Journal of Materials Chemistry BMaterials for biology and medicinersc.li/materials-b

ISSN 2050-750X

COMMUNICATIONJui-Yang Lai, Kevin C.-W. Wu et al.Gelatin-functionalized mesoporous silica nanoparticles with sustained release properties for intracameral pharmacotherapy of glaucoma

Volume 5 Number 34 14 September 2017 Pages 6977–7206

7008 | J. Mater. Chem. B, 2017, 5, 7008--7013 This journal is©The Royal Society of Chemistry 2017

Cite this: J.Mater. Chem. B, 2017,5, 7008

Gelatin-functionalized mesoporous silicananoparticles with sustained release propertiesfor intracameral pharmacotherapy of glaucoma†

Yu-Te Liao,a Chih-Hung Lee,b Si-Tan Chen,b Jui-Yang Lai *bcd andKevin C.-W. Wu *ae

Herein, pilocarpine-loaded gelatin-covered mesoporous silica

nanoparticles (denoted as p/GM) were intracamerally administrated

into the anterior chamber for the reduction of intraocular pressure

(IOP). The in vitro release profile shows that p/GM demostrates a

high release percentage (50%) with a long-lasting release profile

(36 days). The in vivo studies showed the maintenance of IOP in eye

with ocular hypertension for 21 days.

Primary open-angle glaucoma (POAG) is the most common typeof glaucoma, which is characterized by progressive optic neuro-pathy with a high chance of eventual blindness.1 Once theaccumulation of extracellular matrix in the trabecular mesh-work of glaucomatous eyes occurs, the normal aqueous outflowsystem cannot be maintained; this results in ocular hypertension.2

Elevated intraocular pressure (IOP) is known to be an importantrisk factor for the progression of POAG and visual damage.3 Sincethe anterior chamber is a comparatively small area located withinthe eye, one major challenge for glaucoma treatment is to developan effective method to control the IOP.

It is difficult to deliver drugs into the anterior chamber viaoral or intravenous systemic administration routes due to thepresence of blood-ocular barriers.4 Topical drug administrationhas several advantages such as self-administrable therapy, non-invasiveness, and high patient compliance; therefore, it is currentlythe main way to manage ocular diseases. However, the bioavailability

of topical ophthalmic formulations is severely limited by blinking,tear turnover, and naso-lachrymal drainage.4 Another way topromote the efficacy of a drug is invasive treatment involvingintracameral administration. Since intracameral injection canallow direct entry of antiglaucoma medications into the intra-ocular space, this drug administration route is considered to bemore advantageous than topical ophthalmic or systemic drugadministration. In our previous study, we introduced a bio-degradable polymer into the anterior chamber via intracameraladministration and proved that this procedure could pass thedrug carrier through the barrier of cornea.5,6 However, thechallenge of long-lasting treatment, i.e. sustained release, stillremains to be solved.

Sustained release of drug can extend the period of treatment,which can solve the problem of low drug delivery efficacy. Aneasy way to induce sustained release is to release drug viadegradation or decomposition of organic polymers. For example,as reported by Bhattarai et al.,7 solidification of chitosan withgenipin can form an injectable thermosensitive hydrogel. Sutteret al.8 used a recombinant gelatin hydrogel as a protein carrier forthe sustained release of proteins. Another way to extend the drugrelease duration is to coat organic capping agents on inorganicdrug carriers.9–11 In our previous study, we coated alginate on theexternal surface of mesoporous silica nanoparticles (MSNs) tocreate an organic/inorganic hybrid drug delivery system forchemotherapy with sustained release.10 After this, we havedemonstrated that the organic alginate/inorganic alginate/MSN hybrid material exhibits excellent sustained release property,which can extend the release duration up to 40 days. Gelatin-functionalized MSN materials have also been applied for colontumor,12,13 where an enzyme called matrix metalloproteinase-2(MMP-2) is overexpressed and can digest gelatin. Using this uniqueproperty, the drug loaded inside MSN can be slowly released, andthe release duration correlate with the concentration of MMP-2.The anterior chamber is also an MMP-2 rich environment, andKee et al.14 have reported that the activity of MMP-2 is 3.9 timesin the aqueous humor of patients with POAG. Therefore, thecombination of the MMP-2 responsive gelatin-coated MSNs with

a Department of Chemical Engineering, National Taiwan University, No. 1, Sec. 4,Roosevelt Road, Taipei 10617, Taiwan. E-mail: kevinwu@ntu.edu.tw

b Institute of Biochemical and Biomedical Engineering, Chang Gung University,No. 259, Wen-Hwa 1st Road, Kwei-Shan, Taoyuan 33302, Taiwan.E-mail: jylai@mail.cgu.edu.tw

c Department of Materials Engineering, Ming Chi University of Technology,No. 84, Gungjuan Road, Taishan, New Taipei City 24301, Taiwan

d Ophthalmology, Chang Gung Memorial Hospital, No. 5, Fushing Street,Kwei-Shan, Taoyuan 33305, Taiwan

e Division of Medical Engineering Research, National Health Research Institutes,35 Keyan Road, Zhunan, Miaoli County 350, Taiwan

† Electronic supplementary information (ESI) available: Details of the experiment;characterization of GM-x and quantification of in vivo studies. See DOI: 10.1039/c7tb01217a

Received 4th May 2017,Accepted 27th July 2017

DOI: 10.1039/c7tb01217a

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Journal ofMaterials Chemistry B

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intracameral administration could be an efficient and reliableapproach for the sustained release of pilocarpine in the anteriorchamber. Gelatin has been proven as biodegradable and bio-compatible and is widely applied in pharmaceutics and foodindustry,15,16 whereas MSNs have been proven to be very usefulfor many applications such as in catalysis,17–19 metal ioncapture,20 and sensors.21,22 More importantly, they have beenapplied in drug delivery systems (DDS) owing to their excellentbiocompatibility, high surface area, versatile surface functionality,and controllable particle size.23–25

Herein, we fabricated gelatin-functionalized MSNs (denotedas GM) as intracameral drug carriers to extend the drug releasetime and improve ocular bioavailability. As shown in Scheme 1,we first synthesized pilocarpine-loaded MSN@Gels (denoted asp/GM-x) with various amounts of gelatin (x is denoted as mg ofgelatin on per mg of MSN) and directly delivered p/GM-x intothe anterior chamber of glaucomatous eyes. Our aim was toinvestigate the therapeutic efficacy and safety of drug/polymerinjections for glaucoma treatment. The results showed that thegradual degradation of the gelatin polymer by MMP-2 couldlead to a progressive release of pilocarpine from p/GM-x in vitroand in vivo.

The structural and porous properties of the synthesizedsamples were characterized using transmission electron micro-scopy (TEM) and nitrogen adsorption/desorption isotherms. Asshown in Fig. 1a, MSNs with a particle size around 50 nm weresuccessfully synthesized. The nitrogen adsorption/desorptionisotherms of the synthesized MSNs (Fig. S1a, ESI†) indicate ahigh specific surface area (i.e. 1122 m2 g!1) and uniformmesopores (i.e. 2.85 nm), according to the BET and BJHmethods, respectively.26 To control the thickness of the gelatinshell on the MSNs, gelatin at different amounts (i.e. p/GM-x,x = 0, 0.05, 0.1, and 0.5) was used to cross-link with the externalsurface of MSNs through the EDC/NHS chemistry.27 The effectsof gelatin amounts on the specific surface area of MSNwere studied. As shown in Fig. S1b (ESI†), with the increase ofgelatin amount, the specific surface area of the MSN gradually

decreased. This is because the interaction between gelatin andMSN is covalent bonding; this indicates that more gelatincoverage on MSNs would reduce the accessibility of nitrogengas. The complete coverage of gelatin on MSNs can be directlyobserved via the TEM images. As shown in Fig. 1b and Fig. S2(ESI†), it is difficult to see the mesopores of GM-0.05 due to thethickness of gelatin. We then summarized the effects of gelatinamounts on the specific surface area and zeta-potential of theGM samples and the thickness of gelatin. As shown in Table S1(ESI†), as gelatin amounts were increased from GM-0 to GM-0.5,the thickness of the gelatin shell increased from 0 nm to6.65 nm. Another evidence for the successful gelatin coatingwas that zeta potential of GM-x became more positive as the x-value increased. The zeta potential of gelatin is 3.93 mV, whichwould neutralize the negatively charged MSN, making the zetapotential of GM-x more positive. To determine the functionalgroups on the synthesized samples, FTIR spectroscopy wasutilized, and the results are shown in Fig. S3 (ESI†). All gelatin-containing samples (i.e. GM-0.05, GM-0.1, and GM-0.5) showedtwo apparent bands representing amide I (1650 cm!1) and amideII (1545 cm!1),28 indicating the existence of gelatin in thesamples. As the ratio of gelatin to MSN increased, the intensityof these two bands also enhanced; this indicated that the amountof gelatin on MSNs also increased.

To effectively control the IOP, pilocarpine was loaded intoMSNs and then administered to the ocular anterior chamber.The loading capacity of pilocarpine was 95 mg mg!1 of MSN,and the loading capacity of p/GM-x is summarized in Table S1(ESI†). Fig. S4 (ESI†) shows the FTIR spectrum of p/GM-x andpilocarpine, which proves that pilocarpine is indeed loadedinto MSN according to the strong absorption at 1777 cm!1.29

Scheme 1 Synthesis of pilocarpine-loaded gelatin-covered MSNs (p/GM-x)as an ocular drug delivery system to reduce intraocular pressure via intra-cameral administration into the anterior chamber.

Fig. 1 TEM image of the (a) MSN and (b) gelatin-covered MSN (GM-0.05).(c) Cumulative release profile of pilocarpine released from MSN and GM inMMP-2 (50 ng mL!1)-contained BSS solution. The scale bar is 50 nm.

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It has been reported that the minimum effective therapeuticdose of pilocarpine to reduce IOP is 20 mg mL!1 in the anteriorchamber.30 Normally, the volume of the anterior chamber is150 mL,31 indicating that minimum effective therapeutic dose ofpilocarpine in the anterior chamber should reach 3 mg mg!1 ofMSN. Based on this calculation, the amount of pilocarpineloaded in our GM samples is sufficient for long-term glaucomatreatment. The release profiles of pilocarpine from MSN withand without gelatin coating (i.e. p/GM-x and p/MSN) in thepresence of MMP-2 are shown in Fig. 1c. All four samplesshowed burst release initially that became slow after 6 h. Therelease was saturated at about 20% for the p/MSN sample andat about 15% for the p/GM-0.05 sample (Fig. 1c). Many studieshave mentioned that the electrostatic interaction between MSNand guest molecules is the reason for this low release percentage.12,32

However, it was found that the release of pilocarpine fromp/GM-0.05 and p/GM-0.1 continued for up to 36 days, and thetotal release percentage was 50% for p/GM-0.05 and 30% forp/GM-0.1 (Fig. 1c). These results clearly indicate that thep/GM-0.05 sample exhibits sustained release property. The zetapotential of the fragments was !1.25 mV and more negativethan that of gelatin (i.e. 3.93 mV); this suggested thatthe fragments of gelatin contained more negatively chargedmoieties, such as carboxylic group, than gelatin. Gao et al.33

mentioned in their previous study that MSNs were a pH-responsivedrug delivery system. The release percentage of drugs from MSNsunder acidic environment was higher than that under neutral orbasic environment. As gelatin was digested by MMP-2, the micro-environment around MSNs became acidic, and this triggered therelease of pilocarpine from MSNs. Since MMP-2 is richly availablein the anterior chamber, we propose that the p/GM-0.05 samplewill have the same sustained release behavior when gelatin isgradually digested by MMP-2. More importantly, the initial burstrelease of pilocarpine from the p/GM-0.05 sample can provide asufficient concentration of pilocarpine to reduce the IOP in theanterior chamber immediately after the injection, and thesustained release thereafter can provide a long-term treatmentto maintain the IOP at a low level.

We first checked the biocompatibility of the synthesizedsamples before applying them in ocular administration ofpilocarpine. The results of cell viability are shown in Fig. S5(ESI†); it was found that both MSN and GMs are highlybiocompatible with bovine corneal endothelial cell cultures.Herein, an experimental glaucoma model in rabbits induced byinjecting a-chymotrypsin into the posterior chamber was used.Before the intracameral delivery of the pilocarpine-containedsamples, the blockage of the trabecular meshwork and theobstruction of the aqueous outflow result in glaucomatouseye with an abnormal IOP. Then, three samples (i.e. p/MSN,p/GM-0.05, and p/GM-0.5) were intracamerally injected througha 30-gauge needle via minimally invasive procedures for drugdelivery (Video, ESI†).

To confirm the efficacy of the gelatin-coated MSN for glaucomaDDS, slit-lamp biomicroscopy was used for the anterior segmentdiagnostics.15 Fig. 2 shows the representative slit-lamp bio-microscopy images of glaucomatous eyes after intracameral

injection of various pilocarpine-containing samples. In thep/MSN, p/GM-0.05, and p/GM-0.5 groups, the cornea was clearand the anterior chamber was quiet (no cells or flare) duringthe follow-up period from 12 h to 3 weeks. In addition, therewas no cataract, corneal neovascularization, and stromal diseaseand no signs of ocular inflammation. Herein, for the first time, ithas been demonstrated that the intracamerally injected MSN-based materials for minimally invasive pilocarpine delivery donot cause any anterior segment tissue response; this indicatesexcellent ocular biocompatibility of DDS. On the other hand, inthe Ctrl groups, the glaucomatous eyes receiving BSS only had adeeper anterior chamber. By contrast, distinct levels of anteriorchamber narrowing were observed for different pilocarpine-containing MSN samples. As an antiglaucoma medication,pilocarpine is a miotic that can increase aqueous outflow andopen the trabecular meshwork via contraction of the ciliarymuscles. As shown in the front view of Fig. 2, both the p/MSNand p/GM-0.5-injected eyes had similarly large pupil sizes as theCtrl eyes at postoperative 21 days. Note that there was anobvious pupillary constriction after intracameral injection ofthe p/GM-0.05 sample; this proved the long-term pharmacologicalaction of the released drug. The anterior chamber depth (i.e. thedistance between the central anterior corneal epithelium and theanterior crystalline lens capsule) was further quantified fromthe images, and the results are shown in Fig. S6 (ESI†). The anteriorchamber depths in all the three samples (i.e. p/MSN, p/GM-0.05,and p/GM-0.5) were shallower than that in the Ctrl groups at thepostoperative 12 h (P o 0.05) (Fig. S6, ESI†). Since an elevation inIOP is highly correlated with an increase in the anterior chamberdepth, our data demonstrate that both p/MSN and p/GM would

Fig. 2 Time-course slit-lamp biomicroscopy images of rabbit eyesinjected with p/MSN, p/GM-0.05, and p/GM-0.5. The observation timesare 12 hours (H12), 2 days (D2), and 3 weeks (W3) after pilocarpineadministration. The front view of rabbit eyes is observed 3 weeks afterpilocarpine administration. The scale bar is 4 mm.

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have IOP reducing effects. On the postoperative day 2, nosignificant difference in the anterior chamber depth was foundbetween the Ctrl and p/MSN groups (P 4 0.05) (Fig. S6, ESI†)probably due to the blockage of pilocarpine inside MSNs. Bycontrast, at the same time point, the glaucomatous rabbitsreceiving p/GM-0.05 and p/GM-0.5 samples had significantlylower anterior chamber depth than the BSS controls (P o 0.05).It was also noted that at postoperative 3 weeks, only the p/GM-0.05-injected eyes demonstrated anterior chamber narrowingamong the samples evaluated herein (Fig. S6, ESI†). Overall, theresults indicated that p/MSN lost its ability to reduce IOP after12 h, whereas p/GM-0.5 could extend the duration of pupillaryconstriction in response to the released drug for at least 2 days.For the p/GM-0.05, the anterior chamber depth of glaucomatousrabbits gradually returned to normal within 3 weeks. Accordingto the drug concentration profiles of the pilocarpine-containingsamples (Fig. S7, ESI†), the amounts of released drug in bothp/MSN and p/GM-0.5 groups were dramatically decreased(below 3 mg mg!1 of MSN) after the burst release. However, inthe case of the p/GM-0.05 sample, the drug concentration wasmaintained at a level greater than the minimum effectivetherapeutic dose of pilocarpine throughout the 3 weeks.

Since the drug molecules are released from the deliverycarrier materials, it is crucial to understand the in vivo residenceof MSN-based carriers after intracameral injection. Herein,inductively coupled plasma mass spectrometry (ICP-MS) wasused to determine the concentration of silicon in the MSN-basedsamples in the aqueous humor. At the postoperative 21 days, nosilicon was detected in the injected p/MSN sample. However, theconcentration of silicon in the p/GM-0.5 and p/GM-0.05 groupswas 9.1 " 0.3 and 7.2 " 0.8 ppm, respectively. It is well knownthat the aqueous humor usually leaves the eye by flowing acrossthe trabecular wall of the Schlemm’s canal. Johnson et al.34

previously compared the effect of particle size on in vivo retentiontime of particles by trabecular meshwork. Their data demonstratethe accumulation of 45% of the particles with a size of 176 nm inthe trabecular meshwork or juxtacanalicular connective tissue.Raju et al.35 further showed that magnetic particles with a size of50 nm would pass through the trabecular meshwork within oneweek of anterior chamber injections, whereas the large-sizedparticles (i.e. 4 mm) could be identified after 5 months liningthe iris and angle. In this study, the MSN and GM samples with asize of around 50 nm would flow into the Schlemm’s canalduring pilocarpine-induced contraction of ciliary muscles.Therefore, the role of particle size in blocking the filtration ofthe trabecular meshwork could account for a tremendous loss ofp/MSN in the anterior chamber at the end of the experiments(i.e. 3 weeks postoperatively). However, it is noteworthy that thep/GM materials have a better residence in vivo; this indicatesthat the functionalization of MSN with gelatin enhances theretention time in the anterior segment tissue.

Because of the excellent bio-adhesiveness of gelatin, we havebeen using gelatin as a delivery carrier for corneal endothelialcell sheet attachment and transplantation.36 For the first time,herein, a strategy has been demonstrated in which a bio-adhesivegelatin polymer is coated on the external surface of MSNs that

generates a new drug carrier with an extended residence time anda long-term drug release in the ocular anterior chamber. This isfurther supported by HPLC analysis. At 3 weeks postoperatively,the concentration of pilocarpine in the Ctrl, p/MSN, p/GM-0.05,and p/GM-0.5 groups was 0, 0, 12.8 " 0.7, and 0.6 " 0.2 mg mL!1

in the aqueous humor, respectively. The difference between thedetected drug amount in vivo of p/GM-0.05 and p/GM-0.5-injectedeyes also reflects the governing role of the ratio of gelatin to MSNfor fabricating drug carriers with a controlled drug releaseproperty.

Via specular microscopy, the corneal endothelium in rabbiteyes at pre-operation and follow-up was observed to examinethe therapeutic efficacy of pilocarpine released from the MSNs.As shown in Fig. 3, the corneal endothelium is a cell monolayer,which maintains the corneal deturgescence and clarity. Thechanges in the morphology and density of corneal endothelium area sensitive indicator of tissue damage and glaucoma diagnosis.37

The corneal endothelial cells on Descemet’s membrane fromnormal rabbits closely packed together and exhibited a typicalhexagonal shape (Fig. 3a-Pre). By contrast, in a rabbit modelof experimental glaucoma, the ocular hypertension causedirregular shape of endothelial cells (Fig. 3a-GL).38 Without treatmentwith MSN-based carriers, glaucomatous eyes receiving BSS onlyshowed an increase in the size of mono-layered cells at 21 dayspostoperatively (Fig. 3a-Ctrl). In the p/GM-0.05 sample, theintracameral injection did not further alter the corneal endothelialmorphology of glaucomatous eyes, whereas both the p/MSN- andp/GM-0.5-injected eyes possessed similar morphological features as

Fig. 3 (a) Typical specular microscopy images of corneal endothelium ofnormal eye (Pre), glaucomatous eye (GL) before operation, control group(Ctrl) and pilocarpine containing carriers injected eye after postoperative21 days. (b) The corneal endothelial cell density measured via typicalspecular microscopy. An asterisk indicates statistically significant differences(*P o 0.05; n = 6) as compared to other five groups. A number indicatesstatistically significant differences (#P o 0.05; n = 6) as compared to Pre,Ctrl, p/MSN, and p/GM-0.5 groups. A plus indicates statistically significantdifferences (+P o 0.05; n = 6) as compared to Pre, GL, and p/GM-0.05groups.

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the Ctrl eyes. Furthermore, as shown in Fig. 3b, the density ofcorneal endothelial cells from p/GM-0.05-injected eyes was2989 " 88 cells per mm2, which was significantly higher thanthat of the Ctrl (2367 " 100 cells per mm2) groups (P o 0.05),but not significantly different from that of the GL (3095 "60 cells per mm2) groups (P 4 0.05). However, for the p/MSNand p/GM-0.5-injected eyes, significantly lower endothelial cellcounts were found as compared to the values following theglaucoma induction. Our findings suggest that among all thegroups studied herein, only p/GM-0.05 is therapeutically activeand efficacious to preserve the cell morphology and density ofcorneal endothelium with no in vivo toxicity during 3 weeks offollow-up evaluations.

In addition to microscopic observation, the IOP and pupildiameter are two important indicators for evaluating the pharma-cological action of the released drug. Fig. 4a shows the IOP profile ofthe four groups. In the Ctrl groups, the glaucomatous eyes withouttreatment with MSN-based carriers maintained a higher level of IOPelevation (20.0–26.5 mmHg above baseline) within postoperative3 weeks. The intracameral injection of various p/GM samples intoglaucomatous rabbits resulted in a significant decrease in theIOP values at the postoperative 12 h. Note that the IOP of p/MSN-and p/GM-0.5-injected eyes dramatically increased after 12 h and2 days, respectively. In contrast, in the p/GM-0.05 case, theIOP remained persistently stable and normal during the wholefollow-up period. Our results also indicate that the change in IOP

induced by the intracameral administration of pilocarpine-containing MSN injections can be correlated with the alterationof the anterior chamber depth, as demonstrated by the slit-lampbiomicroscopic observations. The time-course of the pupildiameter change is shown in Fig. 4b. In the Ctrl groups, therewas no significant decrease in the pupil diameter over timeduring the postoperative examinations. For the glaucomatouseyes receiving the p/MSN and p/GM samples, the released drugelicited a strong pupillary constriction at 6 h postoperatively.Although the intracamerally administered pilocarpine couldprovoke a continued miotic response, the pupil size of p/MSN-and p/GM-0.5-injected eyes was restored to baseline on postoperativeday 1 and 3, respectively. By contrast, in the p/GM-0.05 case, intenseand sustained miosis was observed throughout the entire 3 weeks ofthe experiment, indicating the long-lasting in vivo pharmacologicaleffects. The results shown in Fig. 4 could also be verified by Fig. S7(ESI†), which illustrated that the minimum effective therapeuticdose (10 mg mL!1) of pilocarpine to treat glaucoma for at least 21days was achieved by drug release from the p/GM-0.05 sample only.The difference in the profile of pupil response to pilocarpinecould reflect the importance of the ratio of gelatin to MSN inthe development of intracamerally administered particularformulations.

ConclusionsIn summary, herein, we demonstrate the synthesis of gelatin-functionalized MSNs with a high surface area and a decentparticle size that are suitable for glaucoma treatment viaintracameral administration. The degradation of gelatin viaup-regulated expression of MMP-2 in the anterior chamberprovided a partially acidic microenvironment, inducing thepilocarpine to release from MSNs. We controlled the thicknessof the gelatin coating on MSN to control the drug releasebehavior (i.e. sustained release), thus enhancing the deliveryefficacy. According to the in vitro and in vivo results, thesynthesized p/GM-0.05 sample exhibited a long-lasting releaseprofile and an ability to successively reduce IOP. We suggestthat this novel drug delivery system can also be applied for thetreatment of other intraocular diseases in the future.

Conflicts of interestThere are no conflicts to declare.

AcknowledgementsThis research was supported by the National Science Council ofTaiwan (101-2628-E-002-015-MY3, 101-2623-E-002-005-ET and101-2923-E-002-012-MY3), National Taiwan University (102R7842,102R7740 and 102R104100), Chang Gung Memorial Hospital(CMRPD2C0071-73), and National Health Research Institute(NHRI) of Taiwan (03A1-BNMP14-014 and NHRI-EX106-10311EC). We would like to thank C.-Y. Chien of the Ministry

Fig. 4 (a) The time-course value change of intraocular pressure (IOP)after pilocarpine administration. (b) The decrease of pupil diameter afterpilocarpine administration. Follow-up time point: glaucomatous eye beforeoperation (GL); hour (H) and day (D) after pilocarpine administration. Anasterisk indicates statistically significant differences (*P o 0.05; n = 6) ascompared to the Ctrl group.

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of Science and Technology (National Taiwan University) for theassistance in TEM experiments.

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