Morphology and structure of SF
As shown in Fig. 1B, mixed size SF appeared as spheres or irregular lumps in aqueous solution not reuniting with each other. The SEM images showed abundant pores spreading on the surface of SF, which were in uneven distribution and size. The large number of pores were beneficial to the loading and release of drugs, as the structural basis of drug sustained-release carriers. Besides, I-125 labeled SF was at the origin of TLC, and free I-125 was not detected, which meet the requirements of SF tracer in vivo (Fig. 1C).
Early in vivo distribution (within 40 min)
125I-SF began to diffuse to the whole body immediately after the injection through the tail vein, and the typical in vivo distribution of silk fibroin in the early 40 minutes was shown in Fig. 2A. It could be observed that the early distribution of silk fibroin with four sizes were similar. After injection through the blood, 125I-SF gathered obviously in the gastrointestinal tract and excreted through urinary system. Meantime, there were no visible aggregation in other organs in the early time, such as the thyroid and liver, indicating good labeling stability and half-life of in vivo blood circulation of silk fibroin.
The curve of accumulation in ROI (region of interest) clearly demonstrated the change of 125I-SF in each organ within the early 40 minutes (Fig. 2B), in which accumulation in upper limb of left hind leg serving as the background (the same below). Quantitative analysis results further verified the distribution of 125I-SF in SPECT images. Obviously, most of the SF gathered in the gastrointestinal tract quickly after injection. The gastrointestinal uptake fluctuated at a high level (approximately 120-140 times to the background) during the 40 min. For bladder, the amount of aggregation showed a slow increasing trend and was 1/3-1/4 of the gastrointestinal tract. Meanwhile, the traced SF in liver kept at a low level (the amount was similar to that of the background) all the time. Both the qualitative and quantitative images suggested that gastrointestinal metabolism was the main pathway of silk fibroin super early metabolism.
Primary in vivo biodistribution (within 72 h)
The typical in vivo biodistribution of four 125I-SF in the first 72 h was shown in Fig. 3A. In image of SF200, the gastrointestinal tract was still the main organ where SF gathered. After 3 h, the amount of silk fibroin in the gastrointestinal tract decreased gradually, while there was rarely 125I-SF in the bladder (Fig. 3B (Ⅰ)). Only at 72 h, a little 125I dissociated from the silk fibroin and gathered in the thyroid. Similarly, SF200-450 mainly appeared in the gastrointestinal system and there was a decreasing trend after 10 h. Silk fibroin was still excreting through the urinary system at 24 h post injection (Fig. 3B (Ⅱ)). Meanwhile, almost no accumulation of 125I were found in the thyroid until 72 h. Distribution image of SF450-800in vivo was roughly similar to that of SF200-450 (Fig. 3B (Ⅲ)), but 125I-SF gathered in the bladder until 48 h. In other words, the three kinds of SF shared a similar in vivo distribution and degradation process, however, the rate of intestinal deposition and clearance were significantly delayed by the increase of SF particle size. Besides, only a small amount of 125I was detected in liver for these three size.
Totally different with the other three particle sizes, SF800 distributed almost in the liver after 10 h and maintained until 72 h, instead of gastrointestinal system (Fig. 3B (Ⅳ)). Traced with 125I, the whole liver was fully displayed, which indicated a large amount of 125I-SF was uptaken by the liver. Besides, thyroid was faintly visualized after 48 h. Taking the characteristics of liver deposition, SF800 can be designed as functional materials, such as embolic agents of hepatic artery. More detailed dynamic and timed SPECT images were shown in Fig. S1.
Quantitative images more vividly showed the change of silk fibroin in ROI, Fig. 3C presented ratio of 125I-SF in organs of interest to background within 72 h. For SF200, SF200-450 and SF450-800, the ratio in the gastrointestinal tract were highest and the peak could reached around 140. The bladder had a downward trend from about 10-20 times, while the liver remained a low level of no more than 10 times. In contrast to the other three sizes, the ratio of SF800 in gastrointestinal tract was obviously lower (the peak was close to 50 times). However, the curve of liver appeared a apparent upward trend at 10 h post-injection and arrived the peak (about 280 times) at around 48 h, while was only about 2-3 times before 10 h. Though there was a downward trend, the liver uptake still maintained a high ratio until 72 h.
Deiodization occurred at about 72 hours, and the distribution of silk fibroin could not be truly reflected after 72 hours, so the images were collected only until 72 h. According to the image of distribution within the first 72 h above, smaller sized silk fibroin (SF200, SF200-450, SF450-800) had basically consistent metabolic pathway, when larger sized (SF800) possessed its own characteristics. SF firstly entered the gastrointestinal tract for digestion and absorption, that was consistent with the metabolic pathway of exogenous protein in the body. Under the action of various protein digestive enzymes, it was decomposed into peptides and a small amount of amino acids. Additionally, most of the SF metabolized by the urinary system was excreted at the early stage, and SF could be observed in the bladder at only a few time points after 1 hour. Among the four sizes of silk fibroin, only the SF800 went into the liver within 72 h probably for further metabolism. Like most proteins in the body, silk fibroin need to be metabolized by the liver in order to reduce toxicity or further exert biological functions. The reason why SF with smaller sizes were not shown to enter the liver may be that small-sized proteins were easier to be excreted directly by the blood circulation and urinary system. Above all, this size effect were mainly reflected by differences on the rule of in vivo biodistribution and metabolism, which was conducive to guide drug design. Taking the SF800 as example, it is suitable to serve as a material that needs to stay in the liver for a long time.