Calcitonin-Loaded Thermosensitive Hydrogel for Long-Term

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Calcitonin-loaded thermosensitive hydrogel for long-term anti-osteopenia therapy Yanpeng Liu, Xiaobin Chen, Sheyu Li, Qiang Guo, Jing Xie, Lin Yu, Xinyuan Xu, Chunmei Ding, Jianshu Li, and Jiandong Ding ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b05740 • Publication Date (Web): 22 Jun 2017 Downloaded from http://pubs.acs.org on June 25, 2017

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ACS Applied Materials & Interfaces

Calcitonin-loaded thermosensitive hydrogel for long-term anti-osteopenia therapy Yanpeng Liu,1, † Xiaobin Chen,b, † Sheyu Li,3, † Qiang Guo,4 Jing Xie,1 Lin Yu,2,* Xinyuan Xu,1 Chunmei Ding,1 Jianshu Li,1,* and Jiandong Ding2 1

College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu, 610065, China. E-mail: [email protected] 2 State Key Laboratory of Molecular Engineering of Polymers, Department of Macromolecular Science, Fudan University, Shanghai, 200433, China. E-mail: [email protected] 3 Department of Endocrinology and Metabolism, West China Hospital, Sichuan University, Chengdu, 610041, China. 4 State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, 610041, China. KEYWORDS: Controlled release, peptide drug, salmon calcitonin, thermosensitive hydrogel, osteoporosis.

ABSTRACT: Effective anti-osteopenia therapy can be achieved by designing long-term protein/peptide drug delivery systems for bone trabecula restoration. Here we show that a complex of salmon calcitonin and oxidized calcium alginate (sCT-OCA) was prepared and loaded into a thermosensitive copolymer hydrogel for long-term anti-osteopenia treatment. The triblock copolymer, poly(D,L-lactic acid-co-glycolic acid)-b-poly(ethylene glycol)-bpoly(D,L-lactic acid-co-glycolic acid) (PLGA-PEG-PLGA) exhibited sol-gel transition at body temperature. The sustained release of sCT from the in situ gelling system was determined by both the degradation of the hydrogel and the decomposition of the sCT-OCA complex. This system showed sustained effects in reducing serum calcium and bone trabecula reconstruction in the treatment of glucocorticoid-induced osteopenia in rats for approximately 30 days after a single subcutaneous injection, which may shed light on anti-osteopenia therapy in the future.

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1. Introduction Viscoelastic and cross-linked polymeric hydrogels constitute an important research area in the field of biomedical materials. In particular, some hydrogels can be reversibly formed by physical crosslinking and possess multifunctional properties. For example, as body temperature is suitable for the triggering of gelation in clinic, thermo-induced forming hydrogels have gained increasing attention for controlled drug delivery1. Overall, thermosensitive hydrogels are ideal for minimally invasive therapy since they exist in the sol states with low viscosity at low temperature but turn into gel states near the body temperature, acting as a sustained drug release depot2-5. Various gel systems, including naturally derived and synthetic materials, have been developed for different biomedical applications. Among these, polysaccharides6-8, amino acids9, polypeptides10-12, and block copolymers13-18 of polyethylene glycol (PEG), polycaprolactone (PCL), polylactide (PLA)19 and poly(lactic-coglycolic acid) (PLGA)20, have advantages in controlling the stimuli-responsive gelation and in vivo degradation. In addition, extensive researches exist on localized delivery carriers based on FDA-approved PEG and PLGA polymers, which have proved to be effective carriers for the homogeneous encapsulation of peptide/protein drugs21. There are some challenges to the sustained delivery of peptide/protein drugs such as their instability, immunogenicity, poor permeability across biological membranes, and especially their short half-lives22. Owing to the poor efficiency of the oral or nasal delivery routes, traditional administration of peptide/protein drugs is usually limited to frequent subcutaneous injection, which is inconvenient and subject to poor compliance during prolonged therapeutic regimens. Thus, peptide/protein drugs have been loaded into various delivery systems for sustained release trials23-27. When designing a new pharmaceutical formulation for the sustained release system, a number of issues should be considered beforehand. For example, the drug needs to be released within an appropriate period, and not always “the longer, the better”, because many drugs may cause cumulative toxicity or drug resistance, resulting in 2 ACS Paragon Plus Environment

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therapy failure for preconceived curative effect28. In addition, the interaction between peptide/protein drugs and materials in the system is an important issue. Since many protein/peptide drugs are positively or negatively charged, they are unstable in an uncharged formulation and this may lead to obvious burst release29. In this study, a complex of salmon calcitonin (sCT) and oxidized calcium alginate (OCA) was prepared and then mixed with a thermosensitive polymer (PLGA-PEG-PLGA) solution. Then the system was injected into the Sprague-Dawley (SD) female rats with osteopenia model (the initial stage of osteoporosis) to form a hydrogel for long-acting therapy. sCT is a bioactive peptide containing 32 amino acid residues30. It can regulate serum calcium concentration by a therapeutic effect to improve bone mass and relieve osteoporotic bone pain31. As the isoelectric point (PI) of sCT is 9.3, it is positively charged at physiological pH. OCA is a negatively charged polyelectrolyte and thus we designed a complex of sCT-OCA with electrostatic interaction for stability and affinity-controlled release32. As alginate hydrogels show a slow and uncontrollable degradation33, oxidized sodium alginate with a low degree of oxidation was cross linked with Ca2+ at an optimal ratio to overcome this disadvantage. In addition, PLGA-PEG-PLGA with appropriate molecular weight and block ratio was synthesized. The triblock copolymer hydrogel was expected to support the sustained release of incorporated drug by localizing the gel at the injection site. The in vitro and in vivo hydrogel degradation and release profiles of sCT were evaluated, and the biocompatibility and the efficacy of this system for osteopenia therapy were investigated in vivo. In particular, the long-term anti-osteopenia effect was assessed by measuring the level of serum calcium and bone trabecula reconstruction in rats with glucocorticoid-induced osteopenia (Figure 1).



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Figure 1. Application of the sCT-OCA complex loaded thermosensitive PLGA-PEGPLGA hydrogel for controlled sCT release. The polymer formulation was subcutaneously injected into rats with osteopenia and formed a physical hydrogel in situ at body temperature. Both hydrogel degradation and complex decomposition determined the controlled release process. 2. Materials and Methods 2.1. Materials Salmon calcitonin (sCT) with the sequence of H-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-LeuGly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-SerGly-Thr-Pro-NH2 (disulfide bridge between Cys1 and Cys7) and Calcium Colorimetric Assay Kit (BioVision, K380-250, USA) were purchased from Xieli Biological Chemical Reagent Co., Ltd. (Sichuan, China). Dulbecco's modified Eagle medium (DMEM), streptomycin, penicillin, fetal bovine serum (FBS), heparin sodium and Cell Counting Kit-8 (CCK-8, 4 ACS Paragon Plus Environment

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Dojindo, CK04, Japan) were purchased from Baoxin Biotechnology Co., Ltd. (Chengdu, China). Female SD rats were obtained from Dashuo experimental animals Co., Ltd. (Chengdu, China). Salmon calcitonin ELISA Kit (Lot 201602, USA), Rat IL-1β (Lot E3012-1606-2, China), IL-6 (Lot E3060-1606-1, China) and TNF-α (E3720-1606-1, China) precoated ELISA Kits were obtained from Ruiao Biotechnology Co., Ltd. (Chengdu, China). All chemicals including deuterated reagent were provided from Energy Chemical Co., Ltd. (Shanghai, China) unless otherwise noted. 2.2. Preparation and Characterizations of sCT-OCA Complex sCT was mixed with oxidized alginate at different weight ratios (sCT : oxidized alginate: 1:0.125, 1:0.25, 1:0.5, 1:1, 1:2, 1:4 and 1:8) under stirring at 4 °C for 15 min. 2 mL of the mixture was dropwisely added into 2 mL calcium chloride solution (2 mg/mL) using a 25gauge needle by trace injection pump at the speed of 0.25 mL/min and stirred gently for 30 min at 25 °C. Then, the complex was obtained after centrifugation (5000 rpm, 10 min, 4°C) and re-dispersed in H2O for size and zeta potential measurements by dynamic light scattering (Zetasizer Nano ZS90, Malvern, UK). The supernatant was used to measure the association efficiency

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of sCT via UV-Vis spectrophotometry (UV-1800PC, APADA, China) with the

wavelength at 214 nm.

Association efficiency =

ℎ × 100% ℎ ℎ

The morphologies of sCT-OCA complex at the ratio of 1:0.5 were obtained by TEM (Hitachi H-600, 75 kV). The sample solution was dropped on a copper grid and then negatively stained with 1 wt% phosphotungstic acid. Finally, the prepared sample was photographed after a complete air-drying. 2.3. Synthesis and Characterization of PLGA-PEG-PLGA PLGA-PEG-PLGA with the block ratio of LA19GA8-EG34-GA8LA19 was synthesized through ring-opening polymerization with glycolide, lactide, PEG and the catalyst of stannous 5 ACS Paragon Plus Environment


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actuate. The detailed synthetic route has been published in our previous work35. Briefly, 4.01 g PEG was heated at 130 °C at vacuum condition for 3 h to remove the moisture. Then, the flask was cooled to 100 °C under the argon (Ar) condition. 7.06 g LA, 2.27 g GA and 12 mg Sn(Oct)2 toluene solution was added into the system, and the toluene was removed under vacuum for 15 min. The reaction was stirred under Ar at 150 °C for 12 h. The unreacted monomers were removed under vacuum for 3 h. Finally, the product was rinsed with water for three times. The final purified products were stored at -20 °C after lyophilization. The synthesized copolymers were characterized by 1H-NMR (Avance II-400 MHz) for structural composition (CDCl3 used as NMR solvents), GPC (Agilent 1260) for molecular weight distribution and TGA obtained from 30 oC to 650 °C at a heating rate of 10 °C min-1 under nitrogen fluid protection for structural property. The morphology of PLGA-PEG-PLGA tri-block copolymer was confirmed by TEM observation (FEI,TecnaI G20). The 20 wt% PLGA-PEG-PLGA solution was diluted by deionized water into 1 wt% solution and dropped on a copper grid covered with nitrocellulose. The films on the grid were negatively stained with 1 wt% phosphotungstic acid for 1 min. 2.4. Sol-Gel Transition sCT (1.5 mg/mL) was added to the 20% (w/w) copolymer PBS solution (0.01M, pH 7.4) and stirred gentlely at 4 °C for 15 min. The behavior of sol-gel transition was tested by the tube inverting method 36. The copolymer solutions with assigned concentrations were placed into 2 mL vials. The temperature was increased from 10 to 60 °C. Then the vials were kept in a water bath at a predetermined temperature for 10 min, following by inverting 180° to observe the hydrogel formation within 30 s. A dynamic stress-controlled rheometer (Kinexus Pro, Malvern) was used to investigate the sol-gel transition behavior of the solution. The cone plate (diameter: 60 mm, cone angle: 1°) was at a fixed gap 0.03 mm and an oscillation frequency of 10 rad/s. About 1.5 mL of solution


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was dropped on the plate. An oscillation single frequency stress test mode was setted. Temperature was at the increasing rate of 0.5 °C/min from 10 to 50 °C. 2.5. In Vitro Hydrogel Degradation 0.5 mL sample solution was added into borosilicate vials and equilibrated at 4 °C overnight. Then vials were incubated at 37°C to form gels. 9.5 mL pre-warmed PBS containing 0.025 % NaN3 was added into vials to serve as degradation medium for the gel. The vials was incubated in a shaking water bath at 37 °C and 50 rpm. Then each vial was taken out every 5 days to remove the supernatant and replace the fresh PBS. At predetermined time, 3 vials were taken out and removed the PBS.The remaining gels were lyophilized to reach a constant weight. The degraded hydrogels were analyzed by 1H-NMR and GPC for remaining structural composition. 2.6. In Vitro Cytotoxicity MG-63 cells in DMEM culture solution were seeded in a 96-well cell culturing plate at a cell density of about 5,000 cells per well (100 µL cell culture containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin). The plate incubated under a humidified atmosphere containing 5% CO2 at 37 °C for 24 h for complete spread. Subsequently, MG-63 cells were incubated with OCA, OCA-sCT, tri-block copolymer solution and their degraded solution at various concentrations for 48 h. Then, 10 µL CCK-8 was added into each well. The plate was kept at 37°C for another 3 h. Finally, the absorption of plate was measured at 450 nm on microplate reader. The value in the group of culture medium without co-cultivated materials was defined as 100%. 2.7. Preparation of the Delivery System The tri-block copolymer was dissolved in PBS (10 mM, pH 7.4) to prepared a concentration of 20% (w/w) by stirring at 4°C for 72 h. Then the stirring solution was transferred to room temperature. The sCT or sCT-OCA complexes were then dispersed in the



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aqueous copolymer solutions to form the final formulations. The details about the formulations were mentioned in Table S1. 2.8. In Vitro Release Study Each 2 mL formulation was incubated in a water bath maintained at 37°C for 5 min to form stable hydrogel system. Subsequently, the gels were added into dialysis bags (MWCO 7,000) which were soaked separately in 80 mL pre-warmed PBS containing 0.02 wt% NaN3 and reciprocated mildly at 20 rpm, 37 °C. At predetermined intervals, the release medium (2 mL) was taken out and replaced by fresh buffer. The sCT concentrations were determined by ultraviolet spectrophotometer at 214 nm. The amount of released sCT was calculated from the standard curve. 2.9. Stability Studies of In Vitro Released sCT The conformation stability of released sCT was examined by circular dichroism (CD, Chirascan plus, Applied photophysics, United Kingdom) scanning from 190 to 300 nm at 25°C

by comparing the sCT at different conditions. High-Performance

Liquid

Chromatography (HPLC, LC-15C, Shimadzu, Japan) equipped with an Inertsil Peptide C-18 column (4.6mm × 150mm × 4µm; GL Sciences, Japan). sCT analysis was perfumed using a gradient system: mobile phase A [10% (v/v) acetonitrile in tetramethylammonium hydroxide pentahydrate solution(0.02 mol/L, pH 2.5, adjusting with phosphoric acid) and mobile phase B [60% (v/v) acetonitrile in tetramethylammonium hydroxide pentahydrate solution (0.02 mol/L, pH 2.5, adjusting with phosphoric acid)], to run 30 min gradients from 28% to 52% B at a flow rate of 1.0 mL/min. Absorbance was monitored at the wavelength of 214 nm. The structural integrity of sCT released from polymeric formations was also examined by a Mini-electrophoresis system (VE 180, Tanon). All the solutions prepared for the HPLC experiments were used for SDS-PAGE study. The gels were conducted according to the manufacturer’s protocol. The samples were boiled with SDS loading buffer and were run on a 20% Tris-Glycine gel. The electrophoresis was performed at 30 V for 1 h, and then rise to 100 8 ACS Paragon Plus Environment

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V for 6-8 h. The gel was fixative for 0.5 h (0.5% glutaraldehyde, 30% ethyl alcohol) and stained with 0.2% Coomassie Brilliant Blue G-250 to visualize protein bands (50% methanol, 10% acetic acid) then decolourization. The gel pictures were taken on a luminescent screen. 2.10. Animal experiments Before the sCT treatments, female SD rats (9 weeks old) with original body weight 200±10 g were allowed to acclimatize in normal accommodation for 2 weeks and then randomly divided into different groups (Table S4). The methylprednisolone acetate solution (MPA in methyl cellulose solution) was used to induce rats generating osteopenia disease model by injecting with every other day at the dose of 0.2 mg/kg (except for G0 group) for 4 weeks. Rats in G2 group was treated with sCT PBS solution (1 mg/kg). G3-G5 groups were subcutaneously injected with respectively assigned formulations containing sCT (2 mg/kg) 37. At predetermined time points, Blood samples (250 µL) were collected from the tail vein in tubes containing heparin sodium and centrifuged at 4°C and 3,000 rpm for 10 min to separate serum. The sCT concentration in serum was measured by sCT ELISA Kit. Serum calcium level was tested by Calcium Colorimetric Assay Kit. The bioavailability of sCT was analyzed based on the area under the curve (AUC) of serum sCT level versus time 38. The maximum serum concentration (Cmax) and the time to reach maximum serum concentration (Tmax) were calculated in this curve.

Relative Bioavaliablity =

)*+,-./01234 5-206.783-9 /; )*+?@A ?-.683-9 /;?@A

At indicated time points, rats were sacrificed and their left femora were immediately operated and then fixed in paraformaldehyde solution at 4°C for further analysis. In addition, another group was treated with blank polymer solution (2 mL) without sCT for degradation in vivo. For each rat, 0.5 g of subcutaneous tissues which were closed to hydrogel was cut into fragments, homogenized in normal saline (2 mL). The homogenized solution was centrifuged (5,000 rpm) for 20 min at 4°C. The supernatant samples solution were processed to evaluate 9 ACS Paragon Plus Environment


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the concentrations of interleukin 1β (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor-α (TNF-α) with ELISA Kit protocols. The subcutaneous tissue surrounding the degraded hydrogel was also detected by histochemical staining. Tissue samples were subjected to hematoxylin-eosin (H&E) and Masson’s trichrome staining to identify the degree of inflammation response. All images were taken by general optical microscope. 2.11. Micro-CT analysis The paraformaldehyde fixed right femora of proximal end were vertically immobilized in the specimen holder. Bone specimens were scanned using micro-CT (µCT50, Scanco medical AG, Switzerland; 70 kV, 114 µA) to quantify the trabecular bone. The images consisted of 926 slices with a voxel size of 9 µm in all three axes. The same region of interest (ROI) of bone tissue selected (about 300 slices) for three dimensional architecture. All datasets were pre-processed automatically with a custom-written program. A single global threshold value for image intensity was used for all samples to quantitatively analyze the physical data. 2.12. Biomechanical Analysis of Femora in SD Rats The femur specimens were taken out from SD rats and fixed in 4% paraformaldehyde solution. Then, the specimens were wet with normal saline before the three point bending test (Universal test machine, INSTRON 5980, USA). Then a single femur after measuring external diameter of diaphysis was placed on the holder with 20 mm span to constitute simply-supported bridge. Squeeze head moved down slowly at a speed of 0.01 mm/s until the specimen fractured for mechanical data. 2.13. Data Analysis The data are presented as mean ± standard deviation (S.D.). One-way analysis of variance (ANOVA) and two-way ANOVA (for two variables) with Bonferroni post-test were evaluated using Graphpad Prism 6 software. A p-value of < 0.05 was considered to be statistically significant, unless otherwise indicated.


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3. Results and Discussion 3.1. Design and Optimization of the sCT-OCA Complex The degradation of alginate occurs via a slow and unpredictable dissolution process in vivo39, which is not ideal for stable drug delivery. Thus, we used periodate oxidation to cleave the carbon-carbon bond of the cis-diol groups in the uronate residue to form aldehyde groups. The molar ratio of periodate to alginate was 0.05 eventually with respect to the structural integrity of oxidized sodium alginate and its ability to form an encapsulating complex with Ca2+ 40. FTIR results clearly showed the oxidized sodium alginate with a new weak vibration indicative of C=O at 1727 cm-1 and the degree of oxidation was approximately 3% (Figure S1). The enthalpy of the oxidized sodium alginate was lower as the structural integrity damaged, and the rate of weight loss of oxidized sodium alginate is slower than that of sodium alginate with carbonization releasing CO2 from carboxyl groups at temperatures between 250°C and 550°C (Figure S2 and S3). The size, ζ-potential, and association efficiency of sCT-OCA complexes at different weight ratios were measured using a Zeta-sizer and UV-Vis spectrophotometry (Figure 2 and Figure S4). Owing to the charge interaction, the size of the complex was reduced to a minimum of 105.3 nm and an optimal mass ratio of 1:0.5 (sCT: OCA). At this ratio, sCT could be effectively associated with OCA using calcium chelation to form cross linked complex, and the association efficiency value reached 68.19% with a zeta potential close to neutrality (0.33 mV). The sCT-OCA complex presented a spherical morphology with a narrow size distribution at the 1:0.5 ratio (TEM insert, Figure 2A). Thus, the optimal ratio of 1:0.5 (sCT: OCA) was chosen for subsequent experiments.



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OCA

sCT

Figure 2. Particle sizes (A) and zeta potentials (B) of sCT, OCA, and sCT-OCA complexes at different weight ratios (sCT: OCA; sCT: 1 µmol/mL). The insert TEM image is of sCT: OCA=1:0.5 (scale bar: 250 nm). Data are presented as the mean ± S.D. (n = 3).

3.2. Synthesis and Characterizations of Thermosensitive PLGA-PEG-PLGA Triblock Copolymer Essential properties of the copolymer were reported previously35. The structure was confirmed by 1H NMR (Figure 3A). The signals of -CH3 and -CH in the LA segment appeared at 1.55 and 5.25 ppm; -CH2 of the GA and EG fractions were observed at 4.8 and 3.65 ppm, respectively. The Mn of the copolymer was approximately 5200 Da based on the calculation of 1H NMR data according to 1500 Da (Mn) of PEG. The PLGA-PEG-PLGA exhibited unimodal MW distribution with a value of 1.27, as measured by gel permeation chromatography (GPC) (Figure S5). The triblock copolymer exhibited symmetrical stretching vibrations of C-O-C at 1098 and 1191 cm-1 and C=O vibration at 1763 cm-1 by FTIR (Figure S6). The weight loss of the copolymer subjected to thermogravimetric analysis (TGA) occurred as a two-stage process and the PLGA fraction (68.72% weight loss) was preferentially lost compared to the PEG fraction (30.44% weight loss) (Figure S7). The tubeinversion method was used to determine the sol-gel transformation region of polymeric 12 ACS Paragon Plus Environment

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solutions (Figure 3B). Aqueous solutions of the copolymer at the concentration of 5-20% (w/w) were liquid below the room temperature, and the temperatures increased resulting in gels formation to construct the phase diagram. Similar results for the sol-gel transition temperatures were obtained by rheology (Figure 3C). The in situ gelling mechanism of the PLGA-PEG-PLGA triblock copolymers involves the formation of a suspension of micelles in aqueous solution because of amphiphilic nature of the copolymer, which aggregate in the sol-gel temperature transition region, gradually constructing a porous micellar network with gel-like properties. The formation of spherical micelles was confirmed by TEM (20 nm in diameter, Figure S8). Meanwhile, the diameter of PLGA-PEG-PLGA micelle also detected by DLS (25.25 nm, Figure S9). Hydrophobic interaction of components arises from diminution of the orientational entropy of water. To be specific, the association of hydrophobic moieties decreases the contact area with the water, thus increasing the entropy of the water. According to the second law of thermodynamics, ∆G =∆H-T∆S，whether a reaction proceeds spontaneously may be judged by the diminution of free energy G, of a system which is related to both changes of enthalpy H and entropy S. When temperature T is raised, T∆S increases, thus ∆G would be smaller, which leads to the sol-gel phase transformation at the macroscopic level. Then, further increased temperature leading to precipitation because of breaking down of the network36. Consequently, these data indicated that thermosensitive PLGA-PEG-PLGA copolymer could form an in situ hydrogel near the body temperature that may be suitable as an injectable implant for biomedical applications.



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Figure 3. Characterization of PLGA-PEG-PLGA. (A) 1H NMR spectrum of PLGA-PEGPLGA in CDCl3. (B) Sol-gel phase diagrams of PLGA-PEG-PLGA in PBS (pH 7.4) plotted by the vial-inversion method. (C) Storage modulus G’ and loss modulus G’’ of the PLGAPEG-PLGA in PBS (20 wt%).

3.3. In Vitro Release of sCT The polymeric formulation system showed a quick sol-gel transition at 34°C in PBS (pH 7.4). Images of the three polymeric formulation systems used in these experiments and described in Table S1 are shown in Figure 4A-D, illustrating the sol and gel states obtained at different temperatures. sCT released from the delivery formulations in vitro are presented in Figure 4E. The sCT-OCA complex loaded hydrogel (Formulation-3) exhibited a significant reduction in the initial burst (19.1 ± 1.9%) in the first day, as compared with that of 14 ACS Paragon Plus Environment

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Formulation-2 containing sCT alone (29.7 ± 1.2%) and sCT-OCA complex without hydrogel (Formulation-1, 32.6 ± 1.2%), as shown in the enlarged release profiles of the first day (insert of Figure 4E) . Formulation-1 showed a high initial burst effect with ca. 95% of the total sCT released from the delivery system within 10 days. Formulation-2 also exhibited a high initial burst effect, which may be attributed to the nature of sCT, a hydrophilic peptide of low molecular weight (3,432 Da)41, resulting in the fast diffusion of drug out of the hydrogel matrix. In contrast, continuous release of sCT from Formulation-3 was observed for a long period (over 20 days). The addition of OCA with a low degree of oxidation was successful in suppressing the diffusion of sCT by forming complexes to slow down the release rate of sCT. Overall, these results indicate that complex loaded in hydrogel played a vital role in controlling sCT release. In addition, the release kinetics of sCT from the other formulations summarized in Table S1 demonstrate that the release model for Formulation-3 fits the Ritger-Peppas equation indicating that the incorporated sCT released under passive diffusion and hydrogel erosion had little effect (Slope: k＜0.45) (Table S2). In contrast, polymeric Formulation-2 showed the highest correlation coefficients (r2) for Weibull fitted equation of hydrogel erosion release kinetics.



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Figure 4. In Vitro Release behaviors of sCT systems. (A) Images of the PLGA–PEG– PLGA (20% w/w) at the respective temperature and the injectable sol flowing from a syringe needle. Images of the sCT delivery systems, (B) Formulation-1, (C) Formulation-2 and (D) Formulation-3 at 37 °C. (E) sCT release in vitro from each of the various formulations. Insert is the enlarged release profiles in the first day. Data are presented as mean ± S.D. (n=3). (F) CD spectra of native sCT, sCT following incubation in PBS at 37 oC for 20 days and sCT released from the polymeric formulations after 20th day. (G) and (H) Chromatograms of sCT after release from Formulation-2 and Formulation-3 by HPLC.

3.4. Stability of in Vitro Released sCT sCT mainly adopts freely mobile a random coil structure in aqueous condition, however, well-organized secondary structures, such as α-helix, β-turns, and β-strands, could be interconvert in response to the change of the medium42. In this study, the conformation of sCT released from the formulations was monitored by CD and compared with that of native sCT to detect peptide fibrillation and aggregation to confirm the stability of released sCT. The results presented in Figure 4F showed the CD spectra of the fresh and released sCT from different 16 ACS Paragon Plus Environment

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formulations. The structural contents (α-helix, β-sheet, β-turn and random coil) of sCT were calculated by CDpro software (Table S3). Native sCT showed well-defined negative bands in the CD spectra, with a minimum at approximately 200 nm, indicating the presence of major random coil structure (~34.5%). Significant increases were observed in the percentages of βsheet (∼30.3%) and β-turn (∼23.5%) due to the fibrillation and hydrolysis of native sCT after incubation for 20 days at 37 °C in comparison with the native sCT in PBS. The spectra of released sCT from the two polymeric formulations were similar to that of the native sCT, with the random coil conformations accounting for the greatest proportion with similar values in each case. Thus, the thermosensitive copolymer formulations protect the conformational stability of sCT over the entire release period. High performance liquid chromatography (HPLC) was also used to monitor the chemical integrity of sCT. The analytical results of native and released samples of sCT from both polymeric formulations are presented in Figure 4G and 4H. sCT showed a single peak at a retention time of 19.94 min on the chromatographs. The peak positions of released sCT from Formulation-2 were similar from Day 5 to Day 20, which showed a major peak at ca. 20 min corresponding to native sCT while Formulation-3 displayed a virtually idential signal over the whole time period, indicating that the sCT-OCA complex in the hydrogel was protective against chemical instability. The native and released sCT at different time points were also tested by Tricine SDSPAGE (Figure S10). Molecular weight markers are shown in Lane 1, and the native sCT (Lane 2) exhibited a band upon the 3.3 kDa marker. The SDS-PAGE gel bands of other sCTs were released from Formulation-2 (Lines 3-6) and Formulation-3 (Lines 7-10). The released sCT solutions have the same bands with that of native sCT, indicating that the integrity of the released sCT is retained. 3.5. Degradation of PLGA-PEG-PLGA in Vitro and in Vivo



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The PLGA-PEG-PLGA degradation process is important for the sCT delivery system. Parallel samples of 20% w/w polymeric solutions were pleaced in a shaking water bath at 37 o

C to form gels and were taken out at interval to characterize any degradative changes in

structure. In parallel experiments, when the triblock copolymer solution was injected subcutaneously into rats at room temperature, they gelled rapidly forming irregularly shaped protrusions (Figure 5A). The rats were sacrificed at predetermined time intervals and the remained hydrogels were taken out. The size of the gels was observed to decrease gradually with time. The gels from both in vitro and in vivo experiments were freeze-dried, and dissolved in THF and CDCl3 for GPC and NMR analyses, respectively. The in vivo degradation of hydrogel was faster than the in vitro degradation as shown by GPC (Figure 5B and 5C). In addition, comparison of the 1H NMR spectra (Figure 5D and 5E) revealed that the peak height of the EG fraction gradually decreased at 3.65 ppm after 20 days of incubation both in PBS and body fluid, but the reduction extent was slightly faster in vivo. Although the -CH3 and CH signals of the LA fraction at 1.55 ppm and 5.25 ppm and the -CH2 of the GA fraction at 4.8 ppm decreased in the remained gel, the proportion of the hydrophobic to hydrophilic fractions was increased (Figure 5F). This finding indicated that the PLGA content in the degraded gels increased relatively during degradation process, i.e., there was a preferential loss of PEG segments. The final products of degradation were glycolic acid, lactic acid, and PEG. In this system, the PLGA segment may also degrade in vivo via the glucose anaerobic metabolism pathway to generate the acidic product glycolic acid, the tricarboxylic acid cycle or the aerobic metabolism of glucose43. By contrast, the PEG segment is non-biodegradable but can be cleared from body by kidney44. Enzymolysis, tissue fluid circulation and other biochemical reactions may accelerate hydrogel degradation in vivo, reflecting the observations made in this study.


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Figure 5. Degradation of PLGA-PEG-PLGA. (A) Images of gel degradation in vivo were taken at the indicated time point after subcutaneous injection of PLGA-PEG-PLGA aqueous solutions (20% w/w) into SD rats. (B) and (C) Time-dependent changes in Mpeak and PDI of PLGA-PEG-PLGA in vitro and in vivo by GPC. (D) and (E) Changes in 1H NMR spectra of PLGA-PEG-PLGA in vitro and in vivo with time. (F) Comparison of the fractions, LGA and EG, PLGA-PEG-PLGA determined by 1H NMR following in vitro and in vivo degradation, with time.

3.6. Cytotoxicity of PLGA-PEG-PLGA and OCA In this hydrogel system, we used 20% wt tri-block copolymer solution to form the hydrogel at 34 oC. The hydrogel contained about 80% PBS medium so that the system was permeable 19 ACS Paragon Plus Environment


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and easy for solution exchange. Therefore, the pH of the medium was nearly the same as that of hydrogel interior. Figure 6A showed that the pH of the medium changed during the degradation of PLGA-PEG-PLGA because of the production of glycolic and lactic acids. However, the pH scarcely changed in the medium after 5th day. It implied that the pH kept invariant in the hydrogel system, which is beneficial for the stability of sCT or sCT-OCA complex. Meanwhile, Figures 4F-H proved that the hydrogel could protect the structure stability of sCT over the entire release period. The in vitro cytotoxicity test was performed to evaluate the biocompatibility of the PLGA-PEG-PLGA triblock copolymer. Cell viabilities of MG63 cells cultured in the presence of copolymer were compared to the control cultures without the materials in the culture medium (defined viability 100%). The viability of MG63 cells in the presence of the copolymer was above 80 % but decreasing with increase of triblock copolymer concentration. A similar experimental procedure was carried out using partially degraded solutions of the triblock copolymer after incubation for 20 days, as described previously (Figure 6B). Cell viabilities of MG63 cells were similarly > 80 %. OCA and its degradation products were also tested for their effect in the cell cultures and similar results were obtained (Figure 6C). Meanwhile, MG63 cells cultured with sCT-OCA and sCTOCA/hydrogel also demonstrated good cell viabilities (Firgue S11).

Figure 6. Cytotoxicity of PLGA-PEG-PLGA and OCA. (A) Culture medium pH after 48 h in the presence of PLGA-PEG-PLGA. (B) Viabilities of MG63 cells co-cultured with copolymer, and (C) OCA, and their degraded products (after 20 days in vitro) at the indicated concentrations. Data are presented as mean ± S.D. (n=5). 20 ACS Paragon Plus Environment

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3.7. In Vivo Biocompatibility The PLGA-PEG-PLGA triblock copolymer was injected into the subcutaneous tissues between the dermis and superficial fascia. The surrounding tissue was stained during the degradation process using hematoxylin-eosin (H&E) staining (Figure 7A). The PLGA-PEGPLGA aqueous solution turned into a hydrogel in situ after injection. When the hydrogel just formed in situ at Day 0, there were few inflammatory cells such as macrophages and lymphocytes in the surrounding tissue. The hydrogel only induced a mild, chronic inflammatory response in parallel with its degradative release, as evidenced by a significant reduction of inflammatory cells after 15 days post-injection. Only a small number of inflammatory cells were observed after the almost complete degradation of hydrogel at Day 20, suggesting that the affected region was almost restored to the normal state. No sign of tissue necrosis or edema was found at the administration site during the entire period of implantation, illustrating that hydrogel possessed acceptable biocompatibility. The biocompatibility was further investigated using Masson’s trichrome stain, which stains collagen blue, myofiber and cytoplasm red45. This stain was applied to produce section images similar to those obtained with H&E staining (Figure S12). Acute and chronic inflammatory responses to the hydrogel typically declined at 20 days. Many inflammatory cells infiltrated the site after 5 days’ implantation. When the hydrogel had almost degraded completely, collagen was loosely distributed in the tissue without necrosis or edema. The PLGA-PEG-PLGA triblock copolymers are subject to tissue responses after being implanted into the living tissues of SD rats. Inflammatory cytokines such as IL-1β, IL-6, and TNF-α are involved in the inflammatory process46, and they can be used as biological indicators for characterization of the inflammatory reaction during polymer degradation (Figure 7B-D). We injected PLGA-PEG-PLGA triblock copolymer solutions into the subcutaneous tissue of rats to form gels, which were allowed to degrade in vivo for different time intervals. Then, the tissues surrounding the degraded gels were taken out and 21 ACS Paragon Plus Environment


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homogenized for assay of inflammatory cytokines. IL-1β is an important mediator in the inflammatory response47. Although a series of inflammatory reactions were triggered and the concentration of IL-1β increased from Day 0 to Day 15, the level decreased thereafter. Likewise, IL-6 is secreted by T cells and macrophages to stimulate the immune response and resistant infection48-50. The concentration of IL-6 at Day 20 was lower than any earlier time point after Day 0, confirming that inflammatory reactions were weak at the point when degradation was almost complete. TNF-α is a cell signaling cytokine involved in systemic inflammation51-52. The TNF-α level of tissue surrounding the hydrogel decreased gradually after Day 10. These results suggest that the PLGA-PEG-PLGA hydrogel underwent a normal degradation process in vivo with reversible effects on surrounding tissue, and thus, it is considered biocompatible and suitable for implant applications.


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Figure 7. HE-stained slides and inflammatory cytokines analysis. (A) Optical micrographs of tissues surrounding the hydrogel implants at the indicated number of days after subcutaneous injection of the triblock copolymer into SD rats for the inflammation reaction observation. Magnification 100×. Left column: area near the dermis; right column: area near the dorsal muscle. Inflammatory cells are indicated: Macrophage, blue arrow; Lymphocyte, yellow arrow. (Scale bar: 100 µm) Concentrations of inflammatory cytokines IL-1β (B), IL-6 (C), and TNF-α (D) during the hydrogel degradation process. Data are presented as mean ± S.D. (n = 5, *p < 0.05, **p < 0.01).

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sCT is mainly administrated by subcutaneous injection because of its fast absorption and rapid onset of its physiological activity. In order to test the preventive and therapeutic effect of the sCT-loaded delivery systems on symptom of osteoporosis, female rats were injected with methylprednisolone acetate (MPA) to induce initial stage of osteoporosis, osteopenia 53. MPA induced osteopenia is characterized by a significant drop in weight, rat serum osteocalcin concentration and an increase in the number of osteoclasts in bones, resulting in enhanced serum calcium levels. The MPA-induced osteopenia rats were divided into experimental groups as shown in Table S3. The disease model rats of the G1 group showed the lowest percentage of weight increase among all the groups (Figure S13). The dose of sCT and proportions of each component were as the same as the formulations of sCT used for release in vitro. Blood samples were taken from the caudal vein to evaluate serum sCT concentration and serum calcium at intervals (Figure 8). As shown, there was no significant decrease of serum calcium level observed for the group of MPA induced rats without sCT injection (G1 group). After subcutaneous administration of sCT alone (G2 group), serum sCT concentration rapidly increased and reached a peak concentration (Cmax) of 0.45 ng mL-1 after about 60 min (Tmax) post-administration, and restored thereafter to the basal level within one day. The serum calcium level decreased to ~62.47% of initial calcium level during the first 4 h and was returned to background level gradually within 24 h post-administration. It should be noted that this is an accelerated experiment carried out in a large volume of solution, thus the release time in vitro could only reflect but not exactly same as that of in vivo experiment.. The serum sCT concentration of G3 group (without hydrogel) was maintained up to ~10 days after subcutaneous administration and was accompanied by a reduced serum calcium level in this period. Rats of G4 and G5 groups showed sustained release of sCT for a longer time and significantly (p < 0.05) lower serum calcium levels in comparison to the G1 group. Thus, the released sCT from the polymeric formulations retained its biological activity. In addition, the G5 group achieved the longest controlled released period for about 30 days, 24 ACS Paragon Plus Environment

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and the serum calcium level in rats remained low over the same period. Since the normal serum calcitonin concentration in rats is about 0.2 ng/mL 54, the 0.2-0.5 ng/mL serum sCT concentration shown in this work is meaningful and effective for anti-osteopenia treatment. Consequently, the sCT-OCA complex in the hydrogel of the G5 group had the greatest effect and the highest relative bioavailability as shown in Table S4.

Figure 8. Serum sCT concentration (A and C) and serum calcium level (B and D) in differently treated groups of MPA induced SD rats. Data are presented as mean ± S.D. (n = 5)

3.10. Evaluation of Therapeutic Effect Body weights increased slowly in MPA-induced group (G1 group) over 30 days, in accordance with the symptoms of osteopenia. Micro-Computed Tomography (micro-CT) measurements of bone morphology have excellent resolution and accuracy, and thus have



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been used for the valuation of sCT treatment of osteopenia in MPA-induced rat models of the disease 55,56. The region of interest (ROI) from the total bone scan was selected to construct a three dimensional model at essentially the same section for each sample (Figure 9 and Table S5). Meanwhile, the models were incised 1/4 volume (along the transverse and vertical direction) to observe the internal structure and separation of metaphyseal trabeculae clearly. The trabeculae showed considerable differences between the G0 and G1 groups. As expected, the MPA induced a dramatic loss of trabeculae, resulting in bone dysplasia. Owing to the effect of MPA in the G1 group, the ratio of bone volume fraction to the total volume (BV/TV) (0.36±0.024), the number (Tb. N) (1.99±0.19) and average thickness (Tb. Th) (0.13±0.01) of trabeculae decreased, which led to an increase of separation (Tb. Sp) (0.37±0.06) and lower bone mineral density (BMD) value (669.77±17.23) compared with that of the G0 group. Transient therapy with sCT (G2 group) only showed a small degree of trabecular restoration. By contrast, the sustained release of sCT (G4 and G5 groups) eventually restored trabecular structure following subcutaneous administration. The femoral, 3D reconstruction data (Figure 9C-G) showed that the G5 group demonstrated the most effective treatment for trabecular restoration, both of integrated and compact structure. The therapeutic effect data is consistent with the in vitro and in vivo release profiles of sCT which showed that the sCT-OCA complex was most successful in stabilizing release from the hydrogel. Administration of sCT treatment in the polymer formulation groups particularly led to almost complete recovery of the physiological structure of bone and biomechanical properties in SD rat femora. In order to demonstrate the latter properties, the curve loaddeflection at mid-span was obtained in real time. The evaluation index for bone materials was analyzed and calculated (Table 1 and Figure S14). Compared with the G0 group, the elastic modulus, maximum strength, break strength and break strain of the G1 group were all decreased markedly. Stiffness increased, indicating that the femora became more fragile and showed weaker impact resistance

57

. However, all the parameters of the groups treated with 26

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sCT, especially the G5 group, revealed rehabilitation effect compared to the G1 group. Once again, the G5 group demonstrated the greatest resistance to fracture of any group. The capacity for bone deformation was significantly enhanced as the result of sustained and stable release of sCT.

Figure 9. Analysis and femoral scans of SD rats by micro-CT. (A): the CT scan image of the whole femur as an example; (B): section VOI of the femora after 30 days of subcutaneous injection from the different experimental groups (scale bar: 500 µm). (C-G) Histograms of the different trabecular structural parameters of the femora. ((C) BV/TV, (D) Tb. N, (E) Tb. Th, (F) Tb. Sp, and (G) BMD).



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Table 1. Mechanical parameters of the femora of SD rats in each treatment group. (mean ± S.D., n = 3) Elastic modulus

Maximum strength

Break strength

Break strain

Stiffness

/MPa

/N

/MPa

/%

/N mm -1

0

2741±51

121.9±4.1

82.8±3.5

3.86±0.48

114.6±7.4

1

2447±102

87.1±3.4

58.7±1.6

3.25±0.31

142.2±3.8

2

2200±105

105.2±8.7

67.5±3.6

4.34±0.03

101.8±1.0

3

2887±211

110.3±7.8

69.6±1.4

3.94±0.24

122.9±7.7

4

2187±92

113.2±4.2

72.5±1.8

4.30±0.78

124.5±3.2

5

3148±242

115.6±4.1

79.8±1.3

4.82±0.76

127.9±2.1

Group

4. Conclusions We developed an injectable hydrogel system based on the thermosensitive triblock copolymer PLGA-PEG-PLGA for the sustained release of sCT therapeutics. In combination with these excellent properties of the triblock copolymer gel formulation, the complex of sCT-OCA with appropriate ratio can lead to sustained sCT release via both hydrogel biodegradation and complex decomposition. The released sCT has conformational stability, maintaining secondary structural integrity and bioactivity. We also tested the released sCT for pharmacological activity in reducing serum calcium in vivo and found it to be effective. The femora of MPA-induced osteopenic SD rats were largely restored by treatment with hydrogels releasing sCT. Hence, the PLGA-PEG-PLGA injectable hydrogel loaded with the sCT-OCA complex has great potential for the long-term treatment of osteopenia.

ASSOCIATED CONTENT Supporting Information Preparation and Characterization of oxidized sodium alginate and PLGA-PEG-PLGA triblock copolymer; Optical micrographs of masson-stained slices of surrounding tissues in rats;


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Secondary structure analysis of released sCT by CD; TEM, DLS, SDS-PAGE and cytotoxicity data; Supplementary data for animal experiments. (PDF)

Corresponding authors * Jianshu Li and Lin Yu are co-corresponding authors. E-mail: [email protected]; [email protected]

Notes The authors declare no competing financial interest.

Author Contributions Janshu Li, Lin Yu and Sheyu Li designed the concepts, Yanpeng Liu, Xiaobin Chen and Xinyuan Xu carried out the experiments. Jing Xie and Chunmei Ding discussed and interpreted the results. Yanpeng Liu designed and performed the Micro-CT experiments, with help from Qiang Guo. All authors contributed to the interpretation of the data. Jianshu Li, Lin Yu and Jiandong Ding supervised the manuscript.

†These authors contributed equally.

ACKNOWELEDGEMENT Financial support from the National Natural Science Foundation of China (21534008, 51322303 and 21474019), and State Key Project of Research and Development (grant No. 2016YFC1100404, 2016YFC1100300) are gratefully acknowledged.

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Calcitonin-Loaded Thermosensitive Hydrogel for Long-Term

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