Dual Regulations of Thermosensitive HeparinâPoloxamer Hydrogel

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Dual regulations of thermo-sensitive heparin-poloxamer hydrogel using #-polylysine: Bioadhesivity and Controlled KGF release for enhancing wound healing of endometrial injury He-Lin Xu, Jie Xu, Bi-Xin Shen, Si-Si Zhang, Bing-Hui Jin, QunYan Zhu, De-Li ZhuGe, Xue-Qing Wu, Jian Xiao, and Yingzheng Zhao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b10211 • Publication Date (Web): 15 Aug 2017 Downloaded from http://pubs.acs.org on August 17, 2017

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ACS Applied Materials & Interfaces

Dual regulations of thermo-sensitive heparin-poloxamer hydrogel using ε-polylysine: Bioadhesivity and Controlled KGF release for enhancing wound healing of endometrial injury He-Lin Xu1a*, Jie Xu1a,Bi-Xin Shen1, Si-Si Zhang2, Bing-Hui Jin1, Qun-Yan Zhu1, De-Li ZhuGe1, Xue-Qing Wu2, Jian Xiao1*, Ying-Zheng Zhao1* 1

Department of pharmaceutics, School of Pharmaceutical Sciences, Wenzhou Medical

University, Wenzhou City, Zhejiang Province 325035, China. 2

First Affiliated Hospital, Wenzhou Medical University, Wenzhou City, Zhejiang

Province 325035, China. *

Correspondence to: H-L Xu,

Department of Pharmaceutics, School of

Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou City, Zhejiang Province 325035, China. Email:[email protected] (H-L Xu) *

Correspondence to: J Xiao, School of Pharmaceutical Sciences, Wenzhou Medical

University, Wenzhou City, Zhejiang Province 325035, China. Email:[email protected] (J Xiao) *

Correspondence to: Y-Z Zhao, School of Pharmaceutical Sciences, Wenzhou Medical

University, Wenzhou City, Zhejiang Province 325035, China. Email: [email protected] (Y-Z Zhao) a

The first two authors contributed equally to this work.

Abstract Hydrogel was not only used as an effective support matrix to prevent intrauterine adhesion (IUA) after endometrial injury but also served as scaffold to sustain release of some therapeutics, especially growth factor. However, because of the rapid turnover of the endometrial mucus, the poor retention and bad absorption of therapeutic agents in damaged endometrial cavity were two important factors hindering their pharmacologic effect. Herein, a mucoadhesive hydrogel was described by using heparin-modified poloxamer (HP) as the matrix material and ε-polylysine (EPL) as functional excipient. Various EPL-HP hydrogels formulations are screened by rheological evaluation and mucoadhesion studies. It was found that the rheological

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and mucoadhesive properties of EPL-HP hydrogels were easily controlled by changing the amount of EPL in formulation. The storage modulus of EPL-HP hydrogel with 90µg/mL of EPL (EPL-HP-90) was elevated to be 1.9×105Pa, in accordance with the adhesion force rising up to 3.18N (10-fold higher than HP hydrogels). Moreover, in vitro release of model drug, keratinocyte growth factor (KGF), from EPL-HP hydrogel was significantly accelerated by adding ε-polylysine in comparison with HP hydrogel. Both of strong mucoadhesive ability and the accelerated drug release behavior for EPL-HP-90 made more the encapsulated KGF be absorbed by the uterus basal layer and endometrial glands after 8h of administration in uterus cavity. Meanwhile, the morphology of endometrium in the injured uterus was repaired well after 3days of treatment with KGF-EPL-HP-90 hydrogels. Compared with KGF-HP group, not only proliferation of endometrial epithelial cell and glands but also angiogenesis in the regenerated endometrium was obviously enhanced after treatment with KGF-EPL-HP-90 hydrogels. Alternatively, the cellular apoptosis in the damaged endometrium was significantly inhibited after treatment with KGF-EPL-HP-90 hydrogels. Overall, the mucoadhesive EPL-HP hydrogel with a suitable KGF release profile may be a more promising approach than HP hydrogel alone to repair the injured endometrium. Keywords: Repairmen of endometrial injury; Mucoadhesion; Bioadhesive hydrogel; Keratinocyte growth factor; Polyelectrolyte complexes Introduction Endometrial injury is the most common pathological process in gynecological diseases, which has become a primary factor resulting in intrauterine adhesion (IUA) and infertility1-3. Current therapies for endometrial injury included the surgical synechiotomy, hormonal drugs and an intrauterine device (IUD)4-5. Even though the effective outcome was obtained for these common therapeutic strategies, the recurrence ratio is up to 62.5% with a poor the prognosis in some conditions such as severe injury of endometrial basement membrane, severe IUA and sclerosis of uterine lumen6-7. Repairment and regeneration of the endometrium is essential to reestablish


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anatomy for embryo implantation and the maintenance of pregnancy after endometrial injury. Keratinocyte growth factor (KGF) has been discovered to play multiple important roles in regulating migration and proliferation of epithelial cells and epithelial mesenchymal interactions8-10 in various organs including skin11, prostate12, vagina13 and lung14. Expression of endogenous KGF has also been detected in the endometrium of a variety of species including mouse, rat, human, and macaque15. Endometrial KGF levels were increased by progesterone treatment in the macaque and mouse and are elevated during the luteal phase of the menstrual cycle in women. It was discovered that exogenous KGF stimulate spiral artery growth and inhibit glandular apoptosis during luteal-follicular transition, but did not affect cell proliferation in the endometrium or block menstrual sloughing and bleeding in normal uterus16. In our previous study17, not only the proliferation of endometrial glandular epithelial cells and luminal epithelial cells but also angiogenesis in injured uterus was observed after treatment with KGF. However, the endometrial mucus layer covered on the surface of epithelial cells is constructing a biological barrier to affect the retention and absorption of the therapeutic agents, especially growth factors18-19. A healthy uterus of adult women secretes about 3~4g of endometrial mucus each 4h20. The rapid turnover of mucus usually results in rapid discharge of the administrated therapeutic candidate in uterus cavity. Moreover, the endometrial mucus layer is a dynamic semipermeable barrier that can also capture some foreign substances including therapeutic agents, particulates and pathogens followed by rapid clearance through the physiological secretion of mucus21. Hydrogel delivery systems with high water content similar to biological tissues have been considered for the treatment of endometrial injury. In our previous study, a temperature-sensitive heparin-poloxamer polymer (HP) was synthesized using poly (ethylene oxide)-poly (propylene oxide)-poly (ethylene oxide)copolymers (PEO-PPOPEO) and heparin as original materials22. HP still exhibited a good gelation profile and its solution-gel transition temperature was not compromised compared with PEO-PPO-PEO. Importantly, HP still exhibited a strong affinity to some growth



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factors including FGFs and made them stable23. However, it was also reported that the skin wound healing effect of some FGFs, for example bFGF and KGF, was compromised when it was encapsulated in HP hydrogel, because a strong binding force between HP and these growth factors resulted in a complete release from hydrogel. Moreover, different from skin wound microenvironment, the endometrial mucus glycoproteins (mucins), a core protein in mucous layer, charge negatively at pH of cervical mucus (pH3.9-7.4)during menstrual cycle24. Since heparin is a negative linear polysaccharide, the electrostatic repulsive force between mucins and HP hydrogel resulted in the bad mucoadhelsive force of HP hydrogel against the injured endometrium. Thus, it may be very helpful for HP hydrogel encapsulating FGFs to improve its therapeutic effect by adjusting release profile and enhancing the mucoadhesivity. ε-Polylysine (EPL) (pKa9.3-9.5), which is cationic homo-polyamide at physiological pH25, exhibited a superior bioadhesive property against various bio-surfaces and has been used as an adhesive material for cell culture, because of electrostatic interactions with cell membrane26. EPL as a drug carrier is easily uptake into the cells, and it also help the attached drug transport bio-membrane27-28. The polymeric micelles prepared by a ε-polylysine derivate (EPL-g-Cetyl) were found to be an effective penetration enhancer to improve topical delivery of proteins and peptides27. Moreover, EPL has been also reported to use as a functional component to improve the bioadhesivity of dextran hydrogel through chemical crosslinking reaction in the publication29. Dextran/EPL hydrogels through aldehyde–mediated Schiff base reaction exhibited a strong adhesive strength against cow skin sheets, and possessed self-degradability and low toxicity. Besides, it was also found that simply mixing cationic polymer with a negatively charged polymer can also form bioadhesive polyelectrolyte complexes (PECs)30. Moreover, PECs offer various potential applications in medicine and biotechnology because of the simple preparation procedure and unique mucoadhesive properties31-33. For example, mucoadhesive force of Pluronic-based PECs was significantly improved by adding cationic chitosan26. Some therapeutic candidates could be encapsulated in PECs through interaction with


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its charged component to control their in vivo release profile34. For proof of concept, the cationic EPL may crosslink with the negatively charged heparin block of HP polymer, which may change some important properties such as rheological characteristics, temperature-sensitivity and bioadhesivity. Moreover, it is inferred that these properties may be easily adjusted by changing the amount of EPL in formulation. Series of EPL-HP hydrogels with different EPL concentration were prepared and their rheology and bioadhesive forces were evaluated in this study. The relationship between EPL amount and the release rate of KGF from EPL-HP hydrogel was also carefully explored. In addition, the repairmen and regeneration of the optimal EPL-HP hydrogels encapsulating KGF on injured uterus was also carefully studied by in vitro cell level and in vivo animal experiment in this study. The aim was to design a mucoadhesive delivery system capable of enhancing retention and absorption of KGF in uterus cavity to improve its therapeutic effect on the injured endometrium (as illustrated in Fig 1).

Fig.1 Scheme of thermo-sensitive bioadhesive KGF-EPL-HP hydrogel for injured uterus

2. Material and methods 2.1 Materials Heparin-poloxamer was synthesized according to the method depicted in our



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previous publication35. ε-Polylysine was purchased from Zhengzhou Bainafo Bioengineering Co.,Ltd. KGF was provided by Anhui Xinhuakun Co.,Ltd. Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA, USA).CCK-8kit was purchased from Dojindo (Minato-ku, Japan). Fluorescein isothiocyanate (FITC) was purchased from Tianjin Lianxing Biotechnology Co., Ltd. Antibodies of CK-18; anti-KGF and anti-CD31 were purchased from Abcam (Abcam, CB, UK). 2.2 Preparation of temperature-sensitive KGF-EPL-HP hydrogels KGF-EPL-HP hydrogels was prepared by the following two steps: first, KGF was added to cold HP solution (17%, w/w) and evenly mixed under gentle stirring; secondly, an amount of EPL powder was added to the cold KGF-HP solution and the mixed solution was further agitated to make EPL powders completely dissolve. Series of formulations with the different concentration of EPL (0, 30, 90, 300 and 900µg/ml) was prepared and final concentration of KGF in these formulation was 2.5mg/ml. 2.3 Micromorphology of KGF-EPL-HP Hydrogels The micromorphology, pore size and interconnectivity of series of KGF-EPL-HP hydrogels with different EPL concentration were examined using a scanning electron microscope (SEM; Hitachi, H-7500, Japan). The hydrogels were frozen at -20°C and lyophilized. The lyophilized powder of KGF-EPL-HP was cross-sectioned and sputter coated with gold followed by scanning analysis. The lyophilized powder of KGF-HP was also observed for comparative study. 2.4 Apparent viscosity and gelation time of KGF-EPL-HP hydrogels The apparent viscosity of EPL-HP hydrogels was measured with a coaxial cylinder rheometer (NDJ-8S) using No.1, No.4 rotor and small sample adapter. At temperature from 25 to 45°C, viscosity of EPL-HP hydrogels was detected and then the temperature-viscosity curve was obtained. Series of KGF-EPL-HP hydrogels were experimented for three times. During increasing temperature, the gelation time of cold KGF-EPL-HP solution was determined using the vial tilting method36. No flow within 1minupon inverting the vial was regarded as the gel formed. Each test was performed in triplicate.


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2.5 Rheological Characterization of EPL-HP hydrogels system All experiments were performed using a hybrid rheometer (discovery HR-2) fitted with a flat plate (12 mm) and 25µm gap. The temperature sweep study included a temperature range of 10 to 45°C. Temperature was controlled by a circulating water bath (ViscothermVT2). Oscillatory temperature sweeps for HP hydrogels and EPL-HP hydrogels system were performed at 10 rad/s and 1% strain with a heating range of 1°C from 10 to 45°C (to cover cool temperature, ambient condition and body temperature). Oscillatory frequency sweeps were performed at 1% strain with increasing frequency from 0.1 to 100 Hz. Oscillatory time sweeps for EPL-HP hydrogels were deformed at 1% strain and 10 Hz over 250s. 2.6 FTIR and 1H-NMR Spectroscopy The structures of EPL-HP hydrogels were characterized by FT-IR (670 FT-IR, Nicolet, Madison, WI, USA)over the wave number range of 4500–500 cm−1 with resolution of 4.0 cm−1. EPL-HP and HP lyophilized power was dissolved in D2O and the result was confirmed via 1H-NMR (AVANCEIII 600 MHz, Bruker, Fallanden, Switzerland) to further confirm the chemical structure of EPL-HP different from HP. For each sample, the series FTIR runs were repeated three times, and in most case, the error of double bond conversion was less than 2%. 2.7 Bioadhesive evaluation of KGF-HP-EPL 2.7.1 Measurement of adhesion strength Lap shear tests are commonly used to assess adhesive strength for bonding biological tissue37. As described in former literature38, gelatin solution was uniformly coated on the substrate (white glass) (5 mm×20 mm×50 mm) simulating human tissue to measure the adhesion strength of EPL-HP hydrogels. The tissue adhesion strength was investigated according to the method modified from ASTM F2255-0339. Briefly, EPL-HP solution

was

placed

in

a

constant

temperature

of

37°C

and

transited to hydrogel. 200µl of EPL-HP hydrogel was spread on the substrate and then the two gelatin glass sheets overlapped. The adhesive joint was kept at 37°C for ca.15 min and then the samples were performed on an Instron machine and loaded to failure with a strain rate of 5 mm/min at 37°C. The maximum force versus displacement was



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measured, and the shear stress at break was used to characterize adhesion for each sample. Each test was performed in triplicate. 2.7.2 In vitro mucoadhesion studies Rabbit endometrial tissue obtained from laboratory animal center of Wenzhou medical university was used to evaluate the mucoadhesive properties of KGF-EPL-HP hydrogel against endometrial mucosa. Briefly, after rinsing with normal saline, fresh rabbit uterus was cut longitudinally with a size of 3×2 cm and fixed to the half cut falcon tubes. Later, 200µl of KGF-EPL-HP hydrogel was applied to the mucosal surface. After an incubation of 2min at 37°C, the mucosa was continuously rinsed with PBS at a constant flow of 1ml/min utilizing peristaltic pump to mimic the fluid mucus secreted. The rinsing PBS from the mucosa was collected at different time point. The rinsed KGF amount at each time point was analyzed by a specific KGF enzyme-linked immune-sorbent assay Kit. PBS flowing over the fresh mucosa surface without coating any KGF-EPL-HP hydrogel was served as blank sample. The experiment was performed in triplicate. 2.8 In vitro KGF release from KGF-EPL-HP hydrogel In vitro release behavior of KGF from KGF-EPL-HP hydrogels was performed in both pH release medium including pH7.4 PBS (0.01 M) and pH4.0 acetate buffer (0.01M). Briefly, 1ml of cold KGF-EPL-HP solution was placed in vial and the vial was placed in thermostatic bath at 37°C. After sol-gel transition, 10ml of release medium was gently added to KGF-EPL-HP hydrogel and incubated in shaking incubator at 120 rpm/min. At each pre-set time point, 100µl of released medium was withdrawn and the released KGF amount in medium was analyzed by KGF-ELISA Kit. Meanwhile, equivalence of fresh release medium was supplemented. The cumulative release percentage (%) was determined by dividing the cumulative amount of KGF recovered in the release medium at each time point by the total amount of KGF in 1ml of KGF-HP hydrogel. 2.9 In Vitro Biocompatible Assay 2.9.1 Cytotoxicity test In vitro cell viability of human uterine endometrial carcinoma cell (ECC) after


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exposure to KGF-EPL-HP hydrogels was assessed using CCK-8 kit. The cells were seeded into 96-well plates at a density of 8× 103cells/well and incubated for 24 h in 100µl of DMEM/F12 medium supplemented with 10% FBS at 37°C. The medium was replaced with fresh KGF-EPL-HP hydrogels. Cells were further incubated for 12h or 24h or 48h. After incubation, the supernatant hydrogel was removed and cells were rinsed with sterile PBS two times followed by addition of 10µl of CCK-8. After 2h of incubation, the optical density was measured at 450 nm using a microplate reader (MultiskanMK3, Thermo, USA). The cell viability was calculated using the following equation. The well treated with blank PBS was used as control group. The cell viability was calculated by the following formula.

=

A − × 100% A − A

2.9.2 Cellular uptake of KGF-EPL-HP hydrogel by human uterine endometrial carcinoma cell In vitro cellular uptake of KGF-EPL-HP hydrogel was evaluated to determine whether the encapsulated KGF in EPL-HP hydrogels could be taken up by ECCs. FITC as a fluorescent probe was covalently tagged to amino terminal of KGF through the method reported in a previous paper17,

40

. ECCs was seeded at a density of

1×106cells/well in 6well culture plates and allowed to adhere for 24 h. And then, the culture medium was discarded and replaced with free FITC-KGF solution or FITC-KGF-HP hydrogels or FITC-KGF-EPL-HP-90 with an equivalent concentration of FITC-KGF (80ng/ml). After 4h of incubation, ECCs was digested by trypsin, washed and suspended with PBS at 4 ℃ for 3 times. Finally, the fluorescence signal was detected by flow cytometer for quantitative analysis (FACSCalibur FCM, Becton Dickinson, San Jose, CA). The average cellular fluorescence intensity was also analyzed by Image pro plus 6.0. 2.10 Animal experiments All animal procedures were approved by the Laboratory Animal Ethics Committee of Wenzhou Medical University. Female Sprague-Dawley rats (230-250 g) were purchased from Shanghai, China. The intrauterine mechanical injury model was



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established according to the reported method41. Briefly, vaginal smears were obtained daily between 08:00-10:00 AM to confirm estrus cycles. Rats with four consecutive4-d estrus cycles were screened to establish the animal model. Rats were anesthetized by an intraperitoneal injection of 4% chloral hydrate (0.1ml/10g body weight) and placed in a prone position. The rats were operated on through a 0.5-cm longitudinal incision on the uterus above its bifurcation, and then the endometrial lining of the middle and upper was scraped using a mini endometrial curette. The cold KGF-EPL-HP solution was perfused to the injured site. Rats in the model group was received the same surgical procedures and were given saline. 2.10.1 Absorption of KGF-EPL-HP hydrogel by uterus: Evaluation of the retention of KGF in injured endometrium was performed in vivo. After the treatment on 8h and 3 days, the rats were sacrificed and the uterus was excised, fixed immediately in 4% paraformaldehyde, and observed by immunohistochemical staining. Primary antibody rabbit polyclonal KGF (ab9377, 1:200, Abcam), and tagged secondary antibody goat anti-rabbit were used. Under a light microscope (Nikon ECLIPSE Ti-S; Ruikezhongyi, Beijing, China), the distribution of KGF loaded by HP or EPL-HP hydrogels in the treated uterus area was viewed and imaged. 2.10.2 In Vivo Endometrium Regeneration of KGF-EPL-HP hydrogel Rats with uterine injury were randomized into four groups as follows: model group, EPL-HP group, KGF-HP hydrogel and KGF-EPL-HP-90 hydrogel. The dose of KGF was treated with 2.5mg/ml and 30µl of various formulations were perfused to injured site in uterus cavity. After 3 days of treatments, all but 6 rats from each group were sacrificed and the hearts were perfused with saline followed by 4% paraformaldehyde, and then the uterus was collected and frozen in refrigerator. Histology analysis: The uterine tissues were fixed in 4% paraformaldehyde overnight, embedded in paraffin, and sectioned at 5µm of thickness. Sections were stained by H&E. Under a light microscope (Nikon ECLIPSE Ti-S; Ruikezhongyi, Beijing, China), morphologic recovery of the injured intrauterine was observed and imaged. Microscopic morphology of injured uterus cells under Transmission electron


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microscope (TEM): The injury uterus tissues were fixed in 2.5% (w/v) glutaraldehyde solution overnight, post-fixed in 2% (v/v) osmium tetroxide and blocked with 2% (v/v) uranyl acetate. Following dehydration in a series of acetone washes, tissues were embedded in Araldite for coronal sections. Semi-thin section and toluidine blue staining were performed by observing the injured site. Finally, ultra-thin sections of at least six blocks per sample were cut and observed under transmission electron microscopy (JEM 1400). 2.11 Immunohistochemical staining: Sections were pretreated with 3% H2O2 and heated to antigen recovery, and after washing, the samples were blocked using 5% bovine serum albumin (BSA) for 30 min at room temperature. Primary antibody rabbit polyclonal anticytokeratin (ab9377, 1:75, Abcam), anti-CD31 (ab28364, 1:300, Abcam), and anti-Ki67 (ab16667, 1:200, Abcam), and tagged secondary antibody goat antimouse or goat antirabbit were used. The tissue was treated with a DAB chromogen kit (ZSGB-BIO, Beijing, China) and then counterstained with hematoxylin (Solarbio) for 2 min. Sections were analyzed, and images were captured using a Nikon ECLPSE 80i (Nikon, Japan). Apoptotic cell deaths in uterus tissues were also detected by using TUNEL kit according to manufacturer instructions followed by observation under microscopy (Nikon ECLIPSE Ti-S; Ruikezhongyi, Beijing, China) 2.12 Statistical analysis As for in vitro and in vivo tests, data were expressed as averages with standard deviations. Statistical analysis of all data was performed: one-way analysis of variance (ANOVA) followed by Tukey’s test with Graph Pad Prism 5 software (GraphPad SoftwareInc., La Jolla, CA, USA). P value was considered statistically significant when

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