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A mussel-inspired film for adhesion to wet buccal tissue and efficient buccal drug delivery

Administration of drugs via the buccal route has attracted much attention in recent years. However, developing systems with satisfactory adhesion under wet conditions and adequate drug bioavailability still remains a challenge. Here, we propose a mussel-inspired mucoadhesive film. Ex vivo models show that this film can achieve strong adhesion to wet buccal tissues (up to 38.72 ± 10.94 kPa). We also demonstrate that the adhesion mechanism of this film relies on both physical association and covalent bonding between the film and mucus. Additionally, the film with incorporated polydopamine nanoparticles shows superior advantages for transport across the mucosal barrier, with improved drug bioavailability (~3.5-fold greater than observed with oral delivery) and therapeutic efficacy in oral mucositis models (~6.0-fold improvement in wound closure at day 5 compared with that observed with no treatment). We anticipate that this platform might aid the development of tissue adhesives and inspire the design of nanoparticle-based buccal delivery systems.


Response to Review
We are grateful to the reviewers for their constructive comments and suggestions. Your comments are extremely important and helpful for us. We have carefully revised the paper according to the reviews' comments. Our point-by-point address to the comments is marked below in blue and the changes are also highlighted in the revised manuscript.

Reviewer #1 (Remarks to the Author):
In this work, the authors designed catechol-bearing PVA/DOPA films incorporated with different PLGA NPs for buccal delivery. The nanoparticles and the films were carefully and thoroughly characterized both in vitro and in vivo. The findings also showed promising results. However, the manuscript needs to be further improved to suit to nature communications.
Response: We thank the reviewer for his/her positive evaluation of our manuscript and constructive comments/suggestions, which have helped us improve the manuscript substantially. The detailed point-by-point responses are below and we have also revised the manuscript accordingly.

Major comments
1. A literature review on the previous works on mucoadhesive catechol-bearing materials should be added in the introduction. The novelty of the current work over the previous published articles should also be discussed and highlighted.

Response:
We thank the reviewer for this suggestion. We have added a brief literature review about the previous works on mucoadhesive catechol-bearing materials in the introduction. The novelty of the current work over the previous published articles was also discussed and highlighted in both the introduction and discussion as follows: "Inspired by this functional group from mussels, enormous efforts has been devoted to the development of catechol-functionalized adhesives via modifications of a variety of mucoadhesive polymers, including catechol-modified poly(ethylene glycol) (PEG) 11,12 , chitosan 5,13 , hyaluronic acid (HA) 14 , alginate 15 , etc. However, most of those studies focused on tissue adhesives for skin, and reports on formulations that exhibit excellent mucoadhesive properties in the wet oral environment and how they interact with the oral mucosa are still very limited. Although Xu et al. 5 reported a catechol-chitosan mucoadhesive hydrogel for buccal drug delivery, they did not investigate the ability of the drug to be transported across the epithelial barrier, and their hydrogel provided sustained drug release for only 3 h. Therefore, attempts should also be made to develop drug carriers that could be transported across the epithelial barriers with a controlled and prolonged drug release profile.
Recently, nanoparticles (NPs) have shown great promise for improved transport through the mucus barrier and can be tuned to support controlled or sustained release behaviour [16][17][18] . Among various strategies, surface modification of NPs with PEG has emerged as a popular strategy to enhance the mucus-penetrating ability of NPs [19][20][21] . Nevertheless, the hydrophilic and neutral surface properties of PEG may serve as a barrier for further cellular uptake of NPs 22 . Therefore, strategies that could achieve both excellent mucus-penetrating ability and cellular uptake across the epithelial barriers are also in high demanded." "In this study, we reported a biologically inspired mucoadhesive film combined with NPs for improved mucoadhesion and drug availability. This composite mucoadhesive film offers superb advantages over materials reported in previous studies: (1) strong adhesion in the wet environment of the oral cavity to enable an adequate residence time; (2) tunable size, thickness, and erosion rate in the form of a thin film to facilitate mechanical matching of tissues and potential applications in various kinds of diseases that demand different dosage intervals; (3) the ability to be transported across epithelial barriers; and (4) a controlled and prolonged drug release profile." 2. Since the title of the article is about mussel-inspired film, the authors should add more details on adhesive phenomenon of the mussel and the role of DOPA in the adhesion process in the introduction of the manuscript.

Response:
We thank the reviewer for the insightful comments. As the reviewer suggested, we have added more details on adhesive phenomenon of the mussel and the role of DOPA in the adhesion process in the introduction of the manuscript as follows: "Marine mussels, which are well known for their remarkable underwater adhesion ability, have attracted widespread attention and are a potential source of an ideal tissue adhesive in the biological field 8,9 . The rapid and robust adhesion of mussels could be attributed to the presence of the mussel adhesive proteins, which are abundant in the catecholic amino acid 3,4-dihydroxyphenylalanine (DOPA) 8,9 . The catechol group of DOPA has an excellent maneuverability during crosslinking because it forms either covalent or noncovalent bonds. First, it can form noncovalent complexes, as in metal bidentate coordination and hydrogen bonding. In addition, the catechol groups oxidize easily to form o-quinone in oxidative or alkaline environments. The oxidized o-quinone is highly susceptible to forming covalent bonds with nucleophiles such as thiols and amines of proteins on the tissue surface via Michael addition or Schiff base reactions. In addition, o-quinone can also form di-dopa crosslinks via phenol radical coupling [8][9][10] ."     UV spectra indicate the covalent interaction occurred between DOPA and mucin.
Would FTIR be more suitable for this investigation?

Response:
We thank the reviewer for this comment. According to previous studies, the present study adopted the UV-vis spectra to investigate the covalent interaction occurred between DOPA and mucin. Kim et al. monitored the covalent formation between catechol modified chitosan (Chi-C20.5) and mucin by means of UV-vis spectra.
They demonstrated that the slightly shifted absorption peak position of Chi-C20.5-mucin complex was due to the covalent crosslinking between mucin and catechol "First, the catechol-mediated covalent reaction between different PVA-DOPA and mucin was monitored by means of UV-vis spectroscopy 13,39,40 . As shown in Fig. 3c and Supplementary Fig. 3b, there was no obvious difference between mucin and PVA-mucin at different concentrations.
However, a slight shift could be observed in the three kinds of PVA-DOPA-Mucin complexes. In addition, the absorption spectra of PVA-DOPA-Mucin complexes were also different from those of PVA-DOPA or mucin alone, and the difference was lager with an increased concentration of PVA-DOPA ( Supplementary Fig. 3b), indicating the covalent reaction of catechol with mucin 13,39,40 . We also investigated the interactions between mucin and PVA or PVA-DOPA using FTIR. The pure mucin exhibited amide I and amide II peak positions at 1645 and 1552 cm -1 , respectively (Fig. 3d), and there was no significant difference between PVA and the PVA-Mucin complex at the amide I and II bands. However, for the three kinds of PVA-DOPA, amide II shifted to 1528 cm -1 after reacting with mucin suspension, suggesting the covalent conjugation of catechol with mucin 41 ."  Besides, we totally agree with you that the catechol groups play an essential role for mucoadhesion. As you suggest, we also quantified the catechol groups on PVA-DOPA conjugates to evaluate the potential mucoadhesion properties of the films by means of UV-vis and 1 H-NMR spectroscopy. As mentioned in Question 3, the catechol content was confirmed by the UV-vis spectroscopy, measuring absorbance at 280 nm   Finally, as for the film with the highest content of DOPA showed the highest mucoadhesion properties in the present study, as mentioned in Question 5, the results of UV-vis and 1 H-NMR spectra also quantified the content of catechol groups, which increased with the added DOPA. Therefore, the film with the highest content of DOPA showed the highest mucoadhesion properties. We thank the reviewer for this constructive comment and we have now added discussions on this point in the results and discussion with reference support as follows: "The absorption peaks of catechol at 280 nm in the UV-vis spectra (Fig. 2a) and the peaks in the aromatic regions ( = 6.62, 6.71, 6.78) of the 1 H-NMR spectra (Fig. 2b) 16.0 wt% to 72.0 wt%. Therefore, the catechol groups could be characterized and quantified by the UV-vis and 1 H-NMR spectroscopy." "As stated before, catechol groups play a key role in rapid adhesion and are prone to oxidize under oxidative or alkaline conditions. Thus, the PVA-DOPA polymer in the present study was synthesized under N2 protection, and the film was formed by direct lyophilization and then stored under vacuum before application. The FTIR spectra (Supplementary 1a) first verified the formation of hydrogen-bonding interactions between the PVA matrix and catechol, which resulted in a decreased crosslinking density of the networks with increasing DOPA content, as evidenced by the rheological studies (Fig. 2c). This may be because the -OH and -NH2 groups in the catechol groups formed hydrogen bonds with the -OH of PVA chains and thus increased the distances between molecules. In addition, the increased G′′ (Fig. 2d) also demonstrated the noncovalent crosslinking of the PVA-DOPA film [29][30][31][32] . Then, we also demonstrated that most catechol groups on the PVA-DOPA films remained unoxidized by means of UV-vis and 1 H-NMR spectroscopy. As stated in previous studies, the absorbance peak at 280 nm in the UV-vis spectra was assigned to the catechol groups and revealed that the catechol groups were not oxidized 24,25 . In addition, the absorption peaks at approximately 305 and 400 nm belong to oxidized o-quinone 28,60 . Therefore, despite a slight shoulder peak at approximately 305 nm in the PVA-DOPA5 and PVA-DOPA6, most catechol groups in PVA-DOPA5 and PVA-DOPA6 remained unoxidized, as evidenced by the strong peak at 280 nm (Fig. 2a)   In addition, as mentioned before, the 1 H-NMR and UV-vis results in the current study were obtained by dissolving the lyophilized PVA-DOPA film into D2O and DD water, respectively. And the results both demonstrated that the catechol groups in different PVA-DOPA films largely remained unoxidized ( Fig. R1 and Fig. R2), thus proved the stability of the prepared films. In response to the reviewer's suggestion, we have also pointed it out in the revised manuscript.
8. In the mucous permeation studies, what was the concentration and amount of mucin suspension used? Please specify in the manuscript.

Response:
We appreciate for the reviewer's careful comment. We have added the concentration and amount of mucin suspension and the relevant reference support in the mucus permeation studies in the revised manuscript.

9.
In the exploration of the interaction mechanism between PVA-DOPA film and mucous, the authors mentioned that the UV absorbance increased with increasing DOPA ratio which could be assumed that PVA-DOPA formed catechol-mediated covalent bonding with mucin. I think the molecule of DOPA itself can also absorb UV which higher content of DOPA will also result in higher UV absorbance. The discussion on this aspect should not be discarded.
Response: Thank the reviewer for pointing out this issue. We totally agree with you that higher content of DOPA will result in higher UV absorbance and our results also demonstrated this phenomenon. As shown in Fig. R2, the higher the content of added DOPA, the stronger the absorbance peak at 280 nm in the UV-vis spectra.
As for how could the UV-vis absorbance be used to explore the interaction mechanism between PVA-DOPA film and mucus, the authors have explained in detail in Question 4. That is, after reacting with mucin suspension, a slight shift could be observed in PVA-DOPA-Mucin complexes (Fig. R3a). Besides, the absorption spectra of PVA-DOPA-mucin complexes were also different from those of PVA-DOPA or mucin alone and the difference were lager with the increased concentration of PVA-DOPA ( Fig. R3c-3e), indicating the extent of reaction between PVA-DOPA and mucin   21 . Moreover, densely grafted PEG of 10-40 kDa can also exhibit mucus-penetrating properties 62 . Therefore, in the present study, the PDA chain was densely coated on the surface of PLGA with a thin layer, which reduced the entanglement of chains between PDA and mucin fibers.
In addition, the negatively charged surface of PDA minimized charge interactions with the negative domains of mucins. In conclusion, the PLGA-PDA NPs diffuse rapidly through the mucus layer and exhibit mucus-penetrating properties." 11. Why did the PLGA-PDA NPs demonstrate significantly higher cellular uptake compared to other NPs? What could be the mechanism behind this finding? Please discuss this point in the results and discussion part.

Response:
We appreciate the reviewer for the valuable comments. As stated above, the presence of multiple functional groups (e.g., amino, phenol) endow PDA with

Minor comment
1. There are some typos and grammar mistake that need be to carefully checked. To make it appropriate for publication in nature communications, the English should be check by a native speaker or an English editing service.
about the finding of the work in the abstract. However, due to the word limit of abstract of Nature Communication (within 150 words), we are only able to add the main finding of the work in the abstract as follows: "Administration of drugs via the buccal route has attracted much attention in recent years. However, developing systems with satisfactory adhesion under wet conditions and adequate drug bioavailability still remains a challenge. Here, we propose a mussel-inspired mucoadhesive film.
Ex vivo models show that this film can achieve strong adhesion to wet buccal tissues (up to 38.72±10.94 kPa). We also demonstrate that the adhesion mechanism of this film relies on both physical association and covalent bonding between the film and mucus. Additionally, the film with incorporated polydopamine nanoparticles shows superior advantages for transport across the mucosal barrier, with improved drug bioavailability (~3.5-fold greater than observed with oral delivery) and therapeutic efficacy in oral mucositis models (~6.0-fold improvement in wound closure at day 5 compared with that observed with no treatment). We anticipate that this platform might aid the development of tissue adhesives and inspire the design of nanoparticle-based buccal delivery systems."