric vector after topical application. Methods The grafted copolymer chitosan.graft.cyclodextrin ( CS.g.CD ) was synthesized, and the physicochemical structures of the polymer were investigated using nuclear magnetic resonance spectroscopy ( NMR) and fourier transform infrared spectroscopy ( FT.IR) . A novel CsA eye drop was prepared using the grafted copolymer as carrier material. The physicochemical properties of eye drop,including drug.loading content, osmotic pressure and viscosity were investigated by high performance liquid chromatography.mass spectrometry ( HPLC.MS) ,osmotic pressure gauge and viscometer,respectively. New Zealand albino rabbits were randomly divided into intact cornea CsA group, epithelium debrided CsA group and epithelium debrided control group. The corneal epithelia of the left eyes was debrided in the cornea epithelium debrided group. Cornea irritation test was performed on New Zealand albino rabbits. The aqueous humor was taken and the corneas were collected at 0. 5 hour and 1 hour after instilled. The concentration of CsA was measured by HPLC.MS. Cy5 labeled vector loaded with Coumarin 6 served as model copolymers system, the penetration capabilities of the double fluorescent labeling copolymers system were monitored in vivo using two.photon scanning fluorescence microscopy on murine corneas after topical application. The use and care of the animals complied with Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission. Results The polymer of CS.g.CD was successfully synthesized and confirmed using NMR and FT.IR. The drug loading of CsA in eye drop solution was 0. 06 ％;the osmotic pressure was 305 mOsmol/kg and the viscosity was 36. 5 cP. The CsA drug delivery system had a reversible temperature.sensitive drug release behavior and had no obvious irritation on the eyes of New Zealand rabbits. One hour after treatment,the concentration of CsA in the cornea and aqueous humor of epithelium debrided CsA group was (5. 88±1. 46)μg/g and (149. 19±3. 93)ng/ml,respectively,which was significantly higher than (3. 98±0. 95)μg/g and (30. 25±11. 43)ng/ml in epithelium debrided control group (both at P<0. 05);the concentration of CsA in the aqueous humor of intact cornea CsA group was ( 7. 23 ± 1. 31 ) ng/ml, which was significantly lower than that in epithelium debrided CsA group ( P<0. 05 ) . Polymer vectors were mainly retained in the corneal epithelium, and coumarin 6 gradually diffused into the deep corneal stroma with time. Conclusions The grafted copolymer can load CsA,and the eye drop can effectively overcome the corneal barrier and increase the corneal permeability of CsA.

Promoting effects of macrogol 15 hydroxystearate micelles on corneal absorption of terbinafine hydrochloride / 中华实验眼科杂志

Background Macrogol 15 hydroxystearate (HS15) is a novel soft non-ionic surfactant and is widely used to solubilize the poorly soluble drugs due to high drug loading and enhancing permeability ability to hydrophobic drug.However,the delivering effects to cornea of HS15 on terbinafine hydrochloride (TH),a insoluble antifungal agents,is unclear.Objective This study was to investigate the promoting corneal absorption effects of HS15 micelles (HNMs) on TH.Methods TH-HNMs was prepared by a co-solvent method.The hydrodynamic droplet size,polydispersity index,and Zeta potential of TH-HNMs were measured by using a Zetasizer.The shape of the micelles was observed under the transmission electron microscope.The high performance liquid chromatography (HPLC) was employed to detect the in vitro cumulative releasing level of TH in the TH-HNMs and drug entrapment efficiency.TH-HNMs was topically adninistered in eyes of 5 healthy male New Zealand rabbits to evaluate the ocular irritation response.Ninety rabbits were randomized into experimental group and control group,and 50 μl of 0.5％ TH-HNMs and 0.5％ oily TH were topically administered in the right eyes of the animals in the experimental group and contral group,respectively.The animals were sacrificed 5,15,30,60,90,120,180,240,360 minutes after eye dropping by over anesthetization way and the corneas were harvested,and the TH content in the cornea was detected using HPLC.The study protocal was approved by Life Science Ethic Committee of Henen Eye Hospital.Results The average size and polyolis persital index of TH-HNMs were 13.32 nm and 0.046,respectively,and its average Zeta potential was-0.133 mV.The drug entrapment efficiency was 100％.The release level of TH from the micelles presented a pH-dependent manner.The release level of TH was (95.20±3.20)％ in the phosphate buffer with pH 5.0 and (0.17± 0.01)％ in the phosphate buffer with pH 7.4.The ocular irritation score was 2,and no visible damage was found around experimental eyes after instillation of TH-HNMs.The peak content of TH in the rabbit cornea 5 minutes was (20.26±2.26)pg/g in the experimental group,which was significantly higher than (1.40± 0.44)μg/g in the control group (t =18.926,P=0.000).The area under the curve (AUC)0.360min of drug concentration-time curve in the experimental group was 1 292.25 μg/(g · min),which was 15.6 times more than the control group.Conclusions TH-HNMs is an ideal agent with a simple preparing process,high drug entrapment efficiency,small size and low ocular irritation.Compared with oily TH,TH-HNMs can effectively enhance the corneal absorption of TH.

Absorption and distribution characteristics of terbinafine hydrochloride nanoemulsions in cornea after instillation / 中华实验眼科杂志

Background Nanoemulsions (NEs) is one of the most popular ophthalmic colloidal drug delivery system due to its long-term stability, low toxicity and irritancy, considerable capacity for solubilization of lipophilic drug molecules and great potential in bioavailability improvement.The cornea pathway is the main route of intraocular absorption after topical use of NEs.Though NEs possess numerous physiological and physicochemical advantages,the use of NEs cannot always obtain satisfactory results.Objective This study was to investigate the impacts of epithelium and stroma on the corneal permeation of topical ophthalmic terbinafine hydrochloride nanoemulsions (TH-NEs).Methods TH-NEs was prepared by the self-emulsification method.The size and Zeta potential of the oil droplets in the formulation were analyzed using a dynamic light-scattering particle size analyzer.The high performance liquid chromatography (HPLC) was used for the in vitro release study.Sixty New Zealand albino rabbits were randomly divided into intact cornea group and cornea epithelium debrided group.The cornea epithelium of the left eyes was debrided in the cornea epithelium debrided group.The TH-NEs were instilled into the lower conjunctival sac of left eyes.Six rabbits were executed from each group 15,30,60,120 and 240 minutes after dosing,respectively.The corneas were collected and analyzed by HPLC.The fluorescein diacetate (FDA) was used to label the TH-NEs.Two C57BL/6 mice with left cornea epithelium debrided and 2 normal mice were used for the fluorescence tracing study.The fluorescence distribution of FDA labeled TH-NEs was observed by a two-photon laser confocal scanning microscope 30 minutes and 60 minutes after single instillation.Results The average size and Zeta potential of the oil droplets were 51.37 nm and-0.232 7 mV respectively,and 0.482％ of encapsulated drugs was released from the TH-NEs after 12 hours.The peak concentrations of TH in the intact cornea and epithelium debrided cornea were (17.85 ± 2.79) μg/g and (4.40± 1.75) μg/g respectively, which occurred 15 minutes postdose.The drug concentrations in the intact cornea were significantly higher than that in the debrided cornea 15,30,60 and 120 minutes after dosing, with significant differences between them (t =9.998,8.658,6.903,7.576;all at P=0.000).The fluorescence was observed in the cornea epithelium when the cornea was intact.The fluorescence intensity in the superior layer of corneal epithelium was obviously higher than that in the deep layers of corneal epithelium 30 minutes and 60 minutes after dosing.No fluorescence was observed in the cornea stroma of both eyes.Conclusions The cornea epithelium is the main of absorption and distribution position of TH-NEs.The cornea stroma is the dominating permeation barrier for the intraocular transportation of the TH-NEs.The cornea stroma may stop the permeation of TH-NEs by molecular exclusion mechanism.

Establishment and evaluation of a murine alphavbeta3-integrin-expressing cell line with increased susceptibility to Foot-and-mouth disease virus

Integrin alphavbeta3 plays a major role in various signaling pathways, cell apoptosis, and tumor angiogenesis. To examine the functions and roles of alphavbeta3 integrin, a stable CHO-677 cell line expressing the murine alphavbeta3 heterodimer (designated as "CHO-677-malphavbeta3" cells) was established using a highly efficient lentiviral-mediated gene transfer technique. Integrin subunits alphav and beta3 were detected at the gene and protein levels by polymerase chain reaction (PCR) and indirect immunofluorescent assay (IFA), respectively, in the CHO-677-malphavbeta3 cell line at the 20th passage, implying that these genes were successfully introduced into the CHO-677 cells and expressed stably. A plaque-forming assay, 50% tissue culture infective dose (TCID50), real-time quantitative reverse transcription-PCR, and IFA were used to detect the replication levels of Foot-and-mouth disease virus (FMDV) in the CHO-677-malphavbeta3 cell line. After infection with FMDV/O/ZK/93, the cell line showed a significant increase in viral RNA and protein compared with CHO-677 cells. These findings suggest that we successfully established a stable alphavbeta3-receptor-expressing cell line with increased susceptibility to FMDV. This cell line will be very useful for further investigation of alphavbeta3 integrin, and as a cell model for FMDV research.

Suckling mouse model establishment of myocarditis induced by foot-and-mouth disease vi rus / 中国人兽共患病学报

Interactions between FMDV and cardiac cells are multifaceted and complex ,these interactions leads to pro-teins alterations in cardiac cells inevitably .To understand the pathogenesis of myocarditis after FMDV infection in mice ,the suckling mouse model for myocarditis induced by foot-and-mouth disease virus (FMDV) was established in this study .Suckling mice within 3 days old was selected to infect by FMDV .Myocarditis caused by FMDV in suckling mice was confirmed with clinical symptom monitor .The observation of Hematoxylin and eosin stain (H&E stain) and transmission electron microscopy (TEM) were performed after samples processing .According to conventional polymerase chain reaction (PCR) methods ,prim-ers of VP1 gene was designed ,synthesised and specific FMDV VP1 gene was amplified from the heart muscle of suckling mice . The results indicated that suckling mice appeared low spirit condition ,dyspnea ,and dull reaction within 36 hours after chal-lenge with FMDV .Infiltration of inflammatory cells and dissolution of myocardial fibers were observed with H&E stain and TEM .Special target gene of FMDV was amplified from the heart of infected group .Obvious inflammation in the heart of suck-ling mice caused by FMDV was observed .It's suggested that suckling mouse model for myocarditis induced by FMDV was es-tablished successfully ,which would lay the foundation for researches of myocarditis mechanism in young cloven-hoofed ani-mals .