Nano-Gel Composite as an Injectable and Bioactive Bulking

Apr 16, 2014 - ABSTRACT: Many women around the world are suffering from urinary incontinence, defined as the unintentional leakage of urine by externa...
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Macro/Nano-Gel Composite as an Injectable and Bioactive Bulking Material for the Treatment of Urinary Incontinence Kyung Min Park,† Joo Young Son,† Jong Hoon Choi,† In Gul Kim,‡ Yunki Lee,† Ji Youl Lee,‡ and Ki Dong Park*,† †

Department of Molecular Science and Technology, Ajou University, Suwon 443-749, Republic of Korea Department of Urology, Catholic University, Seoul St. Mary’s Hospital, Seoul 137-701, Republic of Korea

‡

ABSTRACT: 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.

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INTRODUCTION Many women around the world are suffering from urinary incontinence, defined as the unintentional leakage of urine by external pressure. Urinary incontinence is caused by sneezing or coughing and affects a patient’s quality of life.1,2 This disease occurs when tissues surrounding urethral wall, that is, the urethral sphincter, pelvic floor muscle, and nerve, are damaged, weakened, or loosened by aging or childbirth.3,4 Over the past decade, a myriad of therapeutic methods, including surgical repair, drug therapy, and implantation of bulking materials, have been explored to enhance urethral resistance by external abnormal pressure.5,6 Specifically, injectable materials (i.e., particulate or hydrogel systems), based on minimally invasive techniques, have attracted substantial interest as bulking matrices to narrow the urethral lumen.7 Although various kinds of injectable biomaterials have been commercialized and utilized to treat this disease, these passive bulking materials have critical drawbacks when used for long-term periods due to reabsorption and particle migration.7 Indeed, these techniques cannot regenerate the urethral muscle tissue that regulates the exit of urine. With advances in materials science, innovative © 2014 American Chemical Society

approaches based on regenerative medicine to regenerate damaged or weakened urethral tissues have been widely investigated using various biomaterials.8−10 Some research groups demonstrated that injection of cells (i.e., stem cells, progenitor cells, or adult cells) encapsulated with biomaterials at the defect promoted sphincter regeneration and restored the biological function in animal models and clinical applications.11,12 In addition, sustained release of growth factors (GFs) from the bulking materials, such as basic fibroblast growth factor (bFGF) and nerve growth factor (NGF), has been implicated as a promising strategy to promote the regeneration of the urethral muscle and thus recover urethral muscle function.13,14 However, for effective therapeutics, it is still a challenge to overcome the limitations of GF delivery in vivo, such as the very short half-life in vivo.15 Many researchers have endeavored to design biomaterials that allow for the long-term GF delivery and the retention of GF activity. One of the Received: December 5, 2013 Revised: April 15, 2014 Published: April 16, 2014 1979

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N-acetyl protons of heparin), and 3.2 and 4−4.2 (m, protons on the anomeric carbon of heparin). Fabrication of HP Nanogels. The bFGF-loaded HP nanogels (HP/bFGF) were prepared by a direct dissolution method as described previously.19,20 Briefly, the HP conjugate (100 mg) was dissolved in distilled water (49 mL) below 4 °C, followed by the addition of bFGF solution (1 mL, 22 μg/mL). The mixture was incubated at 37 °C and 100 rpm for 12 h to yield nanogel formation through hydrophobic and electrostatic interactions.19 Unloaded GF was completely removed through a salting-out method in which an aqueous PEO solution (10 wt %) was added to the mixture, followed by filtration.20 Finally, the HP/bFGF solution was lyophilized, and the amount of bFGF was quantified using the DuoSet ELISA kit (R&D Systems, Minneapolis, MN, U.S.A.). Characterization of the HP Nanogels. To measure the size of the nanogels, dynamic light scattering (DLS) measurements were performed with a Malvern Zetasizer Nano Z instrument (Malvern Instruments, U.K.) as previously described.19 The nanogel was dissolved in distilled water at 37 °C to a concentration of 0.1 mg/ mL. The hydrodynamic diameter of the nanogels was calculated using a Laplace inversion program (CONTIN). The morphologies of the nanogels were observed using a transmission electron microscope (TEM, CM-20 Philips, Eindhoven, Netherlands) as previously described.19 Specimens were fabricated by dripping a nanogel solution on a copper grid, followed by air drying for 1 min and negative staining with 1 wt % phosphotungstic acid (Sigma-Aldrich). TEM images were acquired at an operating voltage of 300 kV. Preparation of Hybrid Macro/Nanogel Composites. The hybrid hydrogels were prepared by simple mixing of the nanogel and GPT solutions in the presence of HRP and H2O2, which induced enzymatic cross-linking reactions to form a polymer network. To prepare the hybrid hydrogels, 3 wt % GPT solution including 0.025 mg/mL HRP and 1 mg/mL HP/bFGF was mixed with the same volume of 3 wt % GPT solution including 0.025% H2O2. The mixture was gently shaken at 37 °C. All solutions used were dissolved in 0.01 M phosphate buffered saline (PBS) at pH 7.4. The final concentrations of GPT polymer, HRP, H2O2, and GF were 3 wt %, 0.0125 mg/mL, 0.0125%, and 1 μg/mL, respectively. In Vitro bFGF Release. To investigate the GF release, we fabricated 100 μL of hybrid hydrogels in 1 mL microtubes as described above. The gels were incubated with 1 mL of 0.01 M PBS including 1% bovine serum albumin (BSA, Sigma-Aldrich), and we collected 1 mL of the medium and replaced the same volume of the medium at predetermined intervals. The samples were stored at −80 °C before the ELISA (BD System) analysis according to the manufacturer instructions. In Vivo Animal Studies. To evaluate the urethral muscle regenerating-ability of the hybrid hydrogels, we selected Sprague− Dawley rat urinary incontinent animal model as described previously.21 Briefly, the urinary incontinent rats were prepared by denervation of the sciatic nerve. One week after the denervation, we injected the hybrid hydrogels into the periurethral submucosa, with microscopic guidance to minimize surrounding tissue damage. For in vivo experiments, we prepared different types of hybrids, and all solutions were sterilized by filtering using a syringe filter with a 0.2 μm pore size membrane. The hybrids were injected at the defect using a dual syringe kit with 26-gauge needles. All animal studies were approved by the Animal Care Committee of the Catholic University of Korea, and all procedures were performed according to the appropriate guidelines. Histological and Immunohistochemical Analysis. After the leaking pressure point (LPP) measurement, the specimens were vertically cut into 4 μm slices and subjected to immunohistochemistry for α-smooth muscle actin (SMA), as previously described.21 To quantify SMA positive (SMA+) area, we analyzed the image using ImageJ software and calculate degree of SMC regeneration as percentages of SMC+ area in healthy urethral tissue. LPP Measurement and Contractility Test. To evaluate the physical urethral sphincter function, after 4 weeks of implantation, we performed the LPP measurement using a vertical tilt/intravesical pressure clamp model.21 Briefly, the feces in the distal colon and

representative approaches is to incorporate heparin molecules into the biomaterials. These molecules can induce sustained release of GFs and keep their structural stability through structural specific interaction and electrostatic interactions between GFs and heparin molecules, as well as increase their affinity for cell receptors.16 For effective treatment of urinary incontinence, it is required that the materials play a role as an immediately passive support and a bioactive tissue regeneration source. These materials may provide effective therapeutic tools in promoting the healing of urinary incontinence through a physical bulking effect and biological recovery of urethral muscle function. In our previous studies, we developed in situ cross-linkable gelatin-poly(ethylene glycol)-tyramine (GPT) hydrogels as an injectable material for regenerative medicine and drug delivery.17,18 We also reported self-assembled heparin-pluronic (HP) nanogels as a sustained protein delivery carrier.19 In this study, we report a hybrid macro/nanogel composed of a GPT macrogel and a HP nanogel that can serve as an injectable and bioactive bulking material. We hypothesize that injectable macrogel (GPT) can play a role as a physical bulking reagent and the nanogels (HP) allows sustained release of GFs from the matrix to promote urethral tissue regeneration. We demonstrate that incorporation of a GF-loaded HP nanogel into a GPT hydrogel allows for sustained release of GF from the hybrid matrix. Moreover, the bioactive bulking material promoted the regeneration of the urethral muscle and recovered the biological function of the urethra. Thus, we expect that our approach can provide an advanced therapeutic technique for the treatment of urinary incontinence as well as applications for regenerative medicine.

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MATERIALS AND METHODS

Materials. Gelatin (type A from porcine skin, >300 bloom), poly(ethylene glycol) (PEG, molecular weight = 4000 g/mol), horseradish peroxidase (HRP, type VI, 250−330 units/mg solid), 4dimethylaminopyridine (DMAP), p-nitrophenylchloroformate (PNC), hydrogen peroxide (H2O2), and succinic anhydride (SA) were supplied from Sigma-Aldrich (St. Louis, MO, U.S.A.). Tyramine (TA) and heparin sodium (150 unit/mg) were obtained from Acros (Pittsburgh, PA, U.S.A.). Pluronic F127 (Plu, molecular weight = 12600 g/mol) was purchased from BASF Korea (Seoul, Korea). Triethylamine (TEA) and dimethyl sulfoxide (DMSO) were purchased from Kanto Chemical Co. (Tokyo, Japan). All chemicals and solvents were used without further purification. Synthesis of GPT and HP Conjugates. The GPT polymer was synthesized as previously described.17 Briefly, we synthesized GPT using a two-step conjugative reaction sequence: (1) we modified PEG using PNC to give an amine reactive PEG (PEG−PNC); (2) we then coupled tyramine to PEG−PNC without any catalysts in DMSO and then mixed with gelatin solution, followed by dialysis and lyophilization. We also synthesized HP conjugate as previously reported.19 For the synthesis of HP, we first modified the terminal hydroxyl groups of Plu with SA (molar ratio of Plu to SA = 1:2.2) using DAMP and TEA as catalysts to yield carboxylated Plu (Plu-(COOH)2). The HP polymer was synthesized by coupling heparin and Plu-(COOH)2 through a carbodiimide-mediated reaction, followed by dialysis and lyophilization. The chemical structures were characterized by 1 H NMR spectrometry (Bruker AMX-500 NMR spectrometer, Billerica, MA, U.S.A.). 1H NMR (400 MHz, D2O) of GPT: δ 4.8 (m, proton on the anomeric carbon of gelatin), 0.8−4.6 (m, alkyl protons of gelatin), 3.5−3.8 (s, ethyl protons of PEG), and 6.8 and 7.1 (d, aromatic protons of TA). 1H NMR (400 MHz, D2O) of HP: δ 3.65 (m, ethyl protons of PEO in Plu), 1.12 (d, propyl protons of PPO of Plu), 1.9 (s, 1980

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Figure 1. Regeneration strategy and fabrication of hybrid macro/nanogel composites. (a) Schematic representation of the urethral muscle regeneration using an injectable and bioactive bulking hydrogel. (b) bFGF-loaded HP nanogel formation through hydrophobic interactions. bFGF is loaded into the HP nanogel through structural and electrostatic interactions between the growth factor and the heparin moieties. The bFGF-loaded HP nanogel is dispersed in GPT polymer solution before the injection, and the macro/nanogel composite is formed via an enzymatic reaction, yielding either carbon−carbon bonds at the ortho positions or carbon−oxygen bonds between the ortho carbon and the phenoxy oxygen. rectum were evacuated by gentle massage via a midline abdominal incision after relaxation of the rats, and we sutured around the proximal end of the distal colon to avoid any further migration of feces that would affect the LPP measurement. The rat was then mounted on a tilt table and placed in a vertical position. The intravesical pressure was increased in 1−3 cm H2O increments until leakage was observed, and the leak point was determined as the LPP. We also performed a contractility test to confirm the physical function and regeneration surrounding the urethral tissue according to a previously described procedure.21 Briefly, we sacrificed the animals 4 weeks after the injection. We then dissected, trimmed, weighed, and detubularized the explantation in a spiral fashion to produce a 1 × 10 mm2 tissue strip. The strip was placed on an isometric force transducer (FT03; Grass Instruments, Quincy, MA, U.S.A.). Next, the strip was stimulated at 32 Hz with 1 ms pulses at 80 V using an S48 stimulator (Grass Instruments) for 30 s. We monitored the contraction of the strip caused by the electric field stimulation using an isometric force transducer and recorded the signals using a commercial data acquisition system (PowerLab; AD Instrument, Mountain View, CA, U.S.A.). Statistical Analysis. We performed the in vitro bFGF release study in triplicate, with duplicate readings, and all measurements of the in vivo study, including the LPP measurement and contractility test, were processed in triplicate to guarantee the reliability of the results. We performed statistical analysis using GraphPad Prism 4.02 (GraphPad Software Ind., La Jolla, CA, U.S.A.). We also used this software to determine the significance. Significance levels, determined using post-tests, were set at *p < 0,05, **p < 0.01, and ***p < 0.001.

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carrier, with unique properties such as a heparin moiety and a hydrophobic self-assembled nanostructure.19,22,23 Thus, we chose the HP nanogel as the GF carrier, which allowed for sustained release of GF through specific interactions between the heparin moiety and GF. We synthesized the HP nanogels by coupling carboxylated Pluronic to the hydroxyl group of heparin via a carbodiimide-mediated reaction and characterized the chemical structure of the conjugate using 1H NMR spectroscopy, showing the specific peaks as described above. We prepared the nanogels via hydrophobic interactions of the polypropylene (PPO) groups of Plu to yield GF-loaded HP nanogels (Figure 1b). We chose bFGF because this GF plays a critical role in the regeneration of the urethral muscle and nerve, resulting in recovery of urethral function.13 bFGF was loaded during HP nanogel formation through interactions between bFGF and the heparin moiety (i.e., electrostatic and steric interactions).24 Thus, most of the GF seems to be located in heparin-containing regions of the nanogel structure. The GF loading amount was approximately 200 ng/mg of HP polymer. To characterize the HP nanogel size and morphology, we performed DLS and TEM analyses. We observed round-shaped HP nanogels (Figure 2a) and a similar size distribution in each sample. Interestingly, we found that bFGF-loaded HP nanogels showed slightly smaller nanogels (7.5−58.8 nm, Figure 2b) compared to HP nanogels (7.5−78.8 nm), indicating that the nanogel size decreased with encapsulated bFGF. This result is consistent with a previous study demonstrating that the interactions between bFGF (pI 9.6) to negatively charged heparin segments through steric and electrostatic interactions induces size reduction due to structural shrinkage.19 In summary, we successfully fabricated HP/bFGF through thermodynamic self-assembly with high loading efficiency (90.8 ± 1.0%, 199.9 ± 2.2 ng/mg of HP polymer) and round-shaped nanosized distribution (from 7.5 to 78.8 nm).

RESULTS AND DISCUSSION

Fabrication of HP Nanogels. We hypothesized that encapsulating HP/bFGF into in situ-forming GPT hydrogels would promote regeneration of the urethral muscle and the nerve surrounding the urethral wall through sustained release of GF (Figure 1a). In our previous study, we demonstrated that a HP nanogel has great potential as a sustained protein delivery 1981

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GPT hydrogel incorporating bFGF in an HP nanocarrier (Figure 3). In the case of the free bFGF, over 70% of the

Figure 3. Sustained release of GF. Cumulative release profiles of bFGF from the GPT/HP hydrogel matrix.

loaded bFGF was released from the hydrogel matrix during 1 day; in contrast, 22% of the total loaded bFGF was released from the GPT/HP gel matrix after 1 day. In addition, after the 22% initial burst, the GPT/HP/bFGF gel matrix exhibited sustained release for 28 days. This sustained release could have occurred due to the specific interactions (steric and electrostatic) between the heparin moiety of the HP nanogels and bFGF. This result clearly demonstrates that our macro/nanogel matrix induces sustained release of GF and has great potential as an injectable and bioactive bulking material for the treatment of urethral incontinence. Enhanced Urethral Muscle Regeneration. To evaluate the effect of the GPT/HP hydrogels on urethral muscle regeneration in vivo, we injected the hybrid hydrogels into animal defects and examined the animals up to 28 days. We first performed immunohistochemistry with α-SMA staining to analyze urethral muscle regeneration (Figure 4). Notably, we found that the GPT/HP/bFGF hybrid composite showed a significantly higher density (84.9 ± 15.7%) of urethral muscle surrounding the urethral wall compared with the other groups, demonstrating that the GPT/HP hybrid hydrogel promoted urethral muscle regeneration via sustained release of bFGF, although this effect may be limited to completely organize urethral muscles compared to healthy tissues. Interestingly, the GPT (without GF) and GPT/bFGF (without carrier) groups exhibited a higher density (GPT, 48.5 ± 5.1%; GPT/bFGF, 49.5 ± 5.9%) of urethral muscle surrounding the urethral walls than those of the nontreated groups (32.8 ± 3.5%), but there was no significant difference between each group. These results demonstrated that the GPT hydrogels affected the regeneration of urethral muscle and that bFGF-loaded GPT hydrogels without carrier exhibited a similar density of urethral muscle compared to GPT hydrogels without GF. This observation was expected due to the foreign body reaction. Implantation of biomaterials induces the foreign body reaction during wound healing, which may allow for tissue ingrowth from surrounding tissue as well as blood vessel invasion into the materials. We anticipate that the healing process is helpful for urethral muscle regeneration, even when the hydrogels are implanted in vivo, although this effect may be limited. The wound healing and foreign body reaction might affect the regeneration of urethral muscle during the implantation. To evaluate the functional recovery of the urethral tissue, we performed LPP and contractility analyses. After 28 days of implantation, we first measured the LPP of the experimental groups (Figure 5). We observed that the mean value of the LPP in the denervation group (30.2 ± 2.5 cm H2O) was significantly

Figure 2. Characterization of self-assembled HP nanogels. (a) TEM images of HP nanogels with or without bFGF. (b) Size distributions of HP nanogels with or without bFGF measured by DLS. Scale bars are 100 nm.

Preparation of GPT/HP Macro/Nano Hydrogels. In our previous study, we developed in situ cross-linkable GPT hydrogels, formed by HRP-mediated cross-linking in the presence of a low concentration of H2O2 for regenerative medicine applications.17,18 For this study, we selected the GPT hydrogel to generate an injectable and regenerative bulking material and combined the GPT hydrogel with the HP nanogel to promote urethral muscle regeneration. We prepared hybrid macro/nanogels through enzymatic cross-linking of a GPT polymer with HP nanogels in the presence of HRP and H2O2, yielding either carbon−carbon bonds at the ortho positions or carbon−oxygen bonds between the ortho carbon and the phenoxy oxygen (Figure 1b). In this study, we selected a gelatin-based material as a macrogel construct because gelatin has cell-response properties such as cell adhesion sites and proteolytic degradability, which are crucial for tissue ingrowth and remodeling for regeneration.25,26 In fact, our previous studies demonstrated that the GPT hydrogels are proteolytically degradable and have excellent cytocompatibility. In addition, we also found that our GPT hydrogels had great tissue compatibility, showing tissue ingrowth without a serious inflammatory reaction after subcutaneous injection into the back of rats.17 Furthermore, GPT hydrogels exhibited tunable properties, including gelation time (5−60 s) and mechanical strength (800−8400 Pa), showing a potential for a wide range of applications. In our pilot study, we injected three kinds of GPT hydrogels (1.8, 5.9, and 8.4 kPa) to confirm their durability in the defect site as it takes over 4−6 weeks to regenerate urethral tissue and recover biological function. Injected GPT hydrogels (1.8 and 5.9 kPa) degraded completely within 2 weeks, whereas GPT hydrogels (8.4 kPa) remained in the injection site. Thus, we selected hydrogels that formed within 10 s to prevent diffusion of the polymer precursor solution. The mechanical strength of the GPT hydrogel was 8.4 kPa to support tissue ingrowth and regeneration for the onemonth implantation required to complete muscle regeneration.21 In Vitro bFGF Release. To investigate the release pattern of bFGF from the GPT/HP hydrogels, we examined the release profile under physiological conditions (pH 7.4). We observed a significantly different release behavior between the GPT hydrogel encapsulating free bFGF (without carrier) and the 1982

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Figure 4. Regeneration of urethral muscle surrounding the urethra. (a) Immunohistochemistry images of α-smooth muscle actin-stained sections around the proximal urethra of the hydrogels after 28 days of implantation: green, urethral muscle; blue, DAPI. Scale bars are 1 mm. (b) Quantitative analysis of urethral muscle regeneration as percentages of SMC+ area in healthy urethral tissue. Significance levels were set at *p < 0.05, **p < 0.01, and ***p < 0.001. Values shown are mean ± SD (n = 3).

Figure 5. Bulking effect of the hydrogel composites. Leak point pressure (LPP) at 28 days of implantation of the macro/nanogel matrix (with or without bFGF). Significance levels were set at *p < 0.05, **p < 0.01, and ***p < 0.001. Values shown are the mean ± SD (n = 3).

Figure 6. Recovery of the biological function of the urethral muscle. Contractile response of the urethral strips at 28 days after implantation of the hydrogels (with or without bFGF). Significance levels were set at *p < 0.05, **p < 0.01, and ***p < 0.001. Values shown are the mean ± SD (n = 3).

lower than that of the healthy group (49.7 ± 3.8 cm H2O), suggesting that the animal defect was appropriately generated by the denervation of the sciatic nerve. The GPT/HP/bFGF group showed a significantly higher LPP level (48.7 ± 3.3 cm H2O) compared with the injury group (30.2 ± 2.5 cm H2O) and GPT group (37.0 ± 2.9 cm H2O) but not the GPT/bFGF group (42.3 ± 6.9 cm H2O). Interestingly, the GPT hydrogel without HP nanogels exhibited a significantly higher LPP level (GPT group, 37.0 ± 2.9 cm H2O; GPT/bFGF group, 42.3 ± 6.9 cm H2O) than that of injury group (30.2 ± 2.5 cm H2O) as GPT hydrogels served as physical bulking materials in defect site. To further understand the recovery of urethral muscle function, we conducted an electrical contractility test after 28 days of implantation (Figure 6). Notably, we found that the GPT/HP/bFGF group showed significantly increased muscle contraction (4.5 ± 0.7 g tension/g tissue) compared with the other defect groups (denervation: 2.6 ± 0.2 g tension/g tissue; GPT: 3.2 ± 0.4 g tension/g tissue; GPT/bFGF: 3.3 ± 0.4 g tension/g tissue). Indeed, the GPT/HP/bFGF group (4.5 ± 0.7 g tension/g tissue) exhibited a contraction similar to that of the normal urethral strip (4.0 ± 0.4 g tension/g tissue). This result can be explained by the fact that sustained release of GF enhanced the regeneration of urethral tissue surrounding the

urethra, which is helpful for the recovery of biological sphincter function. Collectively, our macro/nanohybrid hydrogels, which can allow for sustained release of GF, promoted urethral muscle regeneration, resulting in the recovery of their biological function for the treatment of urethral incontinence.

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CONCLUSIONS The present study reports a hybrid macro/nano hydrogel composite as an injectable and bioactive bulking material for the treatment of urethral incontinence. The hybrid hydrogels were formed via enzyme-mediated cross-linking reactions, with encapsulation of bFGF-loaded HP nanogels. We also demonstrated that incorporation of a heparin-based nanocarrier into the injectable gelatin-based hydrogels allowed for sustained release of GF as well as efficient bulking of the defective urethral tissue, promoting regeneration of urethral muscle surrounding the urethral wall, resulting in the recovery of their biological function. We expect that our approach can provide an advanced therapy for the treatment of urinary incontinence and applications in regenerative medicine. 1983

dx.doi.org/10.1021/bm401787u | Biomacromolecules 2014, 15, 1979−1984

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +82-31-219-1846. E-mail: [email protected]. Fax: +82-31-219-1592. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) grant funded by the Ministry of Science, ICT & Future Planning (NRF-2012R1A2A2A06046885) and Basic Science Research Program through NRF funded by the Ministry of Education (No. 2009-0093826)

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REFERENCES

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dx.doi.org/10.1021/bm401787u | Biomacromolecules 2014, 15, 1979−1984