Fibronectin (FN) plays an essential role in the wound healing process due to its ability to form an extracellular matrix (ECM) and to regulate cellular activities1. In addition to the ability to bind together and form FN fibrils, FN molecules have various recognition and binding sites in their structure, and these sites favor interactions with various cell types, growth factors, cytokines, and other ECM proteins, such as collagen, laminin, and heparin2, 3, 4. Soluble FN is a folded dimeric protein whose subunits are composed of three types of repeating modules, I, II and III, which are variably expressed due to alternative splicing of RNA5. Among the three modules, FN type III (FNIII) is the largest and most common repeat2, 4. Once in contact with the cell surface, FNIII domains will be recognized by integrin proteins on the cell membrane and subsequently undergo an unfolding process caused by cell-traction forces, which induces FN fibrillogenesis3, 6. Unfolding is, therefore, essential in FN functionalization, which is the target of FN-based material fabrication1, 7. Although the cell binding sites within the FNIII module are exposed on the surface, the active domains, which are known to be phosphorylation sites, are usually hydrophobic. These domains are buried within FNIII modules in the folded state due to hydrophobic interactions and are maintained by intramolecular ionic bonds8, 9. Therefore, unfolding FN is crucial not only for enhancing the elasticity of fibronectin fibrils but also for exposing the buried sites that are known to display enzymatic activities and to regulate many cellular activities10.
The use of soluble, compact FN has been successful in improving wound-healing treatments. Some examples of wounds considered thus far are diabetes- and radiation-induced cutaneous wounds in rats or mice11, 12. Corneal epithelial wounds have also been reported to heal more quickly after having been treated using eye drops containing a soluble FN and hyaluronan combination13. Nevertheless, recent reports have revealed that supplying cells with unfolded FN, compared to compact FN, improves the wound-healing efficiency more remarkably. For example, Phong et al. reported an enhanced formation of FN fibrils after denaturation of FN by using urea, leading to improved platelet adhesion14. In 2018, Christophe et al. adopted rotary jet spinning to produce unfolded FN fibers and confirmed that its use effectively enhanced wound healing15. These studies suggest that for higher wound-healing efficiency, FN used in tissue regeneration should undergo certain procedures to induce changes in its conformation. Some methods for doing so include physical binding on different substrates, modifying surfaces with chemical groups, or using gold nanoparticles16, 17, 18, 19. Although unfolded FN has been proven to have improved functions over compact FN, the methods for unfolding FN that have been reported so far have had limited clinical applications due to the complex processes required for the fabrication and chemical modification of the substrate.
In this study, to tackle the above issues, we demonstrate a potential FN-unfolding platform using negatively charged, small unilamellar vesicles (SUVs) for FN delivery. These SUVs are simply produced by using a unique composition of lipids so that the surface of the SUV not only enhances direct binding but also induces the unfolding of FN. The effects of FN delivered by our SUV system were investigated using a variety of cell-based assays to evaluate all aspects of cellular activities, including growth, differentiation, and migration. An in vivo wound-healing model of ulcerative colitis (UC) was adopted to evaluate the sufficiency of the unfolded FN that was delivered. Collectively, our findings indicate that negatively charged SUVs represent a new platform for functional FN delivery.
For critical cellular functions to be accessed, compact FN must be unfolded to expose buried domains20. Hence, our first effort was to determine the composition of lipids that not only enhanced the binding efficiency of FN but also induced conformational changes of FN prior to its delivery to cells (Fig. 1a). The optimized lipid composition of SUVs was found to be zwitterionic:net negative:cholesterol at a ratio of 3:2:1. An appropriate ratio of 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) resulted in a net negative charge on the surface of the SUV. The negatively charged SUVs had strong interactions with FN compared to those with only zwitterionic lipids (Fig. S1). An analogous giant unilamellar vesicle (GUV) system was developed with the same lipid composition but a hundredfold increase in size (20-30 µm on average) (Figs. 1b and c). The analogous GUVs allowed us to directly observe the binding by using confocal imaging. FN tended to form insoluble aggregates when it was incubated with neutral vesicles (Fig. 1b) but was attached evenly on the surfaces of negatively charged GUVs (Fig. 1c). The strongly favorable and selective binding of FN to a negatively charged lipid is obvious in phase-separated GUVs with two lipid phases (Fig. S1a). The liquid order (Lo) phase contained zwitterionic sphingomyelin, and the liquid disorder (Ld) phase contained DOPS and other unsaturated lipids, constructing the negative portion of the membrane. Under the same experimental conditions, FN was observed only in the Ld phase (green fluorescence) and not in the Lo phase of the same GUV (Fig. S1b). This result clearly confirmed that a negatively charged vesicle system selectively enhanced FN binding.
Evaluating the binding of FN on SUVs with an appropriate size for cell delivery was much more sophisticated because SUVs (average radius of 200 nm) are remarkably smaller than GUVs (average radius of 20 µm). Images obtained from cryogenic transmission electron microscopy (Cryo-TEM) showed that the SUVs incubated with FN had a membrane that was significantly thicker (thickness d = 8.3 ± 0.6 nm) than the membrane containing only lipids (d = 4.4 ± 0.6 nm) (Figs. 1d and e). The thickness of the membrane comprises the lipid membrane itself, which is approximately 4-5 nm, and an additional FN layer, if any, on the membrane. Therefore, this result implied that FN indeed bound to the SUVs, which was observed as an increase in the membrane thickness. In addition to performing single-SUV thickness measurements, we measured the size distribution of the entire SUV population by using the dynamic light scattering (DLS) method (Fig. S2). The results showed that the average size of the SUV population increased from 140.3 nm to 178.3 nm and that the curve shifted to the right (larger size), indicating that the size of the SUV had increased after incubation in an FN solution.
In addition to enhancing the binding of FN on the surface, negatively charged SUVs induced conformational changes. The stretching of FN was measured by using fluorescence resonance energy transfer (FRET) with FN labeled with donor-acceptor dyes (FN-DA). Four molar (4 M) guanidinium chloride (GdnHCl), one of the strongest denaturants, was used as a positive control 21 (Fig. S3). Figure 1f shows that the IA/ID ratio of FN-DA incubated with SUVs was comparable to that of FN-DA incubated in 4-M GdnHCl (0.38 ± 0.06 and 0.34 ± 0.13, respectively) and was significantly lower than that of FN-DA incubated in phosphate buffered saline (PBS) used for SUV hydration or sucrose used for GUV hydration (1.18 ± 0.2 and 1.19 ± 0.05, respectively). FRET was also accomplished on GUVs coated with FN-DA; the IA/ID ratio of FN-DA was lower after incubation with GUVs (from 0.92 ± 0.11 to 0.18 ± 0.06 after incubation) (Figs. 1g and h). This result verified that stretching of FN had also occurred on the surfaces of GUVs. Taken together, the negatively charged SUV system is a suitable vehicle for delivering FN in an active, unfolded conformation.
FN has been utilized in cell and tissue cultures to strengthen cell attachment, proliferation, and migration17, 19, 22, 23, 24. We hypothesized that the conformational changes induced by binding to negatively charged SUVs would enhance the above cellular functions of FN to a greater extent than its compact form. We performed in vitro experiments using human neonatal dermal fibroblast (HDFn) cells, as they play essential roles in tissue repair. The fate of FN after having been delivered to HDFn cells was probed with either green (FN-SUV) or red (FN) fluorescent indicators. In both groups, FN fibrils were found to colocalize with their membrane receptor, integrin α5 (Fig. 2a). However, more FN fibrils were produced when FN was delivered by using SUVs. We found an interesting event in which a clump of FN-SUVs rapidly burst and expanded when it came into contact with the HDFn cell surface (Fig. 2b). This event was not observed when using FN without SUVs (data not shown), indicating that SUVs promoted the interaction of FN with the cell surface. As we observed that SUVs unfolded FN in advance of the interaction with the cell membrane, we predicted that this process would subsequently increase the adhesion of cells to the culture substrate. Fig. 2c shows that trypsinized HDFn cells had limited attachment to the culture dish. However, when delivered by using the FN-SUV system, the number of active fibroblasts (seen as spindle-shaped cells with long lamellipodia) increased significantly25. A quantitative comparison of cell attachment among the experimental groups was performed by calculating the total area of attached cells, as shown in Fig. 2d and Video S4. Moreover, the total cell surface area covered by FN was significantly higher when FN was delivered by SUV, implying enhanced FN binding to the cells (Fig. 2e). Although FN promoted cell attachment, stretched FN made the surfaces of GUVs unfavorable for bacteria, particularly Staphylococcus aureus (S. aureus), an organism that often causes opportunistic infections on skin and leads to tissue damage, as described in a previous study26. S. aureus was incubated with different types of GUVs (Fig. S4). A large number of bacteria (white) were found on collagen-coated GUVs (COL-GUVs). In contrast to the case with the bare lipid GUVs, the bacteria were hardly present on the FN-coated GUVs, suggesting that using the stretched FN as a wound healing agent had advantages in preventing unwanted bacterial infection.
When tissue is wounded, released cytokines attract fibroblasts from the surrounding area1. During this time, fibroblasts are activated and migrate towards the wounded area with the recruitment of ECM proteins including FN27, 28. We tested whether the stretching of FN would promote the migration rate of fibroblasts, as it has been shown to enhance ECM formation. Figure 3a shows representative images cut from Videos S5-7, illustrating the movements of HDFn-GFP cells as a function of time. Single cells were tracked, and the average migration speeds (µm/min) were compared among groups. As we expected, the migration speed increased when SUVs (green circles) were used for FN delivery (Figs. 3a and b). This enhancement may result from the formation of a denser FN matrix, which facilitates cell adhesion and migration. The expansion of the FN matrix among groups over time can be seen clearly in Fig. S5 and Video S11.
In vitro scratch assays were used to assess the cell migration speed to evaluate cell migration29. To ensure the reproducibility of the experiments, we set up an incubation chamber in the confocal microscope system and continuously observed a specific wounded area for at least 16 h. Representative images at 0 h and 12.5 h for each group are shown in Fig. 3c; the corresponding time-lapse images are shown in Videos S8-10. The results revealed that among the experimental groups, the gap closed at the highest rate in the FN-SUV-treated group, with 60.3 ± 4.3 % of the gap area being covered by HDFn cells after 15 h compared to only 33.5 ± 4.2 % in the FN-treated group (Fig. 3d). Insignificant proliferation among all groups was observed during the first 24 h, which showed that the cell migration assays were unbiased, with minimal contribution from cell proliferation (Fig. S6).
The healing effects of FN in vivo have been described previously by using a wide range of wound models in different species, such as rabbits, mice and guinea pigs12, 30, 31. Existing evidence from previous studies indicates that in inflammatory bowel-related diseases, such as ulcerative colitis (UC) and Crohn’s disease, the level of FN in the plasma is decreased significantly7, 32, 33. In this present study, the healing performance of FN-SUVs was further assessed in vivo using an UC model for rats. An approved protocol using 4% acetic acid was applied to induce UC in the colon, followed by a 10-day treatment strategy with either FN or FN-SUVs (Fig. S7). After treatment, the entire colons of the rats were removed to compare changes in morphology, wet weight and various tissue damage markers. As shown in Fig. 4a, the FN-SUV-treated group showed remarkably thinner and smoother colon walls with less mucus (less tissue damage) than the FN-treated group. In addition, FN-SUV-treated colons had significantly lower wet weights than those treated with FN alone, indicating that SUV-FN treatment reduced inflammation and swelling in the colons.
Colon tissues after either FN or FN-SUV treatment were further analyzed semi-quantitatively for inflammatory markers, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS). However, the differences in the levels of TNF-α, IL-1β, and IL-6 expression between the FN and FN-SUV groups were insignificant (Fig. S8), whereas COX-2 and iNOS expression was downregulated in rats treated with FN-SUV, suggesting that the unfolding of FN via the SUV may expose the domains that communicate with cells via these two pathways (Figs. 4a and b)34, 35. The inhibition of COX-2 and iNOS expression in our in vivo model clearly showed that inflammation was effectively decreased in the experimental UC model when the FN-SUV treatment was administered36.
Many attempts have been made to unfold FN prior to it reaching the cell. However, an appropriate approach to unfolding FN for tissue regeneration applications is still a challenge. The unfolding of FN requires the breakage of intramolecular bonds and hydrophobic interactions, which can be induced by using either chemicals14, 21 or mechanical forces8, 15. The pH or salt concentration can induce the stretching of FN, but this reversible change is hard to maintain for cell delivery, and the solution must be similar to the physiological environment. The improper removal of a denaturant will otherwise potentially result in negative effects on live cells and tissues21.
The adsorption and unfolding facilitated by chemical modifications raise a risk of cytotoxicity16, 17, 18, 19. On the other hand, approaches using mechanical forces to unfold FN usually exploit solid substrates and polymers with low biocompatibility. The most promising approach might be to produce nanofibers from soluble FN by using mechanical forces15, 37. However, FN nanofiber production remains challenging, as the use of the fabrication technique may not always be possible in laboratories. Nonetheless, FN nanofibers are more appropriate for dressing materials and cutaneous wound treatment than for the treatment of bowel diseases. Despite major efforts to deliver ECM or FN to damaged tissues via surface-modified substrates, the fabrication of these materials remains very complex, and these substrates usually have to be removed from the body after having been applied, which is not appropriate for deep, closed wounds38, 39. In contrast, negatively charged SUVs are composed of phospholipids and cholesterol, which are highly biocompatible and have negligible cytotoxicity40. Accordingly, in our novel system, we managed to develop a negatively charged SUV system that has negligible toxicity40, 41. In addition, the preparation of self-assembled SUVs in aqueous solution followed by the autonomous binding and unfolding of FN was much simpler and afforded higher yields than any other method.
The adsorption and unfolding of FN on liposomes were once reported by Micheal et al. as an enhanced drug delivery approach using liposomes that can avoid the rapid uptake of the reticuloendothelial system42. Both gel-phase and liquid-phase liposomes were considered for FN selective binding, and only liposomes composed of lipids that had high melting temperature (Tm) or existed in the gel phase at room temperature, such as DPPC (Tm = 41°C) or DSPC (Tm = 55°C), were observed to bind and unfold FN42. Interestingly, we have sufficient experimental evidence to prove that FN is able to bind and unfold on liquid-phase SUVs composed of lipids with Tm < 0°C under the condition that a negatively charged lipid (DOPS) is added. Due to the low Tm values of the lipids used, the fabrication of negatively charged SUVs is simplified and can be performed at room temperature. Thus, the negatively charged SUV system has significant advantages over previously used materials in terms of fabrication, cytotoxicity, and biocompatibility.
Collectively, we confirmed that a negatively charged SUV can effectively unfold FN bound to its surface. Subsequently, the SUVs enhanced the effects of FN on HDFn migration and proliferation. In vivo experiments showed that rats with UC that had been treated with FN-SUVs improved faster than rats treated with only soluble FN. The abundant amount of FN in the plasma is an advantage of using FN as a potential tissue regeneration material1, 7, 32. FN also possesses an exceptional property for wound healing, as stretched FN disrupts the binding of S. aureus, a common cause of infections in humans26.
We conclude that negatively charged SUVs are ideal vehicles for functional FN delivery. The system shows the capability to carry and unfold FN with high biocompatibility and safety and can be used as a platform for protein-small molecule drug incorporation, especially for drugs that accelerate wound healing. Further studies are expected to discover the mechanisms underlying the efficacy of the SUV-FN delivery system.