Versatile Layer-By-Layer Highly Stable Multilayer Films: Study of the Loading and Release of FITC-Labeled Short Peptide in the Drug Delivery Field

A viable short FITC-peptide immobilization is the most essential step in the fabrication of multilayer films based on FITC-peptide. These functional multilayer films have potential applications in drug delivery, medical therapy, and so forth. These FITC-peptides films needed to be handled with a lot of care and precision due to their sensitive nature. In this study, a general immobilization method is reported for the purpose of stabilizing various kinds of peptides at the interfacial regions. Utilizing Mesoporous silica nanoparticles can help in the preservation of these FITC-peptides by embedding themselves into these covalently cross-linked multilayers. This basic outlook of the multilayer films is potent enough and could be reused as a positive substrate. The spatio-temporal retention property of peptides can be modulated by varying the number of capping layers. The release speed of guest molecules such as tyrosine within FITC-peptide or/and adamantane (Ad)-in short peptides could also be fine-tuned by the specific arrangements of the multilayers of mesoporous silica nanoparticles (MSNs) and hyaluronic acid- cyclodextrin (HA-CD) multilayer films.


Introduction
Layer-by-layer self-assembly technology is a useful method to prepare multilayer films with a thickness which can be controlled even down to the nanoscale [1][2][3][4][5], and is among one of the most widely used techniques to weave the organic-inorganic multilayer films together [6][7][8][9][10]. Mesoporous silica nanoparticles (MSNs) have been widely used in adsorption and drug delivery due to their unique pore structure, large specific surface area and good biocompatibility [11]. Some of these can directly act as catalysts as well because the ordered mesoporous material can accelerate the diffusion rate of the product. The selectivity is 100% and the conversion rate is up to 90%. Due to the flexibility of the structure and the narrow pore distribution, the doping of metal oxide and other complexes to these ordered mesoporous material matrix makes them better catalysts. Biological macromolecules, such as enzymes, proteins, nucleic acids, etc., have molecular sizes less than 10 nm, while viruses have sizes around 30 nm. However, the ordered mesoporous materials have quite a range of pore sizes between 2 and 50 nm. Due to the non-toxicity of these MSNs, they play an important role in the decomposition and fixation of enzymes, proteins and other substances. The mesoporous silica used for a particular

The Preparation of HA-β-CD Gels
HA-CD gel was prepared according to literature [48]. In a 100 mL Erlenmeyer flask, 0.3 g of sodium hyaluronate and 50 mL of morpholinoethanesulfonic buffer (MES) were added and rapid stirred for 6 h. Subsequently, 0.285 g of NHS and 0.342 g of EDC were added into the Erlenmeyer flask. Then the mixture was stirred for another hour. To the mixture, 0.1686 g of amino cyclodextrin was added, followed by stirring for 24 h. The detailed experimental procedure and the sample codes are listed in Table S1. 1 H NMR spectroscopy was used to determine the degree of grafting of HA-CD. The graft ratio was calculated by using the integral area of the 1 H NMR spectrum. As shown in Figure S1, the peak at 5.10 ppm represents the proton of amino (NH) (5.10 ppm) group and its integral area was 0.11. Therefore, the integral area of each hydrogen atom of the aminocyclodextrin was 0.11/7. Similarly, the peak at 2.05 ppm represents the proton of CH 3 (2.05 ppm) group and its integral area was 1.00. Therefore, the integral area of each hydrogen atom of hyaluronic acid was 1/3. Grafting ratio was obtained by dividing the integral area of each hydrogen atom of amino cyclodextrin by the integral area of each hydrogen atom of hyaluronic acid. So, according to the 1 H NMR (400 MHz, D 2 O) data, the grafting rate of HA-CD was 4.71%.

The Synthesis of Mesoporous Silica Nanoparticles (MSNs)
MSNs were synthetized according to the literature [49]. In brief, an aqueous solution containing of 2.0 g CTAB, 7.0 mL NaOH (2 mol L −1 ) and H 2 O (480 g) was heated for 30 min at 80 degrees with rapid stirring. To this solution, 8.31 g ethyl orthosilicate was added. The white solid appeared within two minutes. The product was further stirred at 80 degrees for two more hours, followed by centrifugation, washing with a lot of ultra-pure water, and drying in vacuum oven for 12 h. The CTAB extraction was carried at 60-degrees Celsius. This white powder was added to a mixture of ethanol (120 mL) and concentrated hydrochloric acid (1.0 mL) and stirred for 8 h at 60 degrees Celsius. The product was then centrifuged and washed with a lot of water and ethanol, followed by drying under vacuum. The diameter of meso-SiO 2 is about 100 nm as seen in TEM images shown in Figure 1c.
The surface area of the MSN is 646.715 m 2 g −1 and the pore diameter of the MSN was about 3.4 nm, which was characterized via the BET instrument.

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In addition, DAS could enter the multilayer films and also enhance the stability of the multilayer 195 films through covalent cross-linking bonding. This effective and facile strategy can play an

Layer-By-Layer Assembly Multilayer
Quartz slides cleaned by piranha solution (concentrated H 2 SO 4 /H 2 O 2 (v:v = 7:3) ) for 10 h were immersed in PAH (aq., MW = 15,000, 1 mg ml −1 , pH = 9) for 25 min in a typical layer-by-layer cycle. They were rinsed in ultrapure water and then dried with nitrogen. After that, the slides were dipped into the SiO 2 solution for 25 min and rinsed and dried as before. The cycle was continued until desirable thickness was obtained and the related coating film will be designated as (PAH/SiO 2 ) n . Generally, the LBL film was assembled in pH = 9 solution. The substrate was immersed in PAH (aq., MW = 15,000, 1 mg mL −1 , pH = 9) for 25 min, rinsed in ultrapure water, and then dried with nitrogen. Subsequently, these slides were soaked into the HA-CD (1 mg mL −1 ) solution for 25 min, rinsed and dried as before so that (PAH/HA-CD) LBL films could be deposited onto the surface of the (PAH/SiO 2 ) n films. The film should be fixed on the quartz substrate to avoid bending during handling so that the good quality of multilayers on the quartz substrate can be guaranteed.

Peptide Loading and Release
1 mg of FITC-labeled peptide solution was dissolved in 10 mL of PBS buffer solution (pH = 7.4) in a 20 mL glass beaker at room temperature. The quartz sheets with the multilayers on the surface were then immersed in the peptide solution for 24 h. The quartz sheets were then removed and the surface was immediately rinsed with deionized water and blown dry with nitrogen. Finally, these quartz sheets were used for release experiments in PBS buffer solution.

Results and Discussions
The surfaces of PAH and DAS are positively charged. PAH and DAS can form covalent cross-linked layers in situ under ultraviolet irradiation. The versatile multilayer films reported here were built by integrating MSN, which act as the storage space of polypeptide of FITC-peptide-Ad and the covalently cross-linking multilayers to increase the stability of the whole system of the multilayer films (Scheme 1). A multilayer film of (PAH/SiO 2 ) 5 /(PAH/HA-CD) 5 was prepared as illustrated in Figure 1a. The formation of this multilayer was monitored by UV-vis spectroscopy. The cartoon as shown in Figure 1b, displays the process of layer by layer self-assembly between MSN and PAH. The MSN used in the experiment were synthesized in our laboratory. The diameters for these MSNs vary from small to 100 nm as shown in Figure 1c. The thickness of the cross-section of multilayer films prepared on the silica substrate was about 140 nm with surface roughness of around 35 nm ( Figure S2). As compared to uncross-linked (PAH/DAS) 5 films, the stability of the (PAH/SiO 2 ) 5 multilayer films is not great and could not be enhanced, and the absorbance of the basic solution remains 32.26% of the original absorbance after the immersion as shown by the UV-vis spectra (Figure 2a,b).
into the SiO2 solution for 25 min and rinsed and dried as before. The cycle was continued until 144 desirable thickness was obtained and the related coating film will be designated as (PAH/SiO2)n.

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Subsequently, these slides were soaked into the HA-CD (1 mg ml −1 ) solution for 25 min, rinsed and 148 dried as before so that (PAH/HA-CD) LBL films could be deposited onto the surface of the 149 (PAH/SiO2)n films. The film should be fixed on the qua 150 rtz substrate to avoid bending during handling so that the good quality of multilayers on the 151 quartz substrate can be guaranteed.   UV irradiation was used to enhance the stability of the multilayer films. (PAH/DAS) 5 multilayer films were subsequently prepared by a self-assembly method on the top of the (PAH/SiO 2 ) 5 /(PAH/HA-CD) 5 multilayers, and cross-link of (PAH/DAS) 5 multilayer films was analyzed using UV spectra. The decrease of the absorbance peak at 340 nm indicates that the DAS decomposes and forms covalent cross-linkages within the (PAH/DAS) 5 films. The stability of these films can be verified by soaking the cross-linked multilayer films into the basic solution. After the treatment with basic solution for 2 h, the peak absorbance of the multilayer films increased by 94.12% (Figure 2a).
The result shows that the covalently cross-linked (PAH/DAS) 5 acts as an outermost film which contributes in the enhancement of the stability of the multilayer films within the MSNs. Based on these results, the presence of (PAH/DAS) 5 multilayer films can enhance the stability of the uncross-linked multilayer films. It was supposed that the nano-net effect resulted in the ability of (PAH/DAS) 5 multilayer films to improve the stability of the films by incorporating silica underneath. In addition, DAS could enter the multilayer films and also enhance the stability of the multilayer films through covalent cross-linking bonding. This effective and facile strategy can play an important role in practical purposes because of its ability in stabilizing different kinds of multilayer films.   In consideration of the above facts, a model multilayer film for the release of FITC-labeled peptides can be employed as a model drug. The host-guest interaction between CD and Ad existed on the three-dimensional scale. The cyclodextrin molecule is hydrophilic from outside and hydrophobic from inside. It indicates that the CD possesses the external hydrophilic surface and the hydrophobic central cavity. Figure 3 shows the short peptides release profiles from cross-linked multilayer films.  Figure S3, ten layers of multilayers were used for experiments to prove that the molecular size had little effect on the release rate. As shown in Figure S3, the release time of both FITC-RGD and FITC-RGD-Ad is about 480 min. As illustrated in Figure 3c, it takes about 480 min for the release of FITC-RGD, and 720 min for FITC-RGD-Ad from (SiO 2 /PAH) 5 /(PAH/HA-CD) 5 /(PAH/DAS) 5 multilayer film. Moreover, it shows that the release profiles are very smooth in Figure 3. Despite the number of multilayers in the film being same, the release speed and release time is different. The delayed effect of the films was caused by the host-guest supramolecular interactions between cyclodextrin and adamantane and also the release process increased with number of the multilayers. In addition, the release time of different kinds of multilayer is summarized in Figure 3d. In the experiment, the controlled variable method was used to study the release of peptide. Figure 4a is the same as Figure 3a. Compared with the release profiles in Figures 3 and 4a, Figure 4b clearly shows that the peptide release speed of FITC-RGD is faster than the release speed of FITC-RGD-Ad. The release time of FITC-RGD and FITC-RGD-Ad is about 210 min and 400 min, respectively. As seen in Figure 4c, the release time of FITC-RGD and FITC-RGD-Ad is 350 min and 470 min, respectively. Delayed effect of the multilayer films resulted from the host-guest supramolecular interactions, which show an increase in the release process with an increasing number of multilayers.     It is well known that tyrosine (Y) is a hydrophobic amino acid. We infer that host-guest interactions between CD and Y will delay the release speed of Y-based peptide. For proof of concept, different kinds of multilayers were immersed into different solution of peptide, as shown in Figure 5.

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In this study, a method has been developed which can raise the stability of multilayer films   10 (PAH/DAS) 5 . Figure 5c shows that it takes around 180 min for the release of FITC-(SGGSGGS) 4 and 260 min for FITC-(SGGYGGS) 4 from (SiO 2 /PAH) 5 (PAH/HA-CD) 15 (PAH/DAS) 5 multilayer films. The more the number of the multilayers in the film, the greater the host-guest interactions. By changing variables and adjusting parameters, it is proved that upon coming in contact with supramolecular forces, host-guest interactions between HA-CD and FITC-(SGGYGGS) 4 delayed the release speed of fluorescent molecules as shown in Figure 5.

Conclusions
In this study, a method has been developed which can raise the stability of multilayer films made up of polymer and inorganic nanoparticles. The covalently cross-linked super stratum was formed on outermost layer of the multilayer films so that the structure of multilayers can be stabilized. The super stratum acts as a nano-net which prevents the diffusion of the nanoparticles from the substrate. It is worth mentioning that the super stratum possesses permeability for small molecules like FITC-RGD, FITC-RGD-Ad, FITC-(SGGYGGS) 4 or FITC-(SGGSGGS) 4 . The difference in permeability of the super stratum towards nanoparticles and small molecules is utilized to synthesize a functional system for drug delivery incorporating mesoporous silica as the molecule reservoir. The construction of functional multilayer films that incorporate inorganic nanoparticles has potential promotional value benefitting from the mild synthesis conditions of the super stratum.