Article pubs.acs.org/Biomac
Long-Term Sustained Release of Salicylic Acid from Cross-Linked Biodegradable Polyester Induces a Reduced Foreign Body Response in Mice Yashoda Chandorkar,† Nitu Bhaskar,† Giridhar Madras,‡ and Bikramjit Basu*,†,§ †
Laboratory for Biomaterials, Materials Research Centre, ‡Department of Chemical Engineering, §Bioengineering Program, Indian Institute of Science, Bangalore 560012, India S Supporting Information *
ABSTRACT: There has been a continuous surge toward developing new biopolymers that exhibit better in vivo biocompatibility properties in terms of demonstrating a reduced foreign body response (FBR). One approach to mitigate the undesired FBR is to develop an implant capable of releasing anti-inflammatory molecules in a sustained manner over a long time period. Implants causing inflammation are also more susceptible to infection. In this article, the in vivo biocompatibility of a novel, biodegradable salicylic acid releasing polyester (SAP) has been investigated by subcutaneous implantation in a mouse model. The tissue response to SAP was compared with that of a widely used biodegradable polymer, poly(lactic acid-co-glycolic acid) (PLGA), as a control over three time points: 2, 4, and 16 weeks postimplantation. A long-term in vitro study illustrates a continuous, linear (zero order) release of salicylic acid with a cumulative mass percent release rate of 7.34 × 10−4 h−1 over ∼1.5−17 months. On the basis of physicochemical analysis, surface erosion for SAP and bulk erosion for PLGA have been confirmed as their dominant degradation modes in vivo. On the basis of the histomorphometrical analysis of inflammatory cell densities and collagen distribution as well as quantification of proinflammatory cytokine levels (TNF-α and IL-1β), a reduced foreign body response toward SAP with respect to that generated by PLGA has been unambiguously established. The favorable in vivo tissue response to SAP, as manifest from the uniform and well-vascularized encapsulation around the implant, is consistent with the decrease in inflammatory cell density and increase in angiogenesis with time. The above observations, together with the demonstration of long-term and sustained release of salicylic acid, establish the potential use of SAP for applications in improved matrices for tissue engineering and chronic wound healing. drug.11 Aspirin and salicylate work by inhibition of cyclooxygenase (COX), which is a key enzyme in the biosynthesis of prostaglandins, and hence cause anti-inflammatory activity.12,13 Apart from its traditional use to relieve inflammation, compelling evidence for the use of salicylic acid in adjuvant cancer therapy has recently emerged.14−16 A limited number of studies on the biocompatibility of polyanhydride-esters containing salicylic acid have been reported earlier.17−21 In one such study to assess their in vivo biocompatibility, polyanhydride-esters were implanted in C57/ B16 mice, and salicylic acid was found to modulate the inflammatory response.21 In a different study, Uhrich and coworkers reported that the local delivery of salicylic acid through polyanhydride-ester aided bone healing in normal and diabetic rats.22 Reduced bone resorption due to salicylic acid release has also been demonstrated in vivo using BALB/c mice.23 Besides such studies, the in vivo biocompatibility of salicylic acid releasing polyester, which has been recently synthesized by us, has not yet been investigated in a systematic manner to
1. INTRODUCTION The performance of implanted biomedical devices can be severely limited by the foreign body reaction, which results in implant encapsulation by a dense, insulating collageneous capsule. This inhibits the effective use of biomaterials in various drug release devices and sensors.1−3 The formation of a fibrous capsule prevents effective molecular, electrical, and mechanical communication between the implant and the body. For this reason, long-term implant devices for drug release are quite rare, and most show poor performance in vivo.1 Biodegradable polymers, like PLGA, can aggravate this problem by producing noxious degradation products that can cause further inflammation in the host tissue.4,5 It is well-known that biomaterials inducing inflammation are more susceptible to infection.6 Hence, reduction of inflammation is important. Different strategies to overcome this host response include the use of antifouling biocompatible coatings as well as angiogenic drugs and steroidal and nonsteroidal anti-inflammatory drugs (NSAID).7,8 One of the approaches investigated by us involved the incorporation of anti-inflammatory agents like salicylic acid in the polymer backbone.9,10 Salicylic acid is a NSAID and is also a deacetylation product of aspirin, which is a commonly used as an anti-inflammatory © 2015 American Chemical Society
Received: November 27, 2014 Revised: December 30, 2014 Published: January 5, 2015 636
DOI: 10.1021/bm5017282 Biomacromolecules 2015, 16, 636−649
Article
Biomacromolecules
Figure 1. Reaction scheme showing the degradation of cross-linked salicylic acid based polyester (SAP) into oligomers and, finally, into monomers as a result of ester hydrolysis, which can take place during in vitro/in vivo degradation.
polymer. A detailed in vitro study on this polyester using a 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry measurements with the C2C12 murine myoblast cell line suggested that it is cytocompatible. A degradation model has also been developed with the aim of understanding ester hydrolysis.10 Against this backdrop, here we demonstrate the favorable in vivo tissue response of this polyester through the subcutaneous implantation of SAP into mice. The host response was compared with that obtained with PLGA, which is one of the most widely used biodegradable polymers with proven biocompatibilty.29 It is worth mentioning that many earlier studies reporting the biocompatibility of a new polymer often used PLGA as a control implant.30,31 In the present work, the host response was determined by sacrificing the animals at various time points postimplantation. The physicochemical analysis of the surface and chemical properties of the implants coupled with detailed histomorphometric analysis to calculate the distribution, density, and thickness of collagen as well as the density of inflammatory cells and neovasculature around the implant site were used to assess the tissue response to SAP.32 This is critical for the assessment of its long-term performance
comprehensively understand the progressive response to it in an animal model. As a clinical practice, systemic delivery of salicylic acid to achieve desired concentrations can be followed. The requirement of a high systemic dose of salicylic acid over a prolonged period has a number of disadvantages, which include gastrointestinal tract bleeding and impaired renal function in elderly patients.24−26 The United States Preventive Services Task Force (USPSTF) considered the harmful effects of a sustained oral dose of aspirin and concluded that the risks outweigh the associated benefits and did not recommend aspirin for chemoprophylaxis.25 The beneficial effects are significant only after a few years;16 hence, systemic delivery of salicylic acid is not a viable option. This emphasizes the need for a device for sustained, in situ slow release of salicylic acid. We have previously reported the in vitro sustained release of salicylic acid from a biodegradable salicylic acid based polyester (SAP).10 In this polymer, the matrix itself acts as a prodrug and releases salicylic acid as a degradation product of hydrolysis. The slow release of salicylic acid from the polymer backbone can be helpful for applications in adjuvant cancer therapy.27,28 Moreover, the incorporation of such a drug in the polymer chain ensures the release of drug throughout the life of the 637
DOI: 10.1021/bm5017282 Biomacromolecules 2015, 16, 636−649
Article
Biomacromolecules
2.4. Weight Loss in Vivo. The in vivo degradation of subcutaneously implanted SAP in mice was investigated by measuring the mass loss of the implant samples. The polymer samples were removed from the explant and dried in a hot air oven at 50 °C until a constant weight was attained. For scanning electron microscopy, the samples were dried for at least 24 h in a desiccator before sputtering them with gold (Denton Vacuum, USA) and were observed under a field-emission scanning electron microscope (FEI Quanta) at an accelerating voltage of 2 kV. The attenuated total internal reflection Fourier transform infrared (ATR-FTIR) spectra were recorded using PerkinElmer Frontier FTIR from 4500 to 500 cm−1. For DSC analysis, the dried polymer explants with a constant weight were heated at a rate of 5 °C/min from −80 to 120 °C for both SAP and PLGA. In both cases, the glass transition temperature was calculated from the second heating cycle. 2.5. Histopathological Examination. The NBF fixed explant tissue samples were dehydrated in a graded series of ethanol and embedded in paraffin blocks. Sections were cut at 5 μm and stained with hematoxylin and eosin (H&E) for analysis of the inflammatory response and with Mason’s trichrome stain (MTS) for the analysis of the fibrous capsule. For each time point, three slides were prepared per animal for each stain. The prepared histology slides were then observed by two observers independently without prior knowledge of the mice from which the section was obtained, under a light microscope (Nikon, LV 100D, Japan), to avoid bias. 2.6. Histomorphometrical Evaluation. The thin sections used for histomorphometrical analysis were analyzed using ImageJ software (version 1.48, National Institutes of Health, USA). Digital analysis was used to measure the area of the region of interest. Different parameters, such as the density of inflammatory cells (neutrophils, monocytes/macrophages, fibroblasts), and blood vasculature and distribution of collagen surrounding the implant, were quantified to measure the tissue response to the implant. The cell density was averaged after measuring the number of cells in a region of interest for at least 5 fields of view per slide. The identification of different cell types was based on their respective morphologies in the H&E sections. Neovascularization was calculated by measuring the total number of blood capillaries in the entire fibrous capsule section and normalized to the area from both the H&E and Mason’s trichrome stained images. The collagen distribution around the implant was measured by calculating the blue pixel coverage using ImageJ software2 in the Mason’s trichrome stained sections. The blue pixel coverage was calculated first for 5 μm at the implant−tissue interface and subsequently at 10 μm intervals throughout the capsule thickness for at least 10 points per field of view for at least 3 images per slide. 2.7. Analysis of Proinflammatory Cytokine Levels. The concentration of proinflammatory cytokines, viz., tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), in blood serum was quantified using commercially available ELISA kits (Platinum ELISA kit, eBioscience, Austria). These assays were carried out as per the manufacturer’s instructions. After addition of the stop solution, the absorbance was measured at 450 nm using a microplate reader (Eppendorf AF2200), and the concentration was determined using a standard curve plotted for each cytokine. 2.8. Statistical Analyses. Statistical analyses were carried out using IBM SPSS Statistics 20 software. Values are presented as the mean ± SE. Data for SAP and PLGA implantation were analyzed by a two-tailed independent (unpaired) Student’s t test, with statistical significance defined as p < 0.05, unless otherwise mentioned. To compare across different time points, one-way anova followed by Tukey’s posthoc test (Dunnett’s C posthoc test in case of nonhomogeneity of variance) was used, and a value of p < 0.05 was considered to be statistically significant.
and integration in various applications, including drug delivery, biocompatible coating, and adjuvant cancer therapy.
2. MATERIALS AND METHODS 2.1. Materials. Salicylic acid and pyridine were obtained from Merck, India, and sebacoyl chloride and mannitol were obtained from TCI Chemicals, Japan and S. D. Fine Chemicals, India, respectively. PLGA (lactic acid/glycolic acid 85:15) with a molecular weight (Mw) of 50 000−75 000 and acetic anhydride were procured from SigmaAldrich. 2.2. Polymer Synthesis. The salicylic acid releasing cross-linked polyester was synthesized as described elsewhere.10 Briefly, salicylic acid was dissolved in pyridine, and sebacoyl chloride was slowly added to this mixture in an ice bath according to the stoichiometric requirement. The reaction mixture was stirred for 3 h at 35 °C and then poured over crushed ice. A pH of 2 was achieved using concentrated HCl. The diacid was activated with acetic anhydride. Mannitol was then added (diacid (COOH) groups/mannitol (OH) groups ratio 0.66), and the reactants were allowed to react at 180 °C under high vacuum (
