Preparation of a New Radiolabeled Biomaterial and Its Biodistribution in Mice

Journal of Bionic Engineering 10 (2013) 514–521

Preparation of a New Radiolabeled Biomaterial and Its Biodistribution in Mice Jinshu Ma1, Zhenning Liu2, Fang Wang1, Qinghai Zhou3, Chao Feng4, Fan Li1 1. Department of Pathogenobiology, Norman Bethune College of Medicine, Jilin University, Changchun 130021, P. R. China 2. Key Laboratory of Bionic Engineering (Ministry of Education), Jilin University, Changchun 130022, P. R. China 3. State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Changchun 130022, P. R. China 4. Department of Neurology, First Hospital, Jilin University, Changchun 130021, P. R. China

Abstract Biomaterials have attracted more attention from biomedical research in recent years. Yet there are still unmet demands for current biomaterials, such as the reduction of local inflammation of the implantation site. Poly-Propylene Carbonate (PPC), a polymer with ester bonds on CO2 backbone, degrades to CO2 and water, which are natural components of human body, yielding less inflammatory response than traditional biomaterials. However, the tensile strength and heat resistance properties of PPC are less ideal. In order to improve the properties of PPC, we have developed a new PPC (M-PPC), modified by mixing with Poly-3-Hydroxybutyrate (PHB). Here, we report the biodistribution profiles of PPC and M-PPC, their biocompatibility and toxicity. 125I-radiolabeled PPC and M-PPC were prepared and their biodistribution in Balb/c mice were investigated. Then acute systemic toxicity and haemolysis assays were conducted to study their toxicity and biocompatibility respectively. Results show that M-PPC has a good potential to be used as bone repair materials because it possesses typical biodistribution pattern in major organs, minimal toxicity and good biocompatibility. Keywords: biomaterial, poly-propylene carbonate, PPC, biodistribution Copyright © 2013, Jilin University. Published by Elsevier Limited and Science Press. All rights reserved. doi: 10.1016/S1672-6529(13)60245-0

1 Introduction Biomaterials have become more commonly used in medical practice to replace or augment a natural function in recent years. Meanwhile biomaterial research has become such an interdisciplinary area that encompasses chemistry, biology, medicine, tissue engineering and materials science, and thus is merging with bionic engineering at molecular level. One common interest of biomaterial field and bionic engineering is to develop new bone substitute that can be used in clinical implantation with better biocompatibility and less toxicity. Traditional biomaterial devices of treating bone injuries, such as artificial joints and implantable plates, usually contain metallic components or ceramics[1], which have elements (e.g. Fe and Si)[2] beyond their normal levels for human bodies and thus are not biocompatible or degradable. Moreover, the relative stiffness of these materials induces profound structural Corresponding author: Fan Li E-mail: [email protected]

changes in underlying bones, which can reduce cortical thickness and density, thus increasing porosity and creating the risk of refracture at implant site when the plate is removed[3,4]. Therefore, many chemical approaches have been reported to design new bioresorable biomaterials, which are based on the major elements of human body (e.g. C, H, O, N, and P) and possess desired mechanical strength[5]. Dr. Rudd and coauthors designed a Phosphate Glass Fiber (PGF) reinforced Poly-Lactic Acid (PLA) composites that have initial mechanical properties surpassing those of traditional devices used in high-load bearing application and have better cortical thickness in regeneration[6,7]. These PGF-PLA materials have also been shown to be degradable in vitro with PBS (Phosphate Buffered Saline) and in vivo in animal model, and thus do not require removal after implantation[8]. Sun et al. created biodegradable Extracellular-Matrices (ECM) nanofibrous scaffolds using poly-L-lactic acid, which not only have tunable porosity and mechanical

Ma et al.: Preparation of a New Radiolabeled Biomaterial and Its Biodistribution in Mice

properties but also possess favorable features to facilitate tissue regeneration[9]. These molecular bionic engineering studies have shown promising potentials for polymers to be used in Guided Tissue Regeneration (GTR) due to their better osteoconductivity. However, the degradation of PLA would reduce pH at implantation site and cause local inflammation. Thus we studied a biodegradable polymer, Poly-Propylene Carbonate (PPC), which has ester bonds on CO2 backbone. The degradation products of PPC are CO2 and water, which are more natural to human body and thus yielding less inflammatory response compared to PLA[10−12]. PPC was first synthesized from carbon dioxide and epoxides by Inoue et al. in 1969[13]. The structure and synthesis of PPC is shown in Scheme 1[14]. PPC could be used as adhesives, solid electrolytes, photoresists and plasticizers[15,16]. It has also been reported that PPC can be biodegraded by both soil burial and buffer immersion[10]. Besides its excellent biocompatibility and biodegradability, PPC also has the advantages of low cost and CO2 recycling. However, the tensile strength of PPC is less ideal and this disadvantage has hindered its commercial application as a bone substitute[17]. Poly-3-Hydroxybutyrate (PHB) has mechanical properties comparable to polypropylene[18], which is better than PPC, and good biocompatibility similar to PPC[19,20]. Thus, in order to improve the mechanical properties of PPC, we mixed PHB into PPC to develop a new modified-PPC (M-PPC), which might be used as a biomaterial in medical application.

Scheme 1 Copolymerization of carbon dioxide and epoxides.

Here, we report the biodistribution profiles of PPC and M-PPC, their biocompatibility and toxicity. 125 I-radiolabeled PPC and M-PPC were prepared and their biodistributions in Balb/c mice were investigated. Then acute systemic toxicity and haemolysis assays were conducted to study their toxicity and biocompatibility. We found that M-PPC has a good potential to be used as bone repair materials.

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2 Materials and methods 2.1 Preparation of PPC and M-PPC PPC was synthesized by Changchun Institute of Applied Chemistry from CO2 (99.8% purity) and Propylene Oxide (PO) (99.5% purity) with a ternary rareearth-metal catalyst system[14] as previously described by Li et al.[17]. It was purified through a dissolution-precipitation procedure using acetone and ethanol. The average molecular weight and Polydispersity Index (PDI) of the purified PPC were 114 kg·mol−1 and 4.05 respectively, which were determined by Gel Permeation Chromatography (GPC). The carbonate linkage was determined as 99% by 1H-NMR. PHB was purchased from Beijing Biological Institute (China) and dried at 50 ˚C in a vacuum oven for 48 h prior to M-PPC preparation. PHB and PPC were blended at a weight ratio of 20:80 in Thermo Haake Rheomix at 170 ˚C, and these blends (modified-PPC) were dried under vacuum at 50 ˚C for 48 h and stored in a sealed desiccator. To make porous M-PPC, modified-PPC and NaCl was mixed at a weight ratio of 80:20 at 140 ˚C and 30 rpm for 10 min in a torque rheometer, and the NaCl in the blends was subsequently washed with deionized water for two weeks. PPC and M-PPC plates about 1 mm in thickness were fabricated by compression molding. 2.2 Radiolabeling with 125I Na125I was provided by Chengdu Gaotong Isotope Corporation (Chengdu, China). Chloramine T method was used to radiolabel M-PPC in organic phase as previously described[21]. Briefly, 10 mg of M-PPC was dissolved in 1 ml acetone in a clean and sterile container, and stirred for 2 min at room temperature. Then 125I (1 mCi in 10 μL) was added and stirred for another 2 min. The labeling reaction was started by adding 20 μL of a 50 mg·mL−1 chloramine T (Merck Chemical Co.) in phosphate buffer (pH 8.0). The reaction was stirred for 3 h and 1 mL of an equal-molar solution of sodium hydroxide in methanol was added into the solution to make M-PPC water-soluble. Subsequently, phosphate buffer (pH 8.0) was added to adjust the volume of the reaction to 10 ml. The reaction was stopped by adding 30 μL of sodium metabisulfite (100 mg·mL−1) and stirred for another 2 min. Free (unbound) 125I was displaced by adding 10 μL of 100 mg·mL−1 potassium iodide in phosphate

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Journal of Bionic Engineering (2013) Vol.10 No.4

buffer (pH 8.0) and the solution was then stirred for a further 2 min. Some mixture was kept for column chromatography and paper electrophoresis in order to calculate the radiolabeling efficiency of PPC and M-PPC. The rest was transferred into a boiled dialysis tubing and dialysed in 1% 5 L sodium chloride solution (pH 7.0), which was changed at least 6 times until there was no free radioactivity could be detected as previously described[21]. The solution after dialysis was aliquoted in 1 mL and then stored at −20 ˚C. The radioactive species of the samples were confirmed using column chromatography with Sephadex G25 (PD10) and paper electrophoresis in barbitone buffer (pH 8.6). For column chromatography, samples were diluted in 0.06 M phosphate buffer (pH 7.0), and 1 mL was applied to PD10 column. The column was eluted with phosphate-buffered saline and collected in 1 ml each to measure the radioactivity. For paper electrophoresis, 50 μL sample was dripped onto 0.5 cm-wide cellulose nitrate paper and then electrophoresis was performed under 13 mA, 400 V for 40 min in 0.08 M barbitone buffer at pH 8.6. The dried papers were then drenched in water and placed in an LP3 tube for radioactivity measurement. 2.3 Biodistribution in mice Female Balb/c mice and adult SPF-grade Kunming white mice were obtained from Experimental Animal Center of Jilin University. Animal experiments were conducted in compliance with local ethics committee’s requirements and approved by Institutional Review Board of Jilin University. All procedures were performed in accordance with the guidelines established by the National Science Council of China. Female Balb/c mice at the age of 2–3 months and the weight of 25 g – 30 g were chosen for the biodistribution experiment. They were divided into three groups with three animals each. Before experiments the animals were deprived of food and water for one day. Each mouse was injected at tail vein with 100 μCi (100 μL) dose of the radiolabeled solution. Animals were sacrificed at different time points (5 min, 15 min, 0.5 h, 1 h, 2 h, and 6 h). Blood was collected by cardiac puncture and main organs such as heart, lung, liver, kidney, and spleen were dissected. The main organs were then washed with normal saline and air-dried before weighing. Radioactivity was measured by

Well-Type Scintillation Counter. The results were expressed as the percentage of Injected Dose (ID) per gram of an organ. 2.4 Acute systemic toxicity in mice Thirty healthy adult SPF-grade white mice with body weight of 17 g – 25 g were randomly divided into experimental, negative control and positive control groups. Mice in the experimental group were injected at tail vein with M-PPC at 50 mL·kg−1 according to International Standard ISO/TR 7405−1984. The same dosage of PBS was given to the negative control group, while the positive control group was injected with the same dosage of PPC. The number of deaths was recorded after 72 h[22]. 2.5 Haemolysis assay Haemolysis assay was performed on all human venous blood samples collected from healthy volunteers at Jilin University. The erythrocytes were isolated, washed and resuspended in PBS at a ratio of 1:10, and then incubated with PPC or M-PPC at 37 ˚C for 4 h. Saponin (Sigma) (25 g·L−1 in PBS) was used as the positive control for haemolysis. Samples were centrifuged at 1000 rpm for 15 min to remove non-lysed erythrocytes. The supernatants were collected and analyzed for released haemoglobin by spectrophotometer at 540 nm. Erythrocyte suspension was added to PBS and saponin to obtain 0% and 100% haemolysis respectively. The precentage of haemolysis was determined by the following equation: haemolysis (%) = (Abs – Abs0) /(Abs100 – Abs0) × 100, where Abs, Abs0 and Abs100 are the absorbance of the test samples, 0% haemolysis and 100% haemolysis are negative control and positive control, respectively[23,24]. Less than 5% haemolysis was set as the non-toxic level for our experiments. 2.6 Statistical analysis Statistical comparison between two groups was analyzed by one-tailed Student’s t-test using statistical software (SPSS 13.0). A difference with p < 0.05 was considered statistically significant.

3 Results and Discussion The microstructures of M-PPC and PPC were observed by Scanning Electron Microscope (SEM). Fig. 1 shows the SEM images of M-PPC. The microstructure

Ma et al.: Preparation of a New Radiolabeled Biomaterial and Its Biodistribution in Mice

of M-PPC has tiny granules with no significant pores, which is similar to the microstructure of PPC (images not shown).

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consistent with the knowledge that these polymers are directly excluded from the gel. In contrast, free 125I are eluted later within the fractions 10 to 12, due to its penetration into the gel. By calculating the areas under the curves and the corresponding peaks of Figs. 3 and 4, the radiolabeling efficiencies were determined as 37% and 36% for 125I-M-PPC and 125I-PPC respectively. Paper electrophoresis was then used to confirm the above radiolabeling efficiencies. The results are in line with the above observation, showing 41% and 44% for 125 I-M-PPC and 125I-PPC, respectively. 15000

CPM

10000

5000

0 0

5

10

15

20

Fraction No.

Fig. 2 Sephadex G-25 chromatography of 125I-labeled M-PPC. Peaks A and B represent eluted 125I-labeled M-PPC and free 125I respectively.

Fig. 1 SEM photographs of M-PPC at different magnifications.

3.1 Radiolabeling PPC and M-PPC with 125I In order to investigate the biodistributions of PPC and M-PPC in animals, we set out to radiolabel PPC and M-PPC with 125I and then confirm their labeling efficiency by column chromatography and paper electrophoresis. As shown in Figs. 2 and 3, the radiolabeled formulations as described in Section 2 were loaded to a Sephadex G-25 column and eluted fractions were collected and measured for radioactivity. In both Figs. 3 and 4, the fractions 4 and 6 represent radioactive polymers eluted within the void volume of the column, which is

Fig. 3 Sephadex G-25 chromatography of 125I -labeled PPC. Peaks A and B represent eluted 125I-labeled PPC and free 125I respectively.

Biodistribution of 125I-radiolabeled PPC and M-PPC Biodistribution is a common method of pharmacology to reveal how a biomaterial or a medicine distributes in patients after clinical implantation or drug 3.2

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Journal of Bionic Engineering (2013) Vol.10 No.4 Table 1 Comparison of the clearance of M-PPC and PPC in mice Ralative radioactivity (%)

Heart

Biodistribution of M-PPC

Biodistribution of PPC

0.25 h

6h

Decline

4.79

2.73

43

Blood

3.81

2.56

33

Kidney

4.02

3.23

20

Liver

24.75

20.01

19

Lung

3.86

3.18

18

Spleen

3.67

2.95

20

Heart

6.13

1.96

68

Blood

2.34

1.66

29

Kidney

4.39

1.85

58

Liver

29.87

15.12

49

Lung

3.07

1.84

40

Spleen

2.10

1.69

20

The biodistribution of radiolabeled PPC at different time points post-intraperitoneal injection is shown in Fig. 5, which exhibits a similar but faster pattern compared to that of M-PPC. PPC also accumulates mainly (~30%) and quickly (0.25 h) in liver, but the metabolism is much faster, which has dropped to 15% at 6 h after the injection which is around 50% of that at 0.25 h. The clearance of PPC from the heart is still the winner among all organs, which is around 70% at 6 h (see Table 1). However, the radioactivity level of blood decays at a slower rate (~30% at 6 h) for PPC compared to 33% for M-PPC, which can be explained by the metabolized PPC by liver that needs to be transported to kidney in circulation for excretion. It should be noted that the radioactivity of kidney has declined faster (58% at 6 h) compared to those of liver, lung and spleen, which are 49%, 40%, and 20% respectively (see Table 1), suggesting that kidney is able to excrete PPC directly.

Relative redioactivity (%)

administration. It is usually performed in an experimental animal with a radioactive isotope to track the compounds of interest, when human subject is less available. We used post-dialysis 125I-radiolabeled PPC and M-PPC, which contains no detectable free 125I (data not shown), to investigate the biodistribution in mice. The 125 I-radiolabeled PPC and M-PPC were administered into Balb/c mice via intraperitoneal injection and the major organs and tissues of the injected mice were collected at different time points to measure their radioactivity. The biodistribution of radiolabeled M-PPC at different time points post-intraperitoneal injection is shown in Fig. 4. It is observed that radiolabeled M-PPC accumulates mainly (> 20%) and quickly (0.25 h) in liver. Although radioactivity can be found in blood, heart, lung, spleen and kidney, their radioactivity levels are much lower (< 5%). Even 6 h after the injection, the level in liver remains around 20% while the levels of other organs have dropped to around 2% – 3%. These results indicate that as an exogenous material, M-PPC is mainly gathered and metabolized by liver, which functions as the major detox organ within a body. Interestingly, while the radioactivity levels of liver, lung, spleen and kidney have declined by around 20% at 6 h after the injection, those of heart and blood have decreased most dramatically, by 43% and 33% respectively (see Table 1), suggesting that M-PPC is cleared off the circulation in a relatively quicker manner. Also the coordinated decay for liver and kidney could imply that M-PPC is hard for kidney to excrete. Otherwise a faster decay in kidney would have been observed.

Fig. 4 Biodistribution of radiolabeled M-PPC in mice. The radioactivity is expressed as the percentage of the total radioactivity administered into the corresponding animal. All values are shown as mean ± SD of three independent experiments (n = 3).

Fig. 5 Biodistribution of radiolabeled PPC in mice.

Ma et al.: Preparation of a New Radiolabeled Biomaterial and Its Biodistribution in Mice

3.3 Acute systemic toxicity of PPC and M-PPC in mice In previous study, we found PPC and M-PPC have no toxicity in vitro cell culture of mouse fibroblasts[27]. Here, in order to examine the acute systemic toxicity for PPC and M-PPC in vivo, we administered these polymers to healthy adult SPF-grade white mice by tail vein injection (50 mL·kg−1 body weight). As shown in Fig. 6, the average body weights for M-PPC, PPC and control groups exhibit no significant difference 3 days after the injection (P > 0.05). 30

Pre-injection Post-injection

25 20 15 10 5 0

Control group

PPC group

M-PPC group

Fig. 6 The average body weights for mice injected with PPC and M-PPC. Average body weights for M-PPC, PPC and control groups exhibit no significant difference 3 days after the injection (P > 0.05).All values and error bars are shown as mean ± SD of three independent experiments (n = 10).

During the 3-day post-injection observation, mice of the control, PPC and M-PPC groups showed no death, lethargy, anorexia or diarrhea. No strong reactions were observed for PPC and M-PPC groups, compared to the saline control group. Thus, we found no significant toxicity for PPC and M-PPC in vivo, which is consistent with our previous in vitro findings in mouse fibroblasts. 3.4 Haemolytic effects of PPC and M-PPC The biodistribution and acute toxicity experiments indicate that M-PPC could be a promising candidate as a

biomaterial. However, its biocompatibility with red blood cells remains unknown. To further characterize the biocompatibility of PPC and M-PPC, in vitro haemolysis assay, a common method to examine potential erythrocyte damage, was carried out with human blood samples. As shown in Fig. 7, the percentages of haemolysis induced by PPC and M-PPC are 2.50% and 2.15% respectively, well much below the standard criterion of 5%. Thus, the haemolytic effects of PPC and M-PPC are negligible, indicating PPC and M-PPC possess a good biocompatibility. Haemolysis rate for M-PPC and PPC (%)

Overall, we have found that both PPC and M-PPC are mainly gathered and metabolized by liver, as liver is the main organ that has the central role in detoxifying and removing toxins of the body[25,26]. Yet, PPC appears to be metabolized much faster than M-PPC, probably due to the presence of PHB in M-PPC. We have also observed that, although both PPC and M-PPC are eventually excreted via kidney, the mechanism could be different. We have inferred that PPC could be excreted directly by kidney while M-PPC could not.

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120 100 80 60 40 20 0

PBS

M-PPC

PPC

Saponin

Fig. 7 Haemolysis rates of PPC and M-PPC in human blood sample. Values are expressed as the percentage of the positive control (Saponin). All values and error bars are shown as mean ± SD of three independent experiments (n = 5).

4 Conclusion We have investigated the biodistribution of PPC and M-PPC, a novel biomaterial and examined their toxicity and biocompatibility. Both PPC and M-PPC are mainly metabolized by liver and exhibit no significant enrichment in other organs. But compared to PPC, M-PPC takes longer time to be cleared by kidney, implying M-PPC would stay within the body longer than PPC. We have also found both PPC and M-PPC have no significant toxicity in vivo and demonstrate an excellent biocompatibility with in vitro red blood cells. In summary, M-PPC is found to be a better candidate than PPC to be used in medical applications with typical distribution pattern, minimal toxicity and good biocompatibility. Other properties of this biomaterial need to be further characterized in future research in order to justify its potential clinical applications.

Acknowledgments This work was supported by National Hig-tech R&D Program of China (863 Program) (Grant no. 2007BAE42B06) and Talent Development Fund of Jilin Province (Grant no. JTF[2012]04)

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