Intraocular Pharmacokinetics of 10-fold Intravitreal Ranibizumab Injection Dose in Rabbits.

Purpose: To investigate intraocular pharmacokinetics of 10-fold dose of intravitreally injected ranibizumab compared with the conventional dose in an experimental model. Methods: Ranibizumab 30 µL at 10 mg/mL (conventional) and 100 mg/mL (10-fold) doses was injected separately into each eye of 28 rabbits. Ranibizumab concentrations in the aqueous humor, vitreous, and retina were estimated at each time period after injection, using enzyme-linked immunosorbent assay. The pharmacokinetic properties of ranibizumab were determined using a one-compartment model in all three ocular tissues. The time-concentration profile and predictive trends were plotted to determine drug efficacy in the retina. Results: Maximum concentrations after conventional and 10-fold dosing were observed in the retina at 1 and 4 days after injection, respectively. The half-life of ranibizumab after conventional and 10-fold dosing did not differ in the anterior chamber and vitreous, whereas the half-life was prolonged approximately twice with the 10-fold dose in the retina (36.74 h vs. 76.85 h) and serum (91.93 h vs. 179.01 h). Similarly, the estimated time for ranibizumab concentration in the retina over 27 ng/mL (minimum effective concentration of ranibizumab) was prolonged approximately twice with the 10-fold dose (1315 h [55 days] vs. 2393 h [100 days]). No adverse effects were observed in either group. Conclusions: The retinal half-life and concentration of ranibizumab in rabbit eyes were increased approximately twice after a 10-fold dose compared with the conventional dose. This finding indicates a possibility to lengthen the injection interval to improve the efficacy of ranibizumab in human eyes. Translational Relevance: Our results highlight the potential for clinical application of a high-dose (10-fold) of anti-VEGF agents to prolong the intravitreal injection intervals, simultaneously improving the drug efficacy.

Therapeutic Effect of Controlled Release of Dual Growth Factor Using Heparin-Pluronic Hydrogel/Gelatin-Poly (Ethylene Glycol)-Tyramine Hydrogel System in a Rat Model of Cavernous Nerve Injury.

The number of cases of erectile dysfunction (ED) caused after radical prostatectomy (RP) prostate cancer treatment is increasing steadily. Although various studies have been conducted for treatment of post-RP ED, there is still a need for more effective methods. A dual growth factor incorporated heparin-pluronic/gelatin-poly(ethylene glycol)-tyramine (HP/GPT) hydrogel, which consists of a basic fibroblast growth factor (bFGF)-loaded HP hydrogel and nerve growth factor (NGF)-loaded GPT hydrogel, can control dose and rate of growth factor release. In this study, we demonstrated that dual growth factor incorporated HP/GPT hydrogel could further improve erectile function in a rat model of bilateral cavernous nerve injury (BCNI). We showed that erectile function was decreased after BCNI, but it was further improved by treatment with a dual growth factor incorporated HP/GPT hydrogel compared with groups treated with single growth factor in a rat model of cavernous nerve injury. Also, we observed an increase in cyclic guanosine monophosphate (cGMP) levels in the dual growth factor group when compared with the groups treated with single growth factor. This effect was associated with greater upregulation of nitric oxide synthase and endothelial nitric oxide synthase expression in the penile tissue of the group treated with dual growth factor incorporated HP/GPT than in the other experimental groups. Apoptosis in the penile tissue treated with the dual growth factor incorporated HP/GPT hydrogel was lower than those treated singly with either bFGF or NGF incorporated GPT hydrogel. Both α-smooth muscle actin and CD31 expression increased in the group treated with dual growth factor incorporated HP/GPT hydrogel when compared to in the other experimental groups. Altogether, our results proved that the sequential and continuous release of growth factors from dual growth factor incorporated HP/GPT hydrogel prevented fibrosis and nerve damage induced by BCNI in the corpus cavernosum, and promoted the recovery of erectile function. Dual growth factor incorporated HP/GPT hydrogel may be a potent clinical application for the treatment of post-RP ED and could potentially be used various biomedical application in tissue regnerative medicine.

Hydrogen Peroxide-Releasing Hydrogels for Enhanced Endothelial Cell Activities and Neovascularization.

Reactive oxygen species (ROS) have been implicated as a critical modulator for various therapeutic applications such as treatment of vascular disorders, wound healing, and cancer treatment. Specifically, growing evidence has recently demonstrated that transient or low levels of hydrogen peroxide (H2O2) facilitates tissue regeneration and wound repair through acute oxidative stress that can evaluate intracellular ROS levels in cells or tissues. Herein, we report a gelatin-based H2O2-releasing hydrogel formed by dual enzyme-mediated reaction using horseradish peroxidase and glucose oxidase (GO x). The release behavior of H2O2 from the hydrogel matrices can be precisely controlled by varying the GO x concentrations. We demonstrate that H2O2-releasing hydrogels with the optimal condition increase transient upregulation of intracellular ROS levels in the endothelial cells (ECs), enhance proliferative activities of ECs in vitro, and facilitate neovascularization in ovo. We suggest that our H2O2-releasing hydrogels hold great potential as an injectable and dynamic matrix for the treatment of vascular disorders as well as in tissue regenerative medicine.

Dual Enzyme-Triggered In Situ Crosslinkable Gelatin Hydrogels for Artificial Cellular Microenvironments.

Horseradish peroxidase (HRP) and hydrogen peroxide (H2 O2 )-mediated crosslinking reaction has become an attractive method to create in situ forming hydrogels. While the crosslinking system has been widely utilized, there are certain issues require improvement to extend their biomedical applications, including creation of stiff hydrogels without compromising cytocompatibility due to initially high concentrations of H2 O2 . A gelatin-based hydrogels formed through a dual enzyme-mediated crosslinking reaction using HRP and glucose oxidase (GOx) as an H2 O2 -generating enzyme to gradually supply a radical source in HRP-mediated crosslinking reaction is reported. The physicochemical properties can be controlled by varying enzyme concentrations. Furthermore the hydrogel matrices provide 3D microenvironments for supporting the growth and spreading of human dermal fibroblasts with minimized cytotoxicity, despite the cells being encapsulated within stiff hydrogels. These hydrogels formed with HRP/GOx have great potential as artificial microenvironments for a wide range of biomedical applications.

Targeted doxorubicin nanotherapy strongly suppressing growth of multidrug resistant tumor in mice.

The rational design of nanomedicine to treat multidrug resistant (MDR) tumors in vivo is described in the study. We prepared multifunctionalized Pluronic micelles that are already well-established to be responsive to low pH and redox in order to systemically deliver doxorubicin (DOX) to MDR tumors. Folic acids (FAs) were introduced on the micelle surface as tumor-targeting molecules. In vitro, the DOX-loaded micelles exerted high cytotoxicity in the DOX-resistant cells by bypassing MDR efflux. Cellular uptake studies clearly demonstrated that FA-conjugated DOX micelles (FA/DOX micelles) were efficiently internalized and accumulated in the MDR cells. In vivo studies indicated significant efficacy of FA/DOX micelles for MDR tumors in mice, and that the volume of tumors was 3 times smaller in this group than that of tumors in the free DOX group, and 8 times smaller than the tumors in the saline group. To the best of our knowledge, this methodology has been recognized to have significantly high efficacy, compared to previously reported DOX nanoparticle formulations. This superior anti-tumor efficacy of FA/DOX micelles in MDR tumor-bearing mice can be attributed to FA-targeted and -mediated endocytosis, inhibition of MDR effect, and subsequent DOX release triggered by dual stimuli (low pH and redox) inside the tumor. Given the promise of the multifunctional micelle mediated delivery on inhibition of MDR tumor growth, FA/DOX micelle platform is a much sought after goal for cancer chemotherapy, especially for cancers resistant to anticancer drugs.

Dual growth factor-loaded in situ gel-forming bulking agent: passive and bioactive effects for the treatment of urinary incontinence.

Stress urinary incontinence (SUI) is one of the major medical problems for adult females and has a devastating effect on their quality of life. The major cause of the development of the SUI is dysfunction of the urethral supporting tissues as a result of aging and childbirth. In this study, in situ gel-forming bulking agent loaded with dual growth factors, nerve growth factor (NGF) and basic fibroblast growth factor (bFGF), was fabricated. The bulking agent consisted of three components; (i) polycaprolactone (PCL) beads, (ii) bFGF-loaded nanogels, and (iii) NGF-loaded in situ gel forming solution. The bulking agent can provide an initial passive bulking effect (from the PCL beads) and regenerate malfunctioning tissues around the urethra (from the sequential and continuous release of growth factors from the hydrogel) for the effective treatment of SUI. The PCL beads were located stably at the applied urethra site (urinary incontinent SD rat) without migration to provide a passive bulking effect. The sequential release of the growth factors (NGF within a week and bFGF for more than 4 weeks) from the bulking agent provided regeneration of damaged nerve and smooth muscle, and thus enhanced biological function around the urethra. From the findings, we suggest that dual growth factor (NGF and bFGF)-loaded in situ gel-forming bulking agent may be a promising injectable bioactive system for the treatment for SUI.

Macro/Nano-gel composite as an injectable and bioactive bulking material for the treatment of urinary incontinence.

Many women around the world are suffering from urinary incontinence, defined as the unintentional leakage of urine by external abnormal pressure. Although various kinds of materials have been utilized to treat this disease, therapies that are more effective are still needed for the treatment of urinary incontinence. Here, we present a macro/nanogel composed of in situ forming gelatin-based macrogels and self-assembled heparin-based nanogels, which can serve as an injectable and bioactive bulking material for the treatment of urinary incontinence. The hybrid hydrogels were prepared via enzymatic reaction in the presence of horseradish peroxidase and hydrogen peroxide. Incorporating a growth factor (GF)-loaded heparin nanogel into a gelatin gel matrix enabled the hybrid gel matrix to release GF continuously up to 28 days. Moreover, we demonstrated that the hydrogel composites stimulated the regeneration of the urethral muscle tissue surrounding the urethral wall and promoted the recovery of their biological function when injected in vivo. Thus, the macro/nanohydrogels may provide an advanced therapeutic technique for the treatment of urinary incontinence as well as an application for regenerative medicine.

In situ SVVYGLR peptide conjugation into injectable gelatin-poly(ethylene glycol)-tyramine hydrogel via enzyme-mediated reaction for enhancement of endothelial cell activity and neo-vascularization.

Tissue engineering therapies require biocompatible and bioactive biomaterials that are capable of encouraging an angiogenic response for effective tissue regeneration. In this study, a SVVYGLR peptide, which functions as a potent angiogenic factor, was conjugated into injectable gelatin-poly(ethylene glycol)-tyramine (GPT) hydrogels in situ to enhance endothelial cell activities and neo-vascularization. SVVYGLRGGY (SV-Y) conjugated GPT (SV-GPT) hydrogels were formed in situ via enzyme-mediated reaction using horseradish peroxidase (HRP) and hydrogen peroxide (H(2)O(2)). The physico-chemical properties were characterized and could be controlled depending on the feed peptide and H(2)O(2) concentration. The concentration of conjugated peptide ranged from 0.37 to 0.81 µmol/mL, and the elastic moduli (G') of the hydrogels were 600-4900 Pa. In vitro cell studies using human umbilical vein endothelial cells (HUVECs) and in vivo subcutaneous injection studies were performed to confirm the effect of the SVVYGLR peptide on HUVEC activity and neo-vascularization. Obtained results demonstrated that the in situ conjugation of SVVYGLR sequences into phenol residues of GPT hydrogels enhanced the activity of HUVECs in vitro and stimulated the formation of new blood vessels in the hydrogel matrices in vivo. From the results, we suggest that in situ conjugation of SV-Y to GPT hydrogels via the enzymatic reaction may be an efficient tool to prepare injectable bioactive hydrogels that can enhance endothelial cell activities and promoting angiogenesis for tissue regeneration.

Synthesis and characterizations of in situ cross-linkable gelatin and 4-arm-PPO-PEO hybrid hydrogels via enzymatic reaction for tissue regenerative medicine.

In situ cross-linkable hybrid hydrogels composed of gelatin and 4-arm-polypropylene oxide-polyethylene oxide (Tetronic) was developed as an injectable scaffold for tissue regeneration. The gelatin was modified by hydroxyphenyl propionic acid (HPA) and the Tetronic was conjugated with tyramines (Tet-TA). The hydrogels were rapidly formed by mixing the polymer solutions containing horseradish peroxidase (HRP) and hydrogen peroxide (H(2)O(2)). The gelation time and mechanical properties of the hydrogels could be controlled by varying the HRP and H(2)O(2) concentrations. In vitro degradation study of the hybrid hydrogels was carried out using collagenase and the prolonged proteolytic degradation was obtained due to the presence of the Tetronic. Human dermal fibroblast (hDFB) was cultured in the hydrogel matrices to evaluate the cyto-compatibility. The encapsulated cells were shown to be highly viable and spread over the gel matrices, suggesting that the hybrid hydrogels have an excellent cyto-compatibility. The hydrogels were also subcutaneously injected in the back of mice and the results demonstrated that the hydrogels were rapidly formed at the injected site. From these results, we demonstrate that the in situ cross-linkable hydrogels formed by hybridization of gelatin and Tetronic via enzyme-mediated reactions hold great promise for use as injectable matrices for tissue regenerative medicine due to their tunable physico-chemical properties and excellent bioactivity.