Evaluation of an injectable thermosensitive hydrogel as drug delivery implant for ocular glaucoma surgery.

In this study, a biodegradable thermo-sensitive hydrogel from poly(trimethylene carbonate)15-F127-poly(trimethylene carbonate)15 (PTMC15-F127-PTMC15) was designed and evaluated as an injectable implant during ocular glaucoma filtration surgery in vivo and in vitro. Mitomycin C (MMC) was loaded into this hydrogel for controlled released to prolong the efficacy and to reduce the long-term toxicity. The properties of the hydrogel were confirmed using 1H NMR and gel permeation chromatography (GPC). Compared to the Pluronic F127 hydrogel, the PTMC15-F127-PTMC15 hydrogel showed a good solution-gel transition temperature at 37°C, a lower work concentration of 5% w/v and a longer mass loss time of more than 2 weeks. The in vitro study showed that the drug could be released from PTMC15-F127-PTMC15 (5% w/v) hydrogel for up to 16 days with only 57% of drug released in the first day. Moreover, the cell toxicity, which was tested via LDH and ANNEXIN V/PI, decreased within 72 h in human tenon's fibroblast cells (HTFs). The in vivo behavior in a rabbit glaucoma filtration surgery model indicated that this hydrogel loaded with 0.1 mg/ml MMC led to a better functional bleb with a prolonged mean bleb survival time (25.5±2.9 days). The scar tissue formation, new collagen deposition and myofibroblast generation appeared to be reduced upon histological and immunohistochemistry examinations, with no obvious side effects and inflammatory reactions. The in vitro and in vivo results demonstrated that this novel hydrogel is a safe and effective drug delivery candidate in ocular glaucoma surgery

Postoperative Histological characteristics evaluations in rabbit models.

<p>The rabbits were divided into four treatment groups: Filtration surgery only group (a). Group injected with 0.1 ml 5% w/v PTMC₁₅-F127-PTMC₁₅/MMC (0.1 mg/ml) hydrogel in filtration surgery (b). Treat with 0.5 mg/ml MMC for 5 minutes during filtration surgery group (c). Group with 0.1 ml 5% w/v PTMC₁₅-F127-PTMC₁₅ injected in filtration surgery (d). (A) In H&E staining, the subconjunctival scarring in group ‘a’ were noticed with fibrocellular scar tissue and with closed sclerotomy, group ‘b’ had loosely arranged subepithelial connective tissues, group ‘c’ had a few inflammatory cells and thin subconjunctival tissue, and group ‘d’ was similar to group ‘a’. (B) Massones Trichrome staining showed group ‘a’ and ‘d’ had more dense collagen tissues in sub-conjunctiva space than group ‘b’ and ‘c’. (C) Immunohistochemical examination of α-SMA illustrated that lesser α-SMA specific myofibroblast were differentiated in group ‘b’ and ‘c’. CE: conjunctival epithelium; TC: Tenon's Capsule;</p

Corneal Endothelial Cell Count (/mm², mean±SD).

<p>Corneal Endothelial Cell Count (/mm², mean±SD).</p

Random divided animal groups.

<p>Random divided animal groups.</p

Postoperative evaluations in rabbit models.

<p>The rabbits were divided into four treatment groups: ‘Control group’ is the filtration surgery only group (a). Group injected with 0.1 ml 5% w/v PTMC₁₅-F127-PTMC₁₅/MMC (0.1 mg/ml) hydrogel in filtration surgery (b). Treat with 0.5 mg/ml MMC for 5 minutes during filtration surgery group (c). Group with 0.1 ml 5% w/v PTMC₁₅-F127-PTMC₁₅ injected in filtration surgery (d). (A) Survival curve of filtering blebs after glaucoma filtration surgery using Kaplan-Meier analysis. Filtering blebs in group ‘a’ (n = 4) and ‘d’ (n = 4) mostly failed within 1 weeks. Group ‘b’ (n = 4) markedly increased the bleb survival period. In group ‘c’ (n = 4), the bleb survived nearly 4 weeks. Kaplan-Meier analysis showed a significant difference in the survival distributions among the four groups. (Log Rank  = 75.121, p<0.01). (B) IOP changes within postoperatively 28 days. The results were obtained and expressed as the mean± SD. There is no significant difference between each group (p>0.05). (C) Proliferating cell nuclear antigen (PCNA) was analyzed in each group at post-operative day 28.(D) Apoptotic cells were indicated by TUNEL assay in each group at day 28.</p

In vitro cell toxicity of the HTFs cocultured with PTMC₁₅-F127-PTMC₁₅ copolymer in different concentration and with F127.

<p>(A) primary cultured HTFs were identified with microscopy and (B) with immunofluorescence. (a) Negative control using DAPI dyed the nucleus (blue) of HTFs. (b) Vimentin antibody (red) was added. to primary HTFs (c) Fibronectin antibody (green) was added. (d) Melt. (C) LDH release assay were used to analyze cell toxicity in different concentration of PTMC₁₅-F127-PTMC₁₅ (3%, 5% and 10% w/v) and in 18% w/v F127 within 3 days. (D) Total apoptosis of HTFs incubated with 0.1 ml 5% w/v PTMC₁₅-F127-PTMC₁₅ or 0.1 ml 18% w/v F127 were detected by Annexin V/PI staining using fluorescence-activated cell sorting (FACS). (E) Apoptotic changes between 5% w/v PTMC₁₅-F127-PTMC₁₅ and 18% w/v F127 at 3 days. LDH  =  lactate dehydrogenase. ‘Control’ one is the group with no treatment.</p

Characterization and the properties of the hydrogel.

<p>(A) GPC analysis (in THF at 40°C); (B) ¹H NMR spectrum of the block copolymer (in CDCl₃); (C) Phase diagrams by invert test; (D) mass lose profiles; E, MMC release kinetics of/from PTMC₁₅-F127-PTMC₁₅ hydrogel (5% w/v) and F127 hydrogel (18% w/v) in PBS (pH = 7.4) at 37°C, MMC (1 mg/ml).</p