s2fieldsofstudy: Biology Chemistry

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Reactive Oxygen Species and Folate Receptor-Targeted Nanophotosensitizers Composed of Folic Acid-Conjugated and Poly(ethylene glycol)-Chlorin e6 Tetramer Having Diselenide Linkages for Targeted Photodynamic Treatment of Cancer Cells

Folic acid-conjugated nanophotosensitizers composed of folic acid (FA), poly(ethylene glycol) (PEG) and chlorin e6 (Ce6) tetramer were synthesized using diselenide linkages for reactive oxygen species (ROS)- and folate receptor-specific delivery of photosensitizers. Ce6 was conjugated with 3-[3-(2-carboxyethoxy)-2,2-bis(2-carboxyethoxymethyl)propoxy]propanoic acid (tetra acid, or TA) to make Ce6 tetramer via selenocystamine linkages (TA-sese-Ce6 conjugates). In the carboxylic acid end group of the TA-sese-Ce6 conjugates, FA-PEG was attached again using selenocystamine linkages to make FA-PEG/TA-sese-Ce6 conjugates (abbreviated as FAPEGtaCe6 conjugates). Nanophotosensitizers were fabricated by a dialysis procedure. In the morphological observations, they showed spherical shapes with small diameters of less than 200 nm. Stability of the aqueous FAPEGtaCe6 nanophotosensitizer solution was maintained (i.e., their particle sizes were not significantly changed until 7 days later). When H2O2 was added to the nanophotosensitizer solution, the particle size distribution was changed from a monomodal pattern to a multimodal pattern. In addition, the fluorescence intensity and Ce6 release rate from the nanophotosensitizers were also increased by the addition of H2O2. These results indicated that the nanophotosensitizers had ROS-sensitive properties. In an in vitro cell culture study, an FAPEGtaCe6 nanophotosensitizer treatment against cancer cells increased the Ce6 uptake ratio, ROS generation and light-irradiated cytotoxicity (phototoxicity) compared with Ce6 alone against various cancer cells. When the folic acid was pretreated to block the folate receptors of the Y79 cells and KB cells (folate receptor-overexpressing cells), the intracellular Ce6 uptake, ROS generation and thereby phototoxicity were decreased, while the MCF-7 cells did not significantly respond to blocking of the folate receptors. These results indicated that they could be delivered by a folate receptor-mediated pathway. Furthermore, an in vivo pulmonary metastasis model using Y79 cells showed folate receptor-specific delivery of FAPEGtaCe6 nanophotosensitizers. When folic acid was pre-administered, the fluorescence intensity of the lungs was significantly decreased, indicating that the FAPEGtaCe6 nanophotosensitizers had folate receptor specificity in vitro and in vivo. We suggest that FAPEGtaCe6 nanophotosensitizers are promising candidates for a targeted photodynamic therapy (PDT) approach against cancer cells.

Introduction
Photodynamic therapy (PDT) has been extensively investigated in the last two decades because it is regarded as a safe candidate for the treatment of cancer patients [1][2][3][4][5]. PDT and its output are normally composed of a photosensitizer, light and oxygen species, and furthermore, photosensitizers are only activated in a specific wavelength of light and then produce reactive oxygen species (ROS) (i.e., PDT produces excessive ROS in the site of irradiation and then inhibits the viability of disease cells) [6]. Therefore, PDT has a toxic reaction only in the site of light irradiation, which means it is able to minimize any adverse effects against normal cells or tissues [3,4,7,8]. For example, Leroy et al. reported that 5-aminolevulinic acid (5-ALA)-based PDT for brain cancer is efficient and safe for highgrade glioma [8]. They argued that glioma patients receiving 5-ALA PDT recorded longer survival times than those without PDT. Furthermore, PDT induces a response rate higher than 90% and no local recurrence in oral squamous carcinoma cells [8]. In spite of these advantages, some drawbacks to photosensitizers still limit the clinical application of PDT. PDT application is hard to apply to cancers located in deep tissues because photosensitizers have no cytotoxic reactions in the absence of light irradiation (i.e., the light irradiation depth is below 15 mm), and PDT alone has efficacies against cancers in the epithelial region or mucous layer [9]. Furthermore, conventional photosensitizers have no specificity against tumors. Thus, they are frequently freely dispersed in the body, and this problem induces long periods of sunshade [3]. To solve these problems, various kinds of photosensitizers have been investigated [2,5,[10][11][12][13]. For example, Mono-L-aspartyl chlorin e6 (NPe6) represented a favorable photosensitivity (i.e., the photosensitivity of NPe6 was resolved within 1 week) [5]. In particular, nanophotosensitizers, which are photosensitizer-incorporated nano-sized carriers, have been suggested as an ideal candidate for PDT [12][13][14]. Various biocompatible polymers or nanomaterials such as natural polysaccharides, dendrimers, iron oxide nanoparticles and synthetic polymers have been investigated to fabricate nanophotosensitizers [12][13][14][15][16]. Chin et al. reported that poly(vinylpyrrolidone) (PVP)-Ce6 formulations accelerate the tumor accumulation of photosensitizers and show fast clearance from the skin of nude mice [11]. Poly-cationic polysaccharides such as chitosan can be used to fabricate Ce6-encapsulated nanophotosensitizers [12]. They argued that nanophotosensitizers can be formed by an ion complex formation between water-soluble chitosan and Ce6 (ChitoCe6). The ChitoCe6 nanophotosensitizers showed superior accumulation in the tumor tissue of nude mice compared with free Ce6 and improved PDT efficacy against gastrointestinal cancer cells [12].
On the other hand, nanocarrier-based drug delivery systems have been spotlighted in last several decades due to their superiority in drug targeting issues [17][18][19][20][21]. Due to their small size, nanocarriers are believed to be an ideal candidate for tumor targeting [17,18]. Since the surface of nanocarriers such as polymeric nanoparticles can be easily decorated with targeting moieties such as monoclonal antibodies, they can then be specifically delivered to the tumor with a minimization of side effects against normal cells [17,18]. Furthermore, nanocarriers based on polymeric nanoparticles or micelles can be designed to stimulate of tumor tissues and then deliver the anticancer drug to the tumor tissues specifically [18][19][20][21]. For example, Lei et al. reported that polymeric micelles having pH and temperature dual-responsive properties accelerate the release rate of anticancer drugs at an acidic pH and a higher temperature [20]. From these points of view, stimuli-responsive nanocarriers have great potential in tumor targeting, because the physiological properties of the tumor microenvironment are quite different from their normal counterparts (i.e., tumor tissues are characterized as having an acidic pH, abnormal redox status, abundant extraor intra-cellular enzymes and elevated expression of various molecular receptors) [22][23][24][25]. Therefore, the abnormal status of a tumor microenvironment provides a tumor targeting opportunity for polymeric nanocarrier-based drug delivery [26]. Interestingly, these statuses for tumor microenvironments can be applied in the design of a nanoparticle architecture sensitive to the ROS level, since the intracellular ROS levels in tumor tissues are significantly higher than those of their normal counterpart [27,28]. For example, Lee et al. reported that chitosan cross-linked with a diselenide group and conjugated with acetyl histidine shows dual sensitivity against the pH and ROS [28]. They argued that nanoparticles sensitively liberate anticancer drugs through an acidic pH and ROS. Those features of nanoparticles accelerate the tumor accumulation of nanoparticles and then efficiently inhibit the metastasis of cancer cells. Furthermore, molecular receptors such as folate or CD44 receptors enable nanoparticles to target cancer cells in a receptor-specific manner [29,30]. Lee et al. reported that folic acid-decorated nanoparticles deliver anticancer agents in a folate-specific manner in in vitro cell culture and in vivo tumor xenograft models [29]. In our previous report, hyaluronic acid (HA) as a hydrophilic segment and hyperbranched Ce6 as a lipophilic segment formed nano-dimensional carriers as nanophotosensitizers through a self-assembling process (i.e., hyperbranched Ce6 formed the inner core of the nanophotosensitizers and the HA exposed on the surface of the nanophotosensitizers) [30]. They argued that nanophotosensitizers composed of HA-hyperbranched Ce6 conjugates via disulfide linkages represent the CD44-specific and redox-sensitive delivery capacity of Ce6's capacity against tumors [30]. Those nanophotosensitizers showed CD44-specific imaging and PDT efficacy against U87MG glioblastoma cells. Sun et al. also reported that folic acid-decorated nanocarriers show folate-receptor-mediated targeting and permit MRI-guided imaging of tumors [14].

Characterization of FAPEGtaCe6 Conjugates
To synthesize FAPEGtaCe6 conjugates, Ce6 was attached to the four carboxyl groups of TA via the diselenide group to make a Ce6 tetramer as shown in Figure 1a. Ce6 was primarily conjugated with selenocystamine to endow ROS sensitivity to the conjugates ( Figure S1a). Ce6-selenocystamine conjugates were conjugated again with TA to produce TA-sese-Ce6 conjugates as shown in Figure 1a. Figure S1 shows the specific peaks of Ce6 and TA (i.e., the specific peaks of Ce6 were confirmed at 1-10 ppm (Figure S1a)), and the peaks of the ethylene protons of selenocystamine were confirmed at 2.8-3.4 ppm. Furthermore, the specific peaks of TA (ethylene proton) were confirmed between 2 and 4 ppm, as shown in Figure S1a.
Otherwise, FA was attached to the amine group of bifunctional PEG as shown in Figure S1b. After that, the remaining carboxylic acid of the bifunctional PEG was attached with selenocystamine again to produce FA-PEG-sese conjugates ( Figure S1b). As shown in Figure S1b, the ethylene protons of PEG were confirmed at 3.4-3.7 ppm, while the specific peaks of FA were confirmed at 1 and 9 ppm. The ethylene group of selenocystamine was confirmed at 2.8-3.4 ppm. Next, four equivalents of the FA-PEG-sese conjugates were conjugated to the carboxyl group of the TA-sese-Ce6 conjugates as shown in Figure 1b. Figure 1c also shows that the 1 H NMR spectra showed all of the components of the FAPEGtaCe6 conjugates, the specific peaks of Ce6, selenocystamine, FA, TA and PEG. These results indicate that the FAPEGtaCe6 conjugates were successfully synthesized. Table 1 shows the characteristics of FAPEGtaCe6.
The theoretical contents were calculated as 16.8% (w/w), and the experimental content of Ce6 was 16.2% (w/w), indicating that the experimental content of Ce6 was not significantly different from that of the theoretical value.   Figure 2b. c Particle size of TA-sese-Ce6 is not determined.

Fabrication and Characterization of Nanophotosensitizers
FAPEGtaCe6 nanophotosensitizers were fabricated by a dialysis procedure. The ultraviolet-visible (UV-VIS) absorption spectra of Ce6 alone and the FAPEGtaCe6 nanophotosensitizers were measured as shown in Figure 2a. As this figure shows, the FAPEGtaCe6 nanophotosensitizers represented similar UV absorption spectra between 600 and 700 nm in DMSO. Furthermore, a specific peak at 664 nm was also observed both for Ce6 alone and the FAPEGtaCe6 nanophotosensitizers, indicating the intrinsic absorption properties of Ce6.
The morphology and particle size were measured via transmission electron microscopy (TEM) and a zetasizer as shown in Figure 2b,c. As illustrated in this figure, the nanophotosensitizers of the FAPEGtaCe6 conjugates showed spherical shapes having small sizes of less than 200 nm. The average particle size was 120.1 ± 37.31 nm, as shown in Figure 2c. These results indicate that the FAPEGtaCe6 conjugates could form nanoparticles in an aqueous environment with a monomodal distribution pattern. Furthermore, the aqueous stability of the FAPEGtaCe6 nanophotosensitizers was maintained for at least 7 days (i.e., the mean particle sizes were not significantly changed for 7 days, even though their average particle sizes were slightly increased, as shown in Figure 2d). Furthermore, neither large aggregates nor precipitants were observed even after 7 days. These results indicate that FAPEGtaCe6 nanophotosensitizers can be stored as an aqueous solution for biological applications. The singlet oxygen (SO) generation efficiency of the nanophotosensitizers was evaluated in aqueous conditions using an SOSG reagent as shown in Figure 2e. The fluorescence intensity of the SOSG was gradually increased according to the increase in irradiation time of both Ce6 alone and the FAPEGtaCe6 nanophotosensitizers. The fluorescence intensity of the nanophotosensitizers in particular was higher than that of Ce6 alone. Furthermore, the fluorescence intensity of the nanophotosensitizers was not significantly increased in the absence of light irradiation. These results indicate that the FAPEGtaCe6 nanophotosensitizers efficiently produced and successfully generated SO in the aqueous solution. The ROS sensitivity of the FAPEGtaCe6 nanophotosensitizers was assessed in the presence of H 2 O 2 as shown in Figure 3, Table S1 and Figure S2. Figure 3, Table S1 and Figure S2 shows that the nanophotosensitizers at 0 mM H 2 O 2 demonstrated results of 117.1 ± 45.18 nm with a monomodal distribution. However, the particle size distribution became broad and multimodal in its pattern when H 2 O 2 was added. When 2.0 mM H 2 O 2 was added, the size distribution of the nanophotosensitizers started to become bimodal in phase. In particular, the particle size distribution of the nanophotosensitizers became a bimodal distribution pattern completely with the addition of 10 mM H 2 O 2 , as shown in Figure 3. These results indicate that the FAPEGtaCe6 nanophotosensitizers had ROS-sensitive properties, and they could be disintegrated according to the oxidative stress.  Figure 4 shows the effect of ROS on the changes in the fluorescence emission spectra and the drug release properties of nanophotosensitizers. As shown in Figure 4a, the intensity of the fluorescence emission spectra was gradually increased according to the increase in the H 2 O 2 concentration. The fluorescence images in Figure 4a also increased according to the H 2 O 2 concentration. Furthermore, the Ce6 release became faster with the addition of H 2 O 2 , while the Ce6 release became significantly slower in the absence of H 2 O 2 . When H 2 O 2 was added to the release media, the Ce6 release rate became faster according to the H 2 O 2 concentration, indicating that the FAPEGtaCe6 nanophotosensitizers had ROS sensitivity, which means the liberation of Ce6 could be controlled by the oxidative stress in the biological system.

Cell Culture and PDT Study In Vitro
The PDT efficacy of the FAPEGtaCe6 nanophotosensitizers was evaluated with Y79 retinoblastoma cells, MCF-7 human breast cancer cells and KB human epithelial carcinoma cells as shown in Figures 5-9. Furthermore, dark toxicity as an intrinsic cytotoxicity against normal cells was assessed with ARPE-19 human retinal pigment epithelial cells in vitro as shown in Figure 6. Prior to assessing the PDT efficacy, the Ce6 uptake ratio was evaluated in vitro as shown in Figure 5. As shown in Figure 5a, the Ce6 uptake ratio for both the free Ce6 and FAPEGtaCe6 nanophotosensitizers was gradually increased according to the increase in Ce6's concentration. In particular, the FAPEGtaCe6 nanophotosensitizers represented a two or three times higher uptake ratio of all cell types, such as Y79 cells (Figure 5a), MCF-7 ( Figure 5b) and KB cells (Figure 5c). Figure 5d shows that treatment with nanophotosensitizers represented a significantly higher red fluorescence intensity in the KB cells than that of the free Ce6. These results indicate that the nanophotosensitizers had a higher efficacy in the intracellular uptake ratio against cancer cells.
Prior to performing the PDT study, the effect of a light dose against the Y79 cells was evaluated. As shown in Figure S3, the viability of the Y79 cells gradually decreased according to the increase in the light dose. The viability of the Y79 cells was not practically affected until 1.5 J/cm 2 (i.e., the cell viability was higher than 90% at 1.5 J/cm 2 and then gradually decreased until 20 J/cm 2 ). However, the temperature was also raised when the light dose was higher than 5 J/cm 2 , and this may have affected the viability of the cells. To minimize this, 2 J/cm 2 was used for the PDT study.
The dark toxicity of the Ce6 and FAPEGtaCe6 nanophotosensitizers was evaluated in vitro using noncancerous normal cell lines (ARPE-19 cells ( Figure 6a Figure 6a,b, the viability of the ARPE-19 cells and HaKat cells stayed higher than 80% until a 5.0-µg/mL Ce6 concentration in both the free Ce6 and FAPEGtaCe6 nanophotosensitizers. These results indicate that the FAPEGtaCe6 nanophotosensitizers had no significant cytotoxicity against normal cells until a 5.0-µg/mL Ce6 concentration, just like with the free Ce6. In the absence of light irradiation, the FAPEG-taCe6 nanophotosensitizers also had low cytotoxicity against cancer cells such as Y79 cells (Figure 6c), MCF-7 cells ( Figure 6d) and KB cells (Figure 6e). Ce6 treatment especially showed that the viability of the Y79, MCF-7 and KB cells was decreased to less than 80% at a 5-µg/mL Ce6 concentration, while the nanophotosensitizer treatment still remained at a cell viability higher than 80%. These results indicate that the nanophotosensitizers had a lower dark toxicity and no acute cytotoxicity against cancer cells in the absence of light irradiation. Furthermore, the intracellular uptake of the Ce6 or nanophotosensitizers was gradually increased according to the treatment time as shown in Figure S4a,b (i.e., when the Ce6 or nanophotosensitizers were exposed to cells for 4 h, the intracellular uptake ratio increased both for Ce6 alone and the nanophotosensitizers, as shown in Figure S4b). In particular, the intracellular uptake ratio of Ce6 alone for 4 h was increased more than two times compared with that at 1.5 h, while the nanophotosensitizer treatment's intracellular uptake ratio increased 30% compared with that at 1.5 h. These results indicated that the Ce6 uptake rate of the nanophotosensitizers was higher and faster than that of Ce6 alone, even though the Ce6 uptake ratio of Ce6 alone also gradually increased according to the course of time.   (Figure 7(Ac)). In particular, ROS generation of nanophotosensitizers in the Y79 cells, MCF-7 cells and KB cells was 3.2 times, 3.1 times and 3.4 times higher than those of Ce6 alone, respectively. These results indicate that the nanophotosensitizers had a higher efficacy in intracellular delivery and ROS generation in cancer cells. As expected, the nanophotosensitizers revealed increased phototoxicity against all cancer cells, as shown in Figure 7(Ba-Bc). The free Ce6 revealed a relatively lower or negligible phototoxicity until a Ce6 concentration of 1.0 µg/mL (i.e., the viability of the cancer cells remained higher than 80% at a Ce6 concentration lower than 1.0 µg/mL). Otherwise, the nanophotosensitizers revealed an increased phototoxicity compared with free Ce6, as shown in Figure 7B (i.e., a decrease in cancer cell viability started from a 0.5-µg/mL Ce6 concentration, and the viability was less than 40% at a 1.0-µg/mL Ce6 concentration). These results indicate that the nanophotosensitizers had improved PDT efficacy against cancer cells compared with the free Ce6. To assess the folate receptor and ROS-mediated targetability of the nanophotosensitizers, free folic acid (FA) was pretreated to the cells 30 min before nanophotosensitizer treatment as shown in Figures 8 and 9. As shown in Figure 8a, FA pretreatment did not significantly affect to the changes in intracellular Ce6 uptake in the treatment of free Ce6 (i.e., the red fluorescence intensity of the cells for the free Ce6 treatment did not significantly change under pretreatment of FA). However, FA pretreatment significantly decreased the red fluorescence in the nanophotosensitizer treatment. Furthermore, the intracellular uptake ratio of each cell also showed that the free Ce6 treatment was not affected by FA pretreatment, while the nanophotosensitizers significantly changed in fluorescence intensity, as shown in Figure 8b; that is, the relative fluorescence intensity significantly decreased under pretreatment of FA at the folate receptor positive cells, such as Y79 cells and KB cells. Interestingly, blocking of the folate receptors of the cancer cells by FA pretreatment was less effective in the folate receptor negative cells, such as MCF-7 cells (i.e., the decrease in fluorescence intensity was smaller in the MCF-7 cells than those of the folate receptor positive cells). These results indicate that the FAPEGtaCe6 nanophotosensitizers could be intracellularly delivered through a folate receptor mediated pathway. Additionally, H 2 O 2 was added to the culture medium to investigate the effect of extracellular ROS on the Ce6 uptake ratio. When H 2 O 2 was added to the FA pretreatment group, the fluorescence intensity of the Y79 cells and KB cells increased, while the fluorescence intensity for the Ce6 treatment was not significantly changed, as shown in Figure 8a,b. These results indicate that the FAPEGtaCe6 nanophotosensitizers could be delivered to the cancer cells via ROS-sensitive mechanisms. The effect of ROS generation and PDT also supported the results of Figure 8, as shown in Figure 9. Figure 9a,b shows that FA pretreatment did not significantly affect the changes in the intracellular ROS level or PDT-mediated cell death in Ce6 treatment. However, FA pretreatment significantly changed the intracellular ROS level and cell death, as shown in Figure 9a,b (i.e., FA pretreatment significantly decreased the intracellular ROS level and cell viability in nanophotosensitizer treatment). Additionally, the decrease in intracellular ROS level and cell death of folate receptor negative cells, such as MCF-7 cells, was relatively lower than those of folate receptor positive cells. These results clearly indicate that PDT efficacy of FAPEGtaCe6 nanophotosensitizers could also be controlled by folate receptors. Figure 9. The effect of pretreatment of FA on the intracellular ROS generation (a) and phototoxicity (b). Cells were treated with Ce6 or nanophotosensitizers in a similar manner to Figure 8. FA (5 mM) was pretreated to the cells 30 min before free Ce6 or nanophotosensitizer treatment. Ce6 concentration was 2 µg/mL. Cells were exposed to free Ce6 or nanophotosensitizers for 2 h and then irradiated at 664 nm (2 J/cm 2 ). *, ** p < 0.01.

In Vivo Pulmonary Metastasis Model of Y79 Cells for Biodistribution Study
The effect of folate receptor blocking was also assessed with a pulmonary metastasis model of Y79 cells, as shown in Figure 10. For biodistribution of the nanophotosensitizers in mouse organs, the Y79 cells were intravenously (i.v.) administered via the tail veins of the mice. FA (10 mg/kg in 0.1 mL PBS) was i.v. injected to block the folate receptor of the Y79 cells 30 min before injection of the nanophotosensitizers. As shown in Figure 10, the fluorescence intensity without pretreatment of FA (FA, 0 mg/kg) showed a significantly higher fluorescence intensity in the lungs than those of the other organs, indicating that nanophotosensitizers could be efficiently delivered to the pulmonary metastasis of Y79 cells, as shown in Figure 10 (upper images). Otherwise, blocking of the folate receptors of the Y79 cells (FA, 10 mg/kg) in the lungs induced a significant decrease in the fluorescence intensity compared with the other organs. These results indicate that the FAPEGtaCe6 nanophotosensitizers were delivered to the folate receptor positive Y79 cells through a folate receptor-mediated pathway. An in vivo pulmonary metastasis model of the Y79 cells also proved that the FAPEGtaCe6 nanophotosensitizers had targetability against the folate receptors of the tumors.

Discussion
The tumor microenvironment has a deep relationship with an elevated redox potential compared with normal tissues and cells [31][32][33][34][35]. In the tumor microenvironment, H 2 O 2 metabolic activity is known to be decreased in tumor cells, and the H 2 O 2 in the tumor microenvironment is able to be accumulated up to 100 µM, while the hydrogen peroxide in normal cells is normally less than 20 nM [32][33][34]. Furthermore, de Sá Junior et al. found that high ROS levels act as a cancer modulator and induce a genotoxic or proapoptotic effect on cancer cells [34]. They argued that these paradoxical characters of ROS guide antitumor therapeutic approaches using various chemical agents (i.e., molecules such as antioxidant chemicals can be used to prevent ROS formation and then prevent carcinogenesis). Practically, the FAPEGtaCe6 nanophotosensitizers produced intracellular ROS in various cancer cells by light irradiation, and an elevated ROS level induced the death of cancer cells, as shown in Figure 7. However, the viability of the cancer cells was not affected by Ce6 or nanophotosensitizers in the absence of light irradiation ( Figure 6). Otherwise, other agents for producing ROS may be used to promote a sudden increase in the ROS and then kill the cancer cells through oxidative stress against the tumor tissues [33][34][35][36]. Low intracellular levels of ROS are required for the signal transduction of normal cells, while high levels of ROS in cancer cells are required to maintain their high proliferation rate and to make the tumor resistant to conventional chemotherapy [35,36]. Therefore, this double-edged sword effect of ROS provides the opportunity to develop therapeutic strategies (i.e., increasing the intracellular ROS concentration until a toxic level can be applied to develop anticancer therapeutic carriers) [35][36][37]. For example, ROS-producing agents such as piperlongumine can be used to induce the apoptotic death of cancer cells via overproduction of intracellular ROS [28,38,39]. Furthermore, photosensitizers are mentioned as a typical therapeutic agent for producing ROS in cancer cells [40]. However, a deficiency of tumor specificity of conventional chemical agents is problematic for application in practical use in humans because these agents also have the potential to elevate ROS levels in normal cells or tissues [40,41]. For example, photosensitizers act on both normal cells and unhealthy cells and make patients sensitive to sunlight for a long duration [42,43]. These drawbacks require a novel delivery system for the specific targeting of cancer with the minimization of photosensitizers against normal cells or tissues. In our results, the FAPEGtaCe6 nanophotosensitizers liberated Ce6 in an ROS-sensitive manner (i.e., the liberation of Ce6 from the nanophotosensitizers was accelerated by the oxidative stress, while Ce6's release rate was significantly lower in the absence of H 2 O 2 than in the presence of H 2 O 2 , as shown in Figure 4). These results must have been due to the ROS-specific disintegration of nanophotosensitizers in the presence of H 2 O 2 , as shown in Figure 3. From these points of view, nanocarrier-based drug delivery systems have been investigated and found to be sensitive against higher intracellular ROS levels [27,28,37]. For example, nanofiber mats designed to be sensitive to ROS can be applied to the site-specific release of ROS-producing agents (i.e., anticancer agent release can be accelerated from the nanofiber mats while being sensitive to the ROS level) [43]. In this study, we designed nanophotosensitizers sensitive to ROS (i.e., nanophotosensitizers that responded and disintegrated according to the concentration of H 2 O 2 , a typical ROS molecule) (Figure 3). Furthermore, Ce6's release from nanophotosensitizers could be accelerated according to the concentration of H 2 O 2 (i.e., Ce6's release rate was increased at higher H 2 O 2 concentrations, as shown in Figure 4). Sun et al. also reported that ROS-sensitive nano-assemblies provide a synergistic anticancer effect on chemotherapy and PDT [44]. They argued that ROS-sensitive nano-assemblies can be used to encourage ROS-mediated PDT and synergistically promote paclitaxel-mediated chemotherapy. In our results, the nanophotosensitizers could be disintegrated in the presence of H 2 O 2 , and Ce6's release rate from the nanophotosensitizers could also be accelerated as shown in Figure 4. These results indicate that the nanophotosensitizers could be specifically delivered to a disease site having high oxidative stress, and thereafter, liberated Ce6 could be preferentially delivered to the disease cells. Since oxidative stress is normally elevated in cancer cells, these peculiarities of cancer cells can be applied in the targeting of anticancer agents [37,45]. We used higher H 2 O 2 concentrations than that of the tumor microenvironment for clarity of the ROS sensitivity of the nanophotosensitizers. As shown in Figures 3 and 4, the H 2 O 2 addition in the nanophotosensitizer solution induced modulation of the particle size distribution. Pandya et al. also reported that paclitaxel-incorporated nanoparticles were degraded by incubation with 5 mM H 2 O 2 , and the particle size was modified [46]. Our group also previously reported that poly(DL-lactide-coglycolide)/poly(ethylene glycol) nanoparticles having diselenide linkages showed H 2 O 2 -dependent degradation, and the antibiotic release rate was dependent on the H 2 O 2 concentration (i.e., nanoparticle degradation was accelerated at 10 mM H 2 O 2 rather than 0 or 1 mM H 2 O 2 , and the ciprofloxacin release rate also accelerated) [47]. This study also showed that the Ce6 release rate was significantly increased when 10 mM H 2 O 2 was added to the release media, as shown in Figure 4. These results indicate that the FAPEGtaCe6 nanophotosensitizers had ROS sensitivity and could respond to the oxidative stress of the tumor microenvironment.
On the other hand, cancer cells are normally characterized as an overexpression of various molecular receptors [48,49]. For example, folate receptors are overexpressed in malignant cells such as Y79 human retinoblastoma cells compared with normal cells such as ARPE-19 cells [50]. This event can also be applied to develop nanomedicine to be sensitive to elevated ROS levels. Alsaab et al. reported that folic acid-decorated nanomicelles represent improved cytotoxicity against retinoblastoma cells, while folate receptor negative normal cells such as ARPE-19 cells are not affected by nanomicelles [51]. In our results, when using non-cancerous normal cell lines such as ARPE-19 and HaKaT cells ( Figure S5), the FAPEGtaCe6 nanophotosensitizers showed a higher intracellular Ce6 uptake ( Figure S5a), ROS generation ( Figure S5b) and phototoxicity ( Figure S5c) similar to cancer cells, even though their gap was smaller than those of cancer cells. These results mean that nanophotosensitizers are also able to be delivered to normal cells in a nonspecific manner. However, an in vivo study using pulmonary metastasis showed that the nanophotosensitizers were specifically delivered to the Y79 cell-metastasized lungs as shown in Figure 10. These results indicate that FAPEGtaCe6 nanophotosensitizers can be delivered to tumor cells in the biological system of the human body. Folic acid-conjugated graphene oxide significantly increased the accumulation of Ce6 in the tumor cells and led to a remarkable photodynamic efficacy on the MGC803 cells upon irradiation [52]. Feng et al. reported that fluorescent on-off nanoprobes were delivered more efficiently to the folate receptor positive HeLa cells than the folate receptor negative NIH-3T3 cells or MCF-7 cells [53]. Our results also show that the nanophotosensitizers of FAPEGtaCe6 could be delivered via the folate receptors of cancer cells, as shown in Figure 8. Furthermore, ROS production, and thereby the PDT efficacy, of the nanophotosensitizers also showed folate receptor-sensitive behavior, as shown in Figure 9 (i.e., when the folate receptors of the cancer cells were blocked, the ROS production and PDT efficacy significantly decreased). Furthermore, the nanophotosensitizers of FAPEGtaCe6 could be delivered via a folate receptor-mediated pathway in the in vivo pulmonary metastasis model of Y79 cells.
We suggest that FAPEGtaCe6 nanophotosensitizers are a promising candidate for the targeted PDT of cancers having folate receptors.

Synthesis of FAPEGtaCe6 Conjugates
For the FA-PEG-COOH conjugates, 88.2 mg of FA dissolved in 10 mL DMSO was mixed with 38.4 mg EDAC and 23 mg of NHS. This solution was stirred for 3 h to activate the carboxyl group of the FA. Following this, 400 mg of NH 2 -PEG-COOH was added to this solution and then magnetically stirred for 24 h. This was introduced into the dialysis membrane (MWCO, 1000 g/mol) and then dialyzed against 1 L of distilled water for 1 day. Distilled water was exchanged in 2-3-h intervals to remove organic solvents. The dialyzed solution was lyophilized for 2 days. The yield of the lyophilized solid was calculated as follows: yield = [weight of lyophilized solid/(weight of FA + weight of PEG)] × 100. The yield of lyophilized solid was higher than 97 % (w/w).
For the FA-PEG-sese conjugates, 242 mg of FA-PEG-COOH dissolved in 10 mL DMSO was mixed with 19.2 mg EDAC and 11.5 mg NHS. This solution was stirred for 3 h to activate the carboxylic end group of FA-PEG-COOH, and after that, more than 5 equivalents of selenocystamine HCl were added with TEA. This solution was magnetically stirred for 1 day. After that, this solution was dialyzed against 1 L of distilled water for 2 days. Distilled water was exchanged in 3-h intervals to remove organic solvents. The dialyzed solution was lyophilized for 2 days.
Regarding the TA-sese-Ce6 conjugates, to synthesize the Ce6-sese conjugates, 59.7 mg of Ce6 dissolved in 10 mL DMSO was mixed with 19.2 mg EDAC and 11.5 mg NHS. This solution was stirred for 3 h to activate the carboxylic end group of Ce6. After that, 32 mg of selenocystamine HCl was dissolved in 5 mL of a DMSO/water mixture (DMSO/water = 4/1 v/v). This solution was magnetically stirred for 9 h. For activation of the carboxylic acid of TA, 10.6 mg TA dissolved in 5 mL DMSO was mixed with 19.2 mg EDAC and 11.5 mg NHS. This solution was magnetically stirred for 6 h. After that, this solution was mixed with a solution of Ce6-sese conjugates and then magnetically stirred for 24 h. Following this, the resulting solution was introduced into the dialysis membrane (MWCO: 2000 g/mol). This was dialyzed against deionized water for 2 days with an exchange of water in 2-3-h intervals to avoid saturation and to remove unreacted chemicals.
The resulting solution was lyophilized for 3 days, and the obtained solid was used for the next synthesis step or stored in a refrigerator (4 • C). The yield of the TA-sese-Ce6 conjugates was calculated as follows: yield = [weight of TA-sese-Ce6/(weight of Ce6-sese + weight of TA)] × 100. The yield of the TA-sese-Ce6 conjugates was higher than 95% (w/w).
For synthesis of the FAPEGtaCe6 conjugates, 91 mg of the TA-sese-Ce6 conjugates was dissolved in 20 mL DMSO and mixed with 4 equivalents of an EDAC/NHS system. This solution was stirred for 6 h to activate the carboxyl group of the TA-sese-Ce6. Following this, 265 mg of the FA-PEG-sese conjugates was added to this solution and then magnetically stirred for 2 days. This solution was introduced into the dialysis membrane (MWCO: 8000 g/mol) and then dialyzed against deionized water for 2 days with an exchange of water in 2-3-h intervals. The resulting solution was lyophilized for 3 days, and a dark green solid was obtained. The yield of the FAPEGtaCe6 conjugates was calculated as follows: yield = [weight of FAPEGtaCe6 conjugates/(weight of TA-sese-Ce6 + weight of FA-PEG-sese)] × 100. The yield of the FAPEGtaCe6 conjugates was higher than 94% (w/w).

Characterization of FAPEGtaCe6 Conjugates
1 H nuclear magnetic resonance (NMR) spectroscopy (500 mHz superconducting Fourier transform (FT)-NMR spectrometer, Varian Unity Inova 500 MHz NB High Resolution FT NMR; Varian Inc, Santa Clara, CA, USA) was used to monitor the synthesis procedure.

Fabrication of Nanophotosensitizers
The FAPEGtaCe6 conjugates (20 mg) were dissolved in 5-mL water/DMSO mixtures (1/4, v/v) and then introduced into the dialysis membrane (MWCO: 8000 g/mol) as reported previously [30,54]. This solution was dialyzed for 1 day with an exchange of water every 2-3 h to remove the organic solvent. The resulting solution was used for analysis or a PDT effect.

Ce6 Contents in the FAPEGtaCe6 Nanophotosensitizers
The contents of Ce6 in the conjugates were estimated as follows. The FAPEGtaCe6 nanophotosensitizers were fabricated as described above and were adjusted to 1 mg/mL. Then, 5 mL of this solution was mixed with 45 mL of phosphate-buffered saline (PBS, 0.01 M, pH 7.4) in the presence of 20 mM H 2 O 2 . This solution was stirred for 48 h and then diluted with DMSO more than 10 times. The Ce6 concentration was measured with an Infinite M200 pro microplate reader (Tecan, Männedorf, Switzerland) (excitation wavelength: 407, emission wavelength: 664 nm). The Ce6 itself dissolved in DMSO and was diluted 20 times with PBS (20 mM H 2 O 2 ). This was similarly used instead of a nanophotosensitizer solution for the standard test. Ce6 content (wt.%) = (Ce6 weight/total weight of nanophotosensitizers) × 100, where the Ce6 contents were about 16.2% (w/w).

Characterization of Nanophotosensitizers
The particle size of the FAPEGtaCe6 nanophotosensitizers was measured with Zetasizer Nano-ZS (Malvern, Worcestershire, UK). For measurement, the concentration of the nanophotosensitizer solution was adjusted to 0.1 % (w/w).
A transmission electron microscope (TEM) (H-7600, Hitachi Instruments Ltd., Tokyo, Japan) was employed to observe the morphology of the nanophotosensitizers. One drop of the nanophotosensitizer aqueous solution was dropped onto the carbon film-coated grid for the TEM. This was dried at room temperature for 3 h. Following this, phosphotungstic acid (0.1%, w/w in deionized water) was used to stain the nanophotosensitizers negatively. The observation was carried out at 80 kV.
The UV absorption spectra of the Ce6 or FAPEGtaCe6 nanophotosensitizers were measured with a Genesys 10s UV-VIS spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

Fluorescence Spectra
A fluorescence emission scan was measured between 500 nm and 800 nm (excitation wavelength: 400 nm) with a multifunctional microplate reader (Infinite M200 pro microplate reader, Tecan, Mannedorf, Switzerland). A similar solution was fluorescently observed with a Maestro 2 small animal imaging instrument (Cambridge Research and Instrumentation Inc., Woburn, MA 01801, USA). The nanophotosensitizers in the PBS was mixed with various concentrations of hydrogen peroxide and then incubated for 4 h at 37 • C.

Singlet Oxygen (SO) Generation of Nanophotosensitizers
SO generation from the Ce6 or nanophotosensitizers in an aqueous solution was measured using 1 mL of Ce6 alone or a nanophotosensitizer solution (5 µg/mL Ce6 equivalent in distilled water, 1% DMSO) [55]. For this solution, an SOSG reagent was added (final concentration: 5 µM). Each solution was irradiated with an expanded homogenous beam (664 nm, SH Systems, Gwangju, Korea) at different time points (0.5, 1, 2, 5, and 10 min). After that, the fluorescence intensity of this solution was measured with a fluorescence spectrophotometer (RF-5301PC, Shimadzu Co., Kyoto, Japan) at a 488-nm excitation wavelength and 525-nm emission wavelength. This procedure was carried out under dark conditions.

Drug Release Study
A Ce6 release study was carried out as follows. First, 5 mg of nanophotosensitizers (5 mg/5 mL deionized water), fabricated as described above, was introduced into a dialysis tube (MWCO: 8000 g/mol). Then, this was put into a 50-mL conical tube with 45 mL PBS (0.01 M, pH 7.4). Hydrogen peroxide was added 3 h later to study the effect of hydrogen peroxide. This was incubated in 37 • C with shaking at 100 rpm. The whole media was taken to evaluate the Ce6 release rate at predetermined time intervals and then replaced with fresh PBS. The Ce6 concentration in the media was estimated with an Infinite M200 pro microplate reader (Tecan) (excitation wavelength: 407 nm; emission wavelength: 664 nm). All experiments were performed in triplicate, and the results were expressed as the mean ± standard deviation (S.D.).

Cell Culture
Y79 retinoblastoma cells and ARPE-19 human retinal pigment epithelial cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). HaCaT human keratinocyte cells as well as KB human epithelial and MCF-7 human breast cancer cells were obtained from the Korean Cell Line Bank (Seoul, Korea). Y79, MCF-7 and KB cells were maintained with RPMI1640 medium (Gibco, Grand Island, NY, USA) at 37 • C in a 5% CO 2 incubator. The HaCaT cells were maintained in DMEM medium (Gibco, Grand Island, NY, USA) at 37 • C in a 5% CO 2 incubator. The ARPE-19 cells were maintained using DMEM/F12 medium (Gibco, Grand Island, NY, USA) at 37 • C in a 5% CO 2 incubator. All media for the cell culture were supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Invitrogen) and 1% penicillin/streptomycin.

PDT Treatment
The Y79, MCF-7 and KB cells (2 × 10 4 cells/well) were seeded in 96 well plates and then exposed to Ce6 or nanophotosensitizers. For the Ce6 treatment, it was dissolved in DMSO and then diluted with serum-free media more than 100 times. The nanophotosensitizers fabricated as described above were diluted with serum-free media. Each treatment was incubated for 2 h in a 5% CO 2 incubator at 37 • C. After that, the cells were washed with PBS twice, replaced with 100 µL serum-free media and adopted for PDT treatment. For PDT treatment, the cells were irradiated at 664 nm using an expanded homogenous beam (SH Systems, Gwangju, Korea) at 2.0 J/cm 2 (the signal was measured using a photo-radiometer (DeltaOhm, Padova, Italy)). The light intensity of the expanded homogenous beam and irradiation time were 3.515 mW/cm 2 and 569 s (2000 mJ/cm 2 ), respectively. After that, the cells were incubated for 24 h in a CO 2 incubator at 37 • C. The viability of the cells was evaluated with an MTT proliferation assay, and 30 µL of an MTT solution (5 mg/mL in PBS) was added to the wells and further incubated for 3 h in a CO 2 incubator. The supernatants were discarded and replaced with 100 µL DMSO. The absorbance (570 nm) was measured with an Infinite M200 pro microplate reader. All of the procedure was performed in dark conditions.
For dark toxicity, the ARPE-19, Y79, MCF-7 and KB cells (2 × 10 4 cells/well) were seeded in 96 well plates and then similarly treated as described above without light irradiation.

Intracellular Uptake of Ce6 or Nanophotosensitizers
The cancer cells (2 × 10 4 cells) were seeded in a 96-well plate and then exposed to Ce6 or nanophotosensitizers for 2 h as described above. The cells were washed with PBS twice and solubilized in 50 µL of lysis buffer (GenDEPOT, Barker, TX, USA). Then, the Ce6 uptake ratio was measured with an Infinite M200 pro microplate reader (Tecan) (excitation wavelength: 407 nm; emission wavelength: 664 nm).
For fluorescence observation, 3 × 10 5 cells seeded in 6 well plates with cover glass were exposed to Ce6 or nanophotosenstitizers for 90 min. The cells were washed with PBS twice, fixed with a 4% paraformaldehyde solution and then immobilized with a mounting solution (Immunomount, thermo Electron Co. Pittsburgh, PA, USA). Observation of the cells was performed with a fluorescence microscope (Eclipes 80i; Nikon, Tokyo, Japan).

ROS Assay
The cells (2 × 10 4 cells) were seeded in a 96-well plate and then exposed to Ce6 or nanophotosensitizers for 2 h as described above. To measure the generation of intracellular ROS, a DCFH-DA assay was employed. The cells were exposed to Ce6 or HAssCe6 nanophotosensitizers in phenol red free RPMI media in the presence of DCFH-DA (final concentration: 20 µM) for 2 h at 37 • C. Following this, the cells were washed with PBS twice, replaced with 100 µL fresh phenol red free RPMI media and then irradiated at 664 nm (2.0 J/cm 2 ). Intracellular ROS generation was estimated with a microplate reader (Infinite M200 pro microplate reader (Tecan); excitation wavelength: 485 nm; emission wavelength: 535 nm).

In Vivo Animal Tumor Imaging
A pulmonary metastasis model using nude BALb/C mice (male, 20 g, 5 weeks old) was prepared using the Y79 cells for in vivo fluorescence imaging. The Y79 cells (5 × 10 5 cells/0.1 mL PBS) were intravenously (i.v.) administered via the tail veins of the nude BALb/C mice. Three weeks later, nanophotosensitizers (10 mg/kg) were sterilized with a 0.8 µm syringe filter and i.v. administered via the tail veins of nude BALb/C mice. The injection volume was 0.1 mL, and 30 min before injection of the nanophotosensitizers, FA (10 mg/kg, in 0.1 mL PBS) was i.v. injected to block the folate receptor of the Y79 tumor. One day later, the mice were sacrificed and dissected to observe the organs and to observe the biodistribution of the nanophotosensitizers with a Maestro TM 2 small animal imaging instrument. For fluorescence observation of each organ, the filter of the Maestro TM 2 small animal imaging instrument was an orange filter set (excitation filter: 586-631 nm; emission filter: 645 nm longpass).

Statistical Analysis
A Student's t test using SigmaPlot ® (SigmaPlot ® v.11.0, Systat Software, Inc., San Jose, CA, USA) was used to estimate the statistical significance of the results and evaluate p < 0.05 as the minimal level of significance.

Conclusions
The FAPEGtaCe6 conjugates were synthesized for the ROS-specific and folate receptorspecific delivery of photosensitizers. The nanophotosensitizers were fabricated by a dialysis procedure. The nanophotosensitizers of the FAPEGtaCe6 conjugates showed spherical shapes and had small particle sizes of less than 200 nm. The nanophotosensitizers showed sensitivity (i.e., they disintegrated with the addition of H 2 O 2 , and the particle size distribution was changed from a monomodal pattern to a multimodal pattern). Furthermore, the addition of H 2 O 2 induced an increase in the fluorescence intensity and Ce6 release rate from the nanophotosensitizers, indicating that the nanophotosensitizers had ROSsensitive properties and the Ce6 release could be controlled by the ROS. The Ce6 uptake ratio, ROS generation and phototoxicity of the nanophotosensitizers were significantly higher than those of the free Ce6 against various cancer cells in vitro. In particular, the nanophotosensitizers showed a folate receptor-mediated delivery capacity against folate receptor-overexpressing Y79 cells and KB cells (i.e., blocking by the folate receptor via pretreatment with an FA-induced decrease in the intracellular Ce6 uptake, decreased ROS generation and, thereby, photoxicity). These results indicate that the nanophotosensitizers of the FAPEGtaCe6 conjugates had folate receptor specificity in vitro. Furthermore, the pulmonary metastasis model using Y79 cells showed a folate receptor-specific delivery capacity for the nanophotosensitizers. When the folate receptor of the Y79 cells in the pulmonary metastatic model was blocked, delivery of the nanophotosensitizers to the lungs was inhibited, indicating that the nanophotosensitizers of the FAPEGtaCe6 conjugates had folate receptor specificity in vitro and in vivo. We suggest that the nanophotosensitizers of FAPEGtaCe6 conjugates are promising candidates for a targeted PDT approach against cancer cells.


s2fieldsofstudy:
Biology
Engineering
Materials Science

text:
Electrospun aligned poly(ε-caprolactone) nanofiber yarns guiding 3D organization of tendon stem/progenitor cells in tenogenic differentiation and tendon repair

Hierarchical anisotropy structure directing 3D cellular orientation plays a crucial role in designing tendon tissue engineering scaffolds. Despite recent development of fabrication technologies for controlling cellular organization and design of scaffolds that mimic the anisotropic structure of native tendon tissue, improvement of tenogenic differentiation remains challenging. Herein, we present 3D aligned poly (ε-caprolactone) nanofiber yarns (NFYs) of varying diameter, fabricated using a dry-wet electrospinning approach, that integrate with nano- and micro-scale structure to mimic the hierarchical structure of collagen fascicles and fibers in native tendon tissue. These aligned NFYs exhibited good in vitro biocompatibility, and their ability to induce 3D cellular alignment and elongation of tendon stem/progenitor cells was demonstrated. Significantly, the aligned NFYs with a diameter of 50 μm were able to promote the tenogenic differentiation of tendon stem/progenitor cells due to the integration of aligned nanofibrous structure and suitable yarn diameter. Rat tendon repair results further showed that bundled NFYs encouraged tendon repair in vivo by inducing neo-collagen organization and orientation. These data suggest that electrospun bundled NFYs formed by aligned nanofibers can mimic the aligned hierarchical structure of native tendon tissue, highlighting their potential as a biomimetic multi-scale scaffold for tendon tissue regeneration.

Introduction
Partial or complete tendon ruptures caused by accidents and sports disrupt collagen alignment, leading to loss of function and reduced mobility (Hasan et al., 2014;Nau and Teuschl, 2015). However, native tendon has a poor endogenous ability to regenerate after injury due to fewer blood vessels and nerves in the tendon tissue (Silver et al., 2003;Longo et al., 2011;Kalson et al., 2015). Alternatively, tissue engineering (TE) strategies have demonstrated potential application for tendon tissue regeneration, and biomaterial scaffolds provide a temporary extracellular matrix (ECM) for cell adhesion and proliferation, which is a key element in guiding tissue regeneration (Chen et al., 2015;Zhang, 2020). Tendon has a hierarchical organization, and the subunits of the tendon are the fascicles (diameters: 100-500 μm), which consist of highly aligned collagen fibers (diameters: 1-20 μm) (Zhang et al., 2006;Carniel and Fancello, 2019;Puetzer et al., 2021). Such hierarchical structure is easily disordered upon injury due to the chaotic arrangement of neocollagen in scar tissue appearing at the site of the defect, thereby weakening the mechanical properties and functionality of the injured tendon (Sharma and Maffulli, 2005). This critical structure-function relationship reflects the design strategy for developing a suitable scaffold to facilitate the capacity of TE tendon to mimic the hierarchical structure of native tendon. The functional recovery of tendon tissue is a critical point in tendon TE scaffold development and is an ongoing challenge.
Recently, various fabrication technologies, such as electrospinning (Wu et al., 2016;Ma et al., 2018;Zhu et al., 2021), self-assembly (Gonzalez-Masis et al., 2020;Yin et al., 2020), and 3D bioprinting (Merceron et al., 2015;Chae et al., 2021;Zhang et al., 2021), have been developed to mimic the structure of native tendon by controlling aligned cellular organization. Among these techniques, electrospinning has been widely used in tendon TE due to its ability to prepare aligned nano-and micro-fibers efficiently and mimic the anisotropic hierarchical structure of native tendon tissue (Sensini and Cristofolini, 2018). In previous studies, continuous electrospun filaments have been made through changing the type of collectors (Dabirian et al., 2007;Bosworth, 2014;Laranjeira et al., 2017;Xie et al., 2021) to prepare a hierarchical structure for tendon tissue (Wu et al., 2017b;Sensini et al., 2019;Sensini et al., 2021). Mouthuy et al. (2015) fabricated a polydioxanone filament using a wire collector and assembled filaments into yarn to mimic the structure of native tendon, but then failed to control the diameter of the filaments. To achieve a similar effect, Sensini et al. (2019) prepared a multi-scale scaffold by rolling aligned nanofiber films to form a unit, then assembled them to make the multiscale scaffold. Similarly, Laranjeira et al. (2017) produced braided and woven scaffolds using different textile techniques to weave together nanofiber threads into fibrils, then fibers, and, ultimately, 3D scaffolds. In addition to the morphological and mechanical features of the scaffold, it is well-established that suitable diameters and functions are extremely important for cell proliferation and differentiation. However, the effects of bundled nanofiber diameter have not been investigated.
Recently, we developed a series of aligned nanofiber yarns (NFYs) based on an advanced wet-dry electrospinning technique to induce 3D cellular alignment and differentiation. We further developed biomimetic anisotropic scaffolds based on these aligned NFYs for various TE applications, including skeletal muscle, cardiac, and nerve TE (Wang et al., 2015;Wu et al., 2017b;Wang et al., 2019). However, due to the similar structure of tendon tissue, the effect of aligned NFYs on tenogenic differentiation in vitro and tendon repair in vivo has remained unclear.
Furthermore, stem cells have been regarded as a promising cell type for tendon regeneration. Among the types of stem cells isolated from different tissue sources, tendon stem/progenitor cells (TSPCs), which were isolated from tendon sources, were thought to be especially suitable for the tendon microenvironment and highly likely to differentiate into tenocytes (Guo et al., 2016). However, a recent study showed that TSPCs tend to undergo chondrogenic/osteogenic differentiation under inflammatory conditions, which ultimately leads to ectopic calcification in tendon tissue (Chen et al., 2019). It was found that local stem/progenitor cells are induced into an erroneous differentiation in the diseased tendon (Hu et al., 2016). Therefore, promoting tenogenic differentiation of TSPCs plays a key role in tendon regeneration, yet remains a challenging prospect. Although several previous studies reported that scaffolds with aligned structures could improve the tenogenic differentiation of TSPCs (Yin et al., 2010), there are still few reports on the design of aligned scaffolds that integrate with nano-, micro-and macro-scale structures and would mimic the hierarchical structure of native tendon to promote tenogenic differentiation of TSPCs and tendon repair.
Herein, we report the development of a series of aligned bundled scaffolds based on NFYs prepared by our previously developed wet-dry electrospinning technique, which demonstrated well-defined nano/microarchitecture that mimicked the hierarchical structure of fascicles and collagen fibers in native tendon tissue ( Figure 1A). TSPCs were seeded on aligned NFYs, and the effects of the nano-and micro-structure of aligned NFYs on the cellular alignment and organization of TSPCs were investigated. After culture for 7 days in differentiation medium, the effects of different NFY diameters on the tenogenic differentiation of TSPCs was also investigated. Furthermore, aligned NFY bundle scaffolds were implanted into the rat tendon defect model to investigate their ability to improve in vivo tendon repair. The electrospun aligned NFYs were able to guide cellular alignment and organization, thereby improving tenogenic differentiation of TSPCs in vitro, which suggests that these aligned NFY bundle scaffolds mimic the hierarchical structure of collagen fascicles and fibers in native tendon tissue and demonstrates great potential for tendon TE applications.

Preparation of PCL aligned nanofiber yarns
The aligned PCL NFYs were prepared via dry-wet electrospinning following our previously reported process (Wang et al., 2015;Wu et al., 2017b;Wang et al., 2019). PCL was dissolved at 100 mg/ml in hexafluoroisopropanol at room temperature for 12 h. The schematic diagram in Figure 1 illustrates the working principle of the electrospinning device (ET-2535, Ucalery, China). In brief, the PCL solution was drawn into a 5 ml syringe with a 21G needle with a length of 15 mm, an inner diameter of 0.5 mm, and an outer diameter of 0.8 mm (Ucalery, China). Injection speed was 1 ml/h using a syringe pump (ET-2535, Ucalery, China). The length of the drum was 21 cm and the diameter was 3 cm. Spinning was induced by adjusting the positive and negative pressure of the electrospinning instrument; the positive voltage was set at 18 kV and the negative voltage at 2 kV. During the electrospinning process, the random nanofibrous mat was received on the surface of a water/ethanol (V/V 8:2) mixture, and the continuous NFYs were then obtained by dragging from the surface of the solution using a rolling receptor to gain. The distance between the needle and surface of the solution was approximately 10 cm, and the distance between the liquid surface The preparation scheme of aligned PCL NFYs and their applications for tenogenic differentiation in vitro and tendon repair in vivo. (A) The complex hierarchical structure of native tendon tissue, consisting of aligned collagen fascicles with aligned fibers. The bundled NFY scaffold mimicked the hierarchical structure of native tendon. (B) Fabrication scheme based on aligned NFYs that mimic native tendon tissue to produce the hierarchical structure of scaffolds through a dry-wet electrospinning technique.
Frontiers in Bioengineering and Biotechnology frontiersin.org and the receptor was approximately 15 cm. While the other conditions were fixed, aligned NFYs with different diameters could be obtained by varying the rotation speed of the receptor, ranging from 2 to 80 mm/min. The aligned NFY samples were collected from the receiver and kept at room temperature for at least 2 days before subsequent experiments.

Morphological characterization and mechanical properties of aligned NFYs
Scanning electron microscopy (SEM, Sigma HD, United States) was used at an accelerating voltage of 2 kV to analyze the microstructure surface chemistry of aligned NFYs; each sample had three groups of replicates. The diameter distribution and alignment properties of nanofibers were counted using the ImageJ software module OrientationJ; each sample type had three replicates. Mechanical testing of single aligned NFYs was performed via dynamic thermo-mechanical analysis (Q800, DMA, United States). First, both ends of the single aligned NFY were clamped on filter paper and then fixed on the upper and lower arms of the instrument. The lengths of the aligned NFYs tested were approximately 2 cm. Uniaxial tensile testing was carried out at a stretching rate of 5 mm/ min until specimen failure. As for the mechanical properties of bound NFYs, we bundled 50 NFYs of each diameter, and then used a universal testing machine (a series of LS, AMETEK, United States) to test the force and strain of these bound NFYs. The lengths of aligned NFYs tested were approximately 14 cm. Uniaxial tensile testing was carried out at a stretching rate of 5 mm/min until specimen failure. More than five parallel samples were measured for each sample of different NFY diameter.

Isolation and culture of TSPCs
The Achilles tendon and patella tendon were obtained to isolate tendon stem/progenitor cells (TSPCs) from 6 to 8 week old Sprague Dawley (SD) rats, which were obtained following the approved guidelines set by the Institutional Animal Care and Use Committee, Southern Medical University. First, the peritendinous connective tissue was carefully removed, and the samples were washed three times with Dulbecco's phosphate buffered saline (DPBS, Gibco, China). The samples were then minced into 2 mm 2 pieces and digested with 3 mg/ml collagenase type I for 30 min at 37°C. Following digestion, the samples were diluted with DPBS, filtered through a 70 μm cell strainer, and then centrifuged at 1,000 rpm for 15 min. Single-cell suspensions were cultured in a cell culture incubator at 5% CO₂ and 37°C in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, China) containing 20% fetal bovine serum (FBS, Gibco, Australia) and 1% penicillin/streptomycin. TSPC colonies were observed under a microscope after 5 days. A previous study has shown that TSPCs have good expression of tenomodulin (TNMD) up to passage 10 (Tan et al., 2012), so cells from passages 3-8 were used in our subsequent experiments.

TSPC culture on aligned NFYs
TSPCs were seeded and cultured on aligned NFYs of varying diameters following several steps. First, the aligned NFY samples were fixed around a 3D-printed rectangle model. Second, the aligned NFY samples were sterilized by immersion in 75% ethanol for 30 min, followed by 30 min of UV irradiation. After drying, the aligned NFYs were soaked in 1% ε-Poly-Llysine solution for 30 min and dried overnight. Finally, the TSPC suspension (0.3 ml, 3×10 5 cells/mL) was dropped onto aligned NFYs and allowed to adhere at 37°C for 4 h. When most cells had adhered to the surface of the NFYs, the aligned NFY samples were peeled off from the PDMS mold and placed into wells of a 6well plate for continued culture for required experiments. The cell culture medium was changed every 2 days.

Viability of TSPCs on aligned NFYs
The cellular activity of TSPCs seeded on the aligned NFYs was evaluated by Live/Dead assay (Beyotime, China) after 24 h and by Alamar Blue assay (Molecular Probes, Invitrogen, China) after 1, 5, and 7 days in culture, according to the manufacturer's instructions. After being seeded on the aligned NFYs of various diameters, cells were cultured for 24 h to allow cell spreading. Cell-laden NFYs (n = 3) were then transferred to a new plate and washed with DPBS to remove the non-adherent cells. Calcein AM stained living cells with green fluorescence, and propidium iodide (PI) stained dead cells with red fluorescence. Before Frontiers in Bioengineering and Biotechnology frontiersin.org adding the working fluid, samples were washed with DPBS and then incubated for 30 min. Fluorescence imaging was carried out using confocal laser scanning microscopy (Nikon A1, China). Cells were incubated in 10% (v/v) Alamar Blue reagent in growth medium at 37°C for 4 h. The fluorescence of the supernatant was measured using a microplate reader (Synergy HT, Bio-Tek Instruments, United States) at an excitation wavelength of 560 nm and an emission wavelength of 590 nm.

Morphological evaluation of TSPCs on aligned NFYs
The morphology and organization of TSPCs on aligned NFYs of various diameters were analyzed via immunofluorescence imaging after 3 days in culture; each sample had three replicates. Cultured cells were gently rinsed twice with DPBS for 3 min and then fixed with 4% PFA for 15 min at room temperature. After fixation, cells were gently rinsed with DPBS three times and then treated with 0.2% Triton X-100 for 45 min. The samples were blocked using 1% BSA for 2 h and then treated with fluorescein isothiocyanate (FITC)phalloidin (Invitrogen, China) conjugated at 4°C overnight and counterstained with 4', 6-diamidino-2-phenylindole (DAPI, Sigma, United States). Fluorescence imaging was carried out using a confocal laser microscope (Nikon A1, China). Images of F-actin fluorescence were processed using the OrientationJ module of ImageJ software and the elongation and orientation of TSPCs on these aligned NFYs were analyzed following the protocol of our previous study (Wang et al., 2015). Cell elongation was determined by measuring nuclear aspect ratios and the angle between the long axis of the cells and the direction of aligned NFYs to generate alignment histograms. Cells cultured on glass slides were the 2D control group.

Tenogenic differentiation of TSPCs on aligned NFYs
TSPCs were cultured on aligned NFYs of various diameters in differentiation medium (growth medium with 50 mg/ml 2phospho-L-ascorbic acid trisodium) to induce tenogenic differentiation for 7 days; each sample had three replicates. After 7 days of induction, the cell-seeded NFYs were carefully rinsed twice with DPBS and then fixed with 4% PFA for 15 min at room temperature. After fixation, the cells were carefully washed twice with DPBS and then treated with 0.2% Triton X-100 for 45 min. After blocking in 1% BSA in DPBS for 2 h, the cell constructs were incubated with rabbit anti-TNMD antibody (ab203676, abcam, China) and rabbit anti-collagen I (Col1a1) antibody (CO20191-01, Invitrogen, China) at 4°C overnight. The cell constructs were then washed with DPBS, and Alexa Fluor 488 (ab150077, abcam, China) conjugated secondary antibody was added and samples were incubated for 45 min at room temperature. After incubation, the cell constructs were washed with DPBS and cell nuclei were counterstained with DAPI for 5 min. Finally, the cell-seeded NFYs were observed under a confocal laser microscope (Nikon A1, China).

Real-time qPCR
For real-time quantitative PCR (RT-qPCR) analyses, total RNA was extracted using Trizol (AG RNAex Pro Reagent, AG, China) following the manufacturer's instructions. For synthesizing cDNA, 0.2-1 μL RNA was used in a reverse transcription reagents kit protocol (Abm, Canada). Three cDNA parallel compositions were obtained. Real-time qPCR was carried out according to the protocol. Briefly, qPCR reactions were first carried out on ice, and each well contained 7.5 μL MagicSYBR Mixture, 0.15 μL ROX, 0.5 μL cDNA, 6.35 μL H 2 O, and 0.6 μL mixed primers. The plates were inserted in an Applied Biosystems QuanStudio ™ 3 Real-Time PCR (Singapore) Instrument for cycling at the following temperature profile: 95°C for 30 s, then 40 cycles of 95°C for 5 s followed by 60°C for 30 s. The primer sequences were as shown in Table 1. Each sample had three groups of replicates.

In vivo rat Achilles tendon repair model and histological evaluation
The animal experiment procedures were approved by the Institutional Animal Care and Use Committee, Southern Medical University. The SD rats (n = 6, 8-weeks-old) were treated with 1% pentobarbital sodium intraperitoneally at 40 mg/kg body weight. After anesthesia, a 6 mm length of the Achilles tendon was removed to make a defect model. Bundles of 50 yarns of NFY-50 were used to prepare the aligned NFY scaffolds to provide mechanical support for the repaired tendon. The NFY-50 scaffolds (50 yarns, length = 0.2 cm) were sutured to the residual Achilles tendon using a non-resorbable suture (5-0 nylon) via the Kessler method of tendon repair. Tendons that had been cut off directly served as the control group. The animals were allowed free movement after surgery. After 6 weeks of implantation, samples from each group were harvested and fixed in 4% PFA. Samples were then dehydrated through a series of increasing concentrations of ethanol, embedded in paraffin blocks, and sectioned at a thickness of 8μm. Sections were stained with hematoxylineosin (H&E), Masson's trichrome, and Picrosirius red. Finally, the stained paraffin sections were observed under an optical microscope (Nikon Nxi, China). The Masson's trichrome images were processed using the OrientationJ module of ImageJ software, with analysis of the orientation Frontiers in Bioengineering and Biotechnology frontiersin.org of neo-collagen in the defect site of each rat. Each group included three replicates.

Statistical analysis
Experiments were run in triplicate for each sample and results are presented as mean ± standard deviation. Quantitative data and measurement of cell aspect ratios and orientation were obtained using ImageJ software, with results including at least 5 images of 3 independent locations within each sample (cell number >100). Statistical differences were obtained through analysis of variance followed by Tukey's significant difference post hoc test. A significance level of 0.05 was applied to indicate significant differences.

Preparation and Characterization of aligned NFYs
The fabrication of aligned NFYs based on a modified electrospinning device is shown in Figure 1B, and the process of fabricating these aligned NFYs is detailed in Supplementary Video S1. Briefly, the PCL polymer solution (100 mg/ml) was extruded by the pump in a high voltage field to prepare a mat of random nanofibers on the surface of a water/ethanol mixture. Subsequently, a rolling receptor pulled the nanofibers mat from the solution's surface to form aligned NFYs. By controlling the rotation rate of the rolling receptor, a series of aligned NFYs with varying diameters were fabricated using the dry-wet electrospinning approach. The microstructure of the aligned NFYs was observed using SEM. The images showed that the aligned NFYs were loose and porous cylinders, and that the bundled NFYs had a hierarchical structure similar to native collagen fibers and fascicles of the tendon (Figure 2A; Supplementary Figure S1A). The nanofiber diameter was controlled, at 732.1 ± 167.22 nm by adjusting the parameters of electrospinning. The diameter of the aligned NFYs decreased dramatically from 100 ± 24 μm to 10 ± 2 μm when the receptor rotation rate increased from 2 to 80 mm/min, respectively ( Figure 2B). The aligned NFYs with diameters of 10 ± 2 μm, 50 ± 10 μm, and 100 ± 24 μm were designated NFY-10, NFY-50, and NFY-100, respectively. NFY-10, NFY-50, and NFY-100 showed the proportion of oriented nanofibers, in the range of ±10 o , to be 95.13%, 88.21%, and 85.66%, respectively ( Figure 2C), suggesting that aligned NFYs have great improvement in axial orientation with increased rotation speed. The loaded-force of aligned NFYs was measured using a single fiber tensile test. The tensile analysis results showed that the force of a single NFY-100 was as high as 5.16 ± 0.04 cN, while NFY-50 was slightly decreased at 3.16 ± 0.16 cN, and NFY-10 was only 1.13 ± 0.13 cN ( Figure 2D). Furthermore, the strain of aligned NFYs increased from 141 ± 41% to 486 ± 17% with the increase of NFY diameter from 10 µm to 100 μm, respectively. As shown in Figure 2E, the loaded-force of the bundled 50 yarns of NFY-10 was 0.44 ± 0.1 N with a stretching speed of 5 mm/min. Comparatively, the loaded-force of the bundled 50 yarns of NFY-100 was 3.31 ± 0.37 N. Notably, the loaded-force of the bundled 50 yarns of NFY-50 was 2.15 ± 0.09 N, which is suitable for tendon tissue with a loaded-force of 2-3 N ( Table 2). These results revealed that nano-and micro-scale NFYs containing highly aligned nanofibers can be successfully prepared using the dry-wet electrospinning approach, with the mechanical properties of NFYs easily controlled by changing their diameters.

Identification and characterization of TSPCs
After 3 days of isolation, a small number of TSPCs with spindle-like morphology were observed. The cells proliferated continuously and formed colony units after 9 days. The tenogenic differentiation capacity of TSPCs was verified by testing the expression of TNMD. As shown in Figure 3B, the TSPCs had obvious expression of TNMD. Furthermore, as shown in Figure 3C, over 92% and 33% of TSPCs were positive for BMSC markers CD90 and CD44, respectively, while they were negative for CD18, which is a surface receptor present on BMSCs (Miura et al., 2005). In addition, the TSPCs did not express hematopoietic cell markers, including CD34 and CD45, which are both highly expressed in tenocytes (Li et al., 2021), further confirming that these tendon-derived cells were TSPCs.

Viability and morphology of TSPCs on aligned NFYs
TSPCs were seeded on single aligned NFYs of various diameters, cell viability was assessed using the Live/Dead assay Frontiers in Bioengineering and Biotechnology frontiersin.org after for 24 h in culture, and cell proliferation was assessed using Alamar Blue reagent after 1, 5, and 7 days in culture. TSPCs cultured on aligned NFYs showed high viability, with few dead cells and excellent elongation ( Figure 4A). The proliferation results showed that all aligned NFY groups had a significant increase in cell growth during the culture period. On the first day of cell culture, there were no significant differences between the aligned NFY groups (p > 0.05). As time in culture increased, the number of TSPCs on the aligned NFYs of various groups showed significant differences (p < 0.05). In particular, the NFY-100 group showed significantly higher cell numbers than the NFY-10 and NFY-50 groups on days 5 and 7 ( Figure 4B). These results demonstrated that TSPC growth increases with increased NFY diameter, and the aligned NFYs, regardless of diameter, have good biocompatibility. The cellular alignment and elongation behaviors of TSPCs on aligned NFYs with different diameters were assessed by F-actin staining after 3 days in culture. The cytoskeleton was stained green using FITC-conjugated phalloidin, and the nucleus was stained blue using DAPI. The images of F-actin staining show that TSPCs spread well on the surface of all aligned NFYs and exhibited aligned and elongated morphologies ( Figures 5A-C). In contrast, cells in the 2D group showed random spindle morphology ( Figure 5D). Furthermore, quantitative analysis of cell alignment was accomplished by measuring the angle between  Frontiers in Bioengineering and Biotechnology frontiersin.org

FIGURE 4
The viability of TSPCs on aligned NFYs with different diameters. (A) Fluorescence imaging of living (green) and dead (red) TSPCs seeded on aligned NFYs and cultured in growth medium for 24 h. (B) The proliferation of TSPCs on NFYs with different diameters, after 7 days, as measured using Alamar Blue. *p < 0.05 compared with the NFY-10 group at day 1, # p < 0.05 compared with the NFY-50 group at day 1, and & p < 0.05 compared with the NFY-100 group at day 1.
Frontiers in Bioengineering and Biotechnology frontiersin.org 09 cell growth direction and the direction of aligned NFYs, including relative alignment in ±10°increments ( Figure 6A). These data show that the orientation ratio of cells within ±10 o on NFY-10, NFY-50, and NFY-100 were 73%, 58%, and 53%, respectively ( Figures 6B-E), while the orientation ratio was only 12% in the 2D group ( Figure 6F). Additionally, cell elongation behavior was estimated by assessing TSPC nuclear aspect ratios, which demonstrated a significant improvement in cellular elongation of the NFY-10 group compared to that of the 2D group ( Figure 6H). Together, these data indicate that aligned NFYs are able to enhance the 3D cellular alignment and elongation of TSPCs, which mimics native collagen fibers with tenocyte alignment. Notably, TSPCs showed better alignment and elongation behaviors when cultured on aligned NFYs with smaller diameters.

Tenogenic differentiation of TSPCs on aligned NFYs
To explore the effects of aligned NFYs on tenogenic differentiation of TSPCs, cells were seeded on aligned NFYs of various diameters and then cultured in differentiation medium for 7 days. TNMD is a marker of the mature tenogenic phenotype. Type Ⅰ collagen (Col1a1) is a major matrix component of native tendons that supports essential mechanical properties of tendon tissue (Goh et al., 2014). Tenogenic differentiation was examined by TNMD immunostaining. Images of TNMD and Col1a1 staining showed that the fluorescence intensity in all of the aligned NFY groups was higher than that of the 2D group, indicating that TSPCs on these aligned NFYs had higher TNMD and Col1a1 expression compared with the 2D group, and that aligned NFYs enhanced tenogenic differentiation of TSPCs, as suggested by enhanced expression of these markers ( Figures  7A,B). Additionally, NFY-50 showed the highest TNMD expression, compared with NFY-10 and NFY-100, which indicates that the best tenogenic differentiation of TSPCs occurred on NFY-50.
We also investigated the effects of cell culture on aligned NFYs on the tenogenic differentiation of TSPCs maintained in growth medium for 7 days. Decorin (DCN) is the most abundant small protein-polysaccharide rich in leucine in the tendon and plays a key role in ensuring precise alignment and stability of collagen fibers related to the phenotype of the tendon. Scleraxis (SCX) is an essential transcription factor for tenogenesis (Bavin et al., 2017). RT-qPCR results showed that aligned NFYs improve the expression of tendon-related genes, including TNMD, DCN, Col1a1, and SCX ( Figure 7C). Gene expression of TNMD and DCN was approximately 30 times and 20 times greater,

FIGURE 5
The top and 3D views of TSPCs with F-actin-stained cytoskeletons (green) and DAPI-stained nuclei (blue) after 3 days of culture on NFY-10 (A), NFY-50 (B), and NFY-100 (C), as well as cells in the 2D group (D). Scale bar = 100 μm. TSPCs cultured on single aligned NFYs with varying diameters showed aligned morphology, contrasting with the morphology of the 2D control group.

Aligned NFYs aid in repair of rat Achilles tendon injury in vivo
The ability of single NFYs to improve the tenogenic differentiation of TSPCs has been verified. Therefore, to investigate the effects of NFYs on improving tendon regeneration in vivo, we implanted 50-yarn NFY bundles into a rat tendon injury model. The NFY-50 group showed significantly better tenogenic differentiation of TSPCs compared with the other NFY groups. Therefore, we used NFY-50 to further investigate effects of NFYs on in vivo tendon repair. Tendon injury usually includes three sequential phases from injury onset to healing: the injured tendon initially becomes inflamed, forming granulation tissue within the injury region to serve as a provisional matrix. Then, type III collagen fibers, fibronectin, and proteoglycans are formed at the injury site and type III collagen fibers are replaced by type Ⅰ collagen fibers at the proliferation stage. Finally, there is alignment of tenocytes and collagen fibers, with type Ⅰ collagen fibers increasing and type III collagen fibers decreasing (Vasiliadis and Katakalos, 2020). In our study, 6 weeks after implantation, partial neo-tissue with a typical white, tendon-like appearance was observed in rats that received the bundled NFY-50 scaffold ( Figure 8A). To identify the type of collagen prominent in the neo-tissue, we used Picrosirius red staining to distinguish collagen types in the tendon defect area. As shown in Figure 8C, the aligned NFY scaffold group formed more collagen I (red), with little collagen Ⅲ (green) remaining; in contrast, the injury only group showed more collagen Ⅲ formation, indicating that aligned NFY scaffolds improve tendon regeneration. H&E staining results ( Figure 8D) showed partial formation of aligned neo-tissue around the NFY-50 scaffold. Bundled NFY-50 scaffolds surrounded by high cell nuclei density indicated that the nanofiber structure of NFY-50 had allowed immune cells and blood infiltration, which may have enhanced tissue remodeling. Frontiers in Bioengineering and Biotechnology frontiersin.org Images taken at high magnification showed partial neo-tissue that was organized and aligned, as it was guided by the aligned micro-and macro-structure and mechanical support of the bundled NFY-50 scaffold. In contrast, the neo-tissue in the injury only group was disordered and showed typical scar tissue without aligned structure. Masson's trichrome staining ( Figure 8E) results also indicated that neo-tissue formation included an abundance of collagen fibers (blue), and showed that the collagen fibers had an aligned microstructure along the direction of the NFY-50 scaffold. These quantitative data Frontiers in Bioengineering and Biotechnology frontiersin.org demonstrate that the orientation ratio of collagen within ±10°in the injury only and NFY-50 groups were 34% ± 4% and 57% ± 6%, respectively. Therefore, the injury only group showed amorphous organization, indicating fibrous scar tissue formation, compared to alignment in the NFY-50 group.

Discussion
Native tendon tissues show a hierarchical structure, where highly-aligned collagen fibers form fascicles, and many fascicles are bound together to form the tendon (Goh et al., 2014). Tenocytes, in particular, lay on the longitudinally aligned collagen fascicles and fibers and exhibit cellular orientation. This hierarchical structure plays a key role in the high tensile strength of native tendon. Therefore, developing a biomimetic TE scaffold with a hierarchical structure that enhanced cellular alignment, elongation, and mechanical strength would improve tendon repair (Vasiliadis and Katakalos, 2020). In preparing the biomimetic scaffold, the first challenge is in providing suitable mechanical support for the newly synthesized ECM at the injury site during tendon tissue regeneration. In this study, we chose PCL polymer as the primary material from which to develop the electrospun NFY due to its suitable mechanical properties, good biocompatibility, and broad use in various biomedical applications (Sun et al., 2006;Hu et al., 2016).
The electrospinning technique is regarded as a promising approach to preparing aligned nanofibers. For instance, previous studies have shown that aligned nanofiber sheets promote cellular elongation and tenogenic differentiation (Watari et al., 2012;Tu et al., 2020). However, those nanofiber sheets have failed to mimic the complex hierarchical structure of native tendon tissue. In contrast, we have reported on a series of NFYs, prepared using a well-developed dry-wet electrospinning technique, that were able to guide 3D cellular alignment and elongation in soft TE applications (Wu et al., 2017b;Wang et al., 2019). Therefore, in this study, we utilized our dry-wet electrospinning system to prepare the aligned PCL NFYs with an abundance of aligned nanofibers that mimicked the hierarchical structure of alignment fascicles and collagen fibers in native tendon tissue (Figure 2; Supplementary Figure S1). The aligned NFYs with smaller diameters had better alignment structure but less robust mechanical properties ( Figure 2D). The rotation rate of the NFY receptor played a key role in the diameter and orientation of NFYs. When the receptor was rotating faster, the random nanofibers would be dragged from the surface of the solution and received on the receptor faster. Therefore, the NFYs would have better alignment structure and smaller diameter with increased rotation rate of the receptor. In addition, a previous study suggested that nanofiber orientation was affected by the rate of the receptor with only one variable (Vimal et al., 2016). The mechanical properties of murine tendons showed 2-3 N according to a previous study (Kurtaliaj et al., 2019). In comparison, we found that the 50-yarn bundles of NFY-50 had suitable force with 2.15 ± 0.09 N, demonstrating suitable mechanical properties for rat tendon tissue repair compared with NFYs of other diameters.
Several types of stem cells isolated from different tissue sources, such as bone marrow, adipose tissue, and tendon, have been applied in tendon TE applications (Bi et al., 2007;Bavin et al., 2015;Wu et al., 2017a;Laranjeira et al., 2017). Among them, TSPCs, which are isolated from tendon sources and differentiate into tenocytes to regulate tendon homeostasis, would be more suitable for the tendon microenvironment (Guo et al., 2016). A previous study revealed promising results from the use of TSPCs in tendon injury repair in vivo (Youngstrom et al., 2016). In our study, we used TSPCs to evaluate the effects of aligned NFYs on tenogenic differentiation in vitro, after successful isolation of TSPCs from rat tendon tissue and confirmatory assessment of tenogenic markers using flow cytometry ( Figure 3). The composition and structure of the ECM regulates the phenotype and differentiation of TSPCs and affects the regeneration of functional tendon tissue (Sheng et al., 2019). Recent studies have investigated the effects of nanofiber diameters on the behavior of tendon cells (English et al., 2015;Jenkins and Little, 2019). However, there are still few reports on investigation of scaffolds with integrated hierarchical structure that induce TSPCs behavior and phenotypes that lead to tenogenic differentiation. In our study, we successfully prepared aligned NFYs of varying diameters, allowing us to study the effects of NFY diameter on cellular response and tenogenic differentiation of TSPCs. The proliferation results showed that the TSPCs on NFY-50 and NFY-100 exhibited higher proliferation rates compared with the other groups ( Figure 4). One of reasons for this phenomenon was greater contact inhibition on NFYs of smaller diameter due to reduced area available for cell growth. In a prior study, Gerard and Goldbeter (2014) created a detailed computational model to interpret contact inhibition, which suggested that the proliferation of cells would be inhibited by the Hippo/YAP pathway rather than culture conditions when cell density reached the threshold. Another reason was higher initial adhesion rate in the groups with NFYs of larger diameters, with the larger diameter providing increased adhesion area and greater area for cell growth. As observed by the results of Alamar Blue staining on day 1 in culture, there was a rising trend in the number of TSPCs on the NFYs with increasing diameters. Moreover, our previous study showed that cell adhesion would be limited by the narrower surface of the aligned NFYs of smaller diameter, which is consistent with the results of the current study. On the other hand, F-actin staining confirmed that the 3D aligned NFYs provided a 3D microenvironment that induced TSPCs to orient in alignment with the NFYs (Figures 5, 6). More importantly, recent studies demonstrated that nanofiber scale could affect cell differentiation (Sheng et al., 2019). For instance, Bashur et al. (2009) showed that electrospun nanofibers exhibited nanoscale fiber diameters similar to those of collagen fibers in native tendon, and that the nanofibers could increase the level of TNMD gene expression. In our study, both RT-qPCR and immunofluorescence staining to assess levels of the tenogenic markers TNMD, DCN, and Col1a1 showed that use of NFYs of 50 μm diameter significantly improved the tenogenic differentiation of TSPCs compared with use of aligned NFYs of other sizes (Figure 7). These results are mainly due to the aligned structure of NFY-50 and it's diameter, which is similar to the collagen fascicles and fibers of rat tendon tissue of approximately 50-300 μm in diameter. Ultimately, aligned NFYs with 3D hierarchical structure consisting of aligned monodisperse nanofibers promoted tenogenic differentiation of TSPCs.
To further investigate the effects of the aligned NFYs on tendon repair in vivo, NFY-50 was chosen for implantation due its superior ability to promote TSPCs tenogenic differentiation and suitable mechanical properties. We bundled 50 yarns of NFY-50 together to prepare the aligned NFY scaffolds to mimic the macrostructure of the tendon tissue and provide mechanical support for the repaired tendon. We then made a defect in the Achilles tendon of a rat model and implanted the scaffold. Tissue was harvested 6 weeks later to obtain sections for H&E, Masson's trichrome, and Picrosirius red staining. NFY-50 scaffolds resulted in better histological structure and regeneration ability compared with the injury only group (Figure 8). Based on histological evaluation, there was obvious degradation of aligned NFYs due to the greater surface area of the nano-scale structure . A previous study showed that the PCL grafts composed of nanofiber sheets had undergone significant degradation, suggesting an advanced cell response when the tendon was repaired during the 6-week period of remodeling (Bosworth, 2014). In our study, NFYs composed of numerous nanofibers showed a higher degradation rate and many macrophages gathered around the scaffold. The main reason for inflammation was the adsorption of protein on the surface of implanted NFY scaffolds due to the nanofibrous structure (Zhou and Groth, 2018). With degradation of the bundled NFY-50, neo-collagen filled in the scaffolds after 6 weeks. The complete tissue sections (Picrosirius red staining) are shown in Figure 8B, demonstrating that the bundled NFY-50 connected the fracture of the tendon as a bridge, and that neocollagen appeared inside the degraded area of the bundled NFY-50 to wrap it externally. Upon degradation of the bundled NFY-50, i.e., the PCL materials, neo-collagen replaced the NFY scaffold to achieve repair of the tendon.
To further distinguish different types of collagen, we used polarized light microscopy (PLM) to image tissue with Picrosirius red staining, where type Ⅰ collagen fibers appeared bright red-yellow, while type Ⅲ collagen fibers appeared green (Rittié, 2017). A recent study revealed that type Ⅰ collagen fibers are associated with mechanical strength (Goh et al., 2014). The type III collagen fibers would be gradually replaced by type I collagen fibers during tendon remodeling, and type I collagen fibers begin to align along the long axis of the tendon (Deng et al., 2014). In this study, the Picrosirius red assay demonstrated that the type Ⅰ collagen fibers were stained bright red-yellow in the NFY scaffold group in contrast with the injury only group, indicating that the bundles of NFY-50 improved mechanical strength and induced neo-collagen alignment of the neo-tendon tissue. There were fewer type Ⅲ collagen fibers in the aligned NFY scaffolds than in the injury only group ( Figure 7D). These results indicate that the aligned NFYs enhance regeneration of tendon tissue and, at the same time, guide the aligned formation of collagen fibers during tendon repair.
Above all, this study confirmed that PCL 3D aligned NFYs prepared by dry-wet electrospinning provide suitable mechanical support and the hierarchical structure guidance for tendon tissue regeneration, and that NFY-50 improves tenogenic differentiation of TSPCs as a biomimetic of similar diameter to collagen fascicles and fibers in native tendon. Bundled NFY scaffolds mimic the macrostructure of tendon tissue and provide mechanical support for the repaired tendon in vivo. There is high potential for use of aligned NFY-50 in development of additional scaffolds that require element units to produce hierarchical 3D aligned scaffolds for the construction of tissue-specific functional architecture, thus advancing regenerative therapies for soft tissue repair.

Conclusion
In this study, highly aligned NFYs of varying diameters were fabricated and characterized to investigate their effects on the behavior of TSPCs. We found that aligned NFYs could mimic the topography and mechanical properties of collagen fascicles and fibers of native tendon. Aligned NFYs induced TSPCs orientation and elongation. Moreover, aligned NFYs enhanced tenogenic gene and protein expression, including that of TNMD; NFYs of 50 μm diameter showed the greatest effect. Taken together, these data indicate that the nanofibrous structure of aligned NFYs is able to induce TSPCs orientation and elongation, and the muti-scale structure of aligned NFYs may improve tenogenic differentiation. To further investigate the effects of aligned NFYs on tendon repair in vivo, 50-yarn bundles of NFY-50 were implanted in rat Achilles tendon models, with the results demonstrating that the aligned hierarchical NFY scaffolds induced neo-collagen orientation Frontiers in Bioengineering and Biotechnology frontiersin.org and cell alignment, and that bundled NFY-50 scaffolds enhanced tendon tissue regeneration. Overall, these results suggest that aligned NFYs of well-defined diameter mimic the complex hierarchical structure of native collagen fascicles and fibers and show excellent potential for use in tendon regeneration applications.

Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics statement
The animal study was reviewed and approved by the Institutional Animal Care and Use Committee, Southern Medical University.