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Drug-Loaded Biocompatible Chitosan Polymeric Films with Both Stretchability and Controlled Release for Drug Delivery

  • Ji Ha Lee*
    Ji Ha Lee
    Chemical Engineering Program, Graduate School of Advanced Science and Engineering, Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima739-8527, Japan
    *Email: [email protected]
    More by Ji Ha Lee
  • Tomoyuki Tachibana
    Tomoyuki Tachibana
    Chemical Engineering Program, Graduate School of Advanced Science and Engineering, Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima739-8527, Japan
  • Hijiri Wadamori
    Hijiri Wadamori
    Chemical Engineering Program, Graduate School of Advanced Science and Engineering, Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima739-8527, Japan
  • Keita Yamana
    Keita Yamana
    Applied Chemistry Program, Graduate School of Advanced Science and Engineering, Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima739-8527, Japan
    More by Keita Yamana
  • Riku Kawasaki
    Riku Kawasaki
    Applied Chemistry Program, Graduate School of Advanced Science and Engineering, Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima739-8527, Japan
  • Shogo Kawamura
    Shogo Kawamura
    Applied Chemistry Program, Graduate School of Advanced Science and Engineering, Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima739-8527, Japan
  • Hinata Isozaki
    Hinata Isozaki
    Applied Chemistry Program, Graduate School of Advanced Science and Engineering, Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima739-8527, Japan
  • Mina Sakuragi
    Mina Sakuragi
    Department of Nanoscience, Sojo University, 4-22-1 Ikeda, Nishi-ku, Kumamoto860-0082, Japan
  • Isamu Akiba
    Isamu Akiba
    Department of Chemistry and Biochemistry, Kitakyushu University, 1-1 Hibikino, Wakamatsu, Kitakyushu808-0135, Japan
    More by Isamu Akiba
  • , and 
  • Akihiro Yabuki*
    Akihiro Yabuki
    Chemical Engineering Program, Graduate School of Advanced Science and Engineering, Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima739-8527, Japan
    *Email: [email protected]
Cite this: ACS Omega 2023, 8, 1, 1282–1290
Publication Date (Web):December 20, 2022
https://doi.org/10.1021/acsomega.2c06719

Copyright © 2022 The Authors. Published by American Chemical Society. This publication is licensed under

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Abstract

Chitosan is a natural polysaccharide with the advantageous qualities of biocompatibility and biodegradability, and it has recently been spotlighted as a soft material for a sustainable society. Advantages such as these are in demand for application in various biomaterials. Although extensive studies have been conducted on the preparation of chitosan films, overcoming the problems of weak mechanical properties remains a significant barrier. In the present study, we developed stretchable doxorubicin-loaded biocompatible chitosan films by adding acetic acid in controlled concentrations. The stretchable properties of doxorubicin-loaded chitosan film at various concentrations of acetic acid were measured. Elongation to the point of breakage reached 27% with a high concentration of acetic acid, which could be described as high stretchability. The release ratio of doxorubicin from chitosan film reached 70% with a high acetic acid concentration. The cytotoxicity of doxorubicin-loaded chitosan films was measured, and cancer spheroids had completely collapsed after 7 days. According to the results of skin permeability testing, use of the doxorubicin-loaded chitosan film is a plausible choice for a drug sealant.

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Introduction

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The excellent qualities of biodegradability, biocompatibility, and bioactivity of polysaccharide-based films provide society with opportunities to achieve many of its sustainable development goals (SDGs) via the use of a product that is naturally abundant and inexpensive. (1−4) The development of biocompatible films with excellent mechanical properties will find broad applications in fields such as food, (5,6) biomedical science, (7,8) and tissue engineering. (9,10) Chitosan is a linear polysaccharide composed of d-glucosamine that is used to produce such films. It is produced by treating the chitin extracted from sources such as crab and shrimp shells, squid skeletons, and mushrooms. A high level of biocompatibility (11,12) makes chitosan available for various functions via chemical treatment. (13−15) Valuable applications to the medical field include artificial skin (16,17) and hydrogel patches. (18,19) Applications involving drug permeation offer particular merit with this product; a specific drug could be encapsulated and administered without invasive treatment of a patient, which would reduce side effects. For other applications, however, films using polysaccharides such as chitosan have a weak polymer network with mechanical properties that must be improved. Application as a biomaterial requires a different range of acceptable mechanical properties that depend on tissue modeling. It is necessary to control the mechanical properties within each range to reduce the side effects to the target organ. In particular, human skin has a tensile strength of ∼5.0 to 30.0 MPa. (20) Therefore, application as a biomaterial must fall within a range that approximates that of human skin. Various methods have been suggested to overcome the weak mechanical properties of chitosan films. (21) The Sun group has introduced double cross-linked chitosan composite films with oxidized tannic acid and ferric ions via the use of a green method. (21) These chitosan composite films have improved the mechanical properties and confer excellent water resistance. To produce them, however, a second process is required that involves a radical reaction with a metal complex. In another development, Chalitangkoon et al. have developed silver-loaded hydroxyethylacryl chitosan/sodium alginate hydrogel films for medical applications. (22) The loading of silver improved the mechanical properties, which reached 8 MPa. Polysaccharide-based films for medical applications, however, continue to suffer from weak mechanical properties. Otherwise, several groups have improved the mechanical properties of chitosan films via the addition of acidic solvents. (23−25) The amine groups of chitosan can be chemically reacted with an acidic solvent to improve the mechanical properties. (26) Qiao et al. have reported the role of organic acid structures in terms of the physical and antioxidant properties of cross-linked chitosan films (27) and have shown that the hydroxyl groups in acid are beneficial to the ductility, thermal stability, and smooth nature of chitosan films. The researchers synthesizing chitosan films under strong acidic conditions have focused on the mechanical properties. Films produced under strong acid conditions, however, have limitations for use as medical materials, which dictates the synthesis of films under conditions using either weak acids such as acetic acid or neutral conditions. For medical applications, it will be necessary to develop films under physiological conditions.
In the present study, the amount of weak acetic acid was controlled to produce chitosan films with mechanical flexibility that could load and deliver cancer-targeting drugs. In addition to improving the mechanical properties of the film, the release behavior of drugs from films with various mechanical properties was investigated.

Materials and Methods

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Materials

Chitosan (CS) solution (2 wt % in water, n = 480) was obtained from Sugino Machine Ltd. Acetic acid (AA, MW = 60 g/mol) and doxorubicin hydrochloride (DOX) were purchased from TCI. These were used without further purification. The skin of a 7-week-old male hairless mouse, from which subcutaneous fat was removed, was purchased from SLC, Inc. The IR spectra of KBr pellets with CA and AA were measured using FT-IR (8400S, Shimadzu Corp.) spectroscopy that ranged from 400 to 4000 cm–1. The optical absorption spectra of the samples were obtained at 298 K using a UV–vis spectrophotometer (V-750, JASCO Corp.).

Preparation of the Hydrogels

A CS solution (2 wt %) of 1 mL was mixed with AA solutions (1.8 M) of 0, 0.01, 0.05, and 0.125 mL to prepare the hydrogel. Mass ratios of the AA/CS solution were 0, 6, 30, and 75, respectively. The cancer drug DOX was dissolved in dimethyl sulfoxide (DMSO) to obtain a DOX solution of 8 g/L. A DOX solution of 50 μL was added to various ratios of hydrogel to prepare DOX-loaded hydrogels, and these were kneaded with a spatula in an ointment container.

Rheological Properties of the Hydrogels

The DOX-loaded hydrogels were placed onto a rheometer plate according to the standard level. The rheological measurements were conducted in dynamic oscillation mode using a dynamic shear rheometer (MCR102; Anton Paar GmbH, Graz, Austria) with a stainless steel cone plate. There was a gap of 1.0 mm between the hydrogel and the plate, which had a diameter of 50 mm, and measurements were conducted at 25 °C. Frequency sweep tests were performed from 1 to 100 Hz. Strain-sweep tests were performed with an increase in the amplitude of oscillation from 1 to 200% of the apparent shear strain at 1 Hz.

Preparation and Mechanical Properties of the Films

DOX-loaded hydrogels of ∼3 mL were dropped onto a polypropylene bottom dish that was 50 mm in diameter. The dish was then allowed to dry at room temperature for 1.5 days to obtain DOX-loaded films with a thickness of ∼100 μm. The average thickness of the films was measured at 10 random locations using an electromagnetic coating thickness tester (LE-200J, Kett Electric Laboratory). Values for the tensile strength (TS) and elongation at the point of breakage (EB) of the DOX-loaded films were gathered using a tensile strength meter (JSV-H1000, Japan Instrumentation System Co., Ltd.) at 25 °C. The DOX-loaded films were cut into a 10 × 30 mm2 (length × width) strip, and both ends of the strip were separately mounted on the analyzer with an initial grip separation of 14 mm, and a gap-stretching rate of 5 mm/min was applied. TS (MPa) and EB (%) were calculated using eqs 1 and 2.
TS(MPa)=FA
(1)
In eq 1, F is the applied load (N), and A is the cross-sectional area (mm2) of the film strip, which was calculated using the average thickness and width of the films.
EB(%)=(L1L0)L0×100
(2)
In eq 2, L0 is the initial length (mm) of the film strip, and L1 is the final length (mm) of the film strip at the point of breakage. The strain–stress curves were plotted for each film. The stretchability of the films was evaluated according to their elongation at the point of breakage.

Release of a Drug from the Film

The release amount of DOX from DOX-loaded films was measured using a UV–vis spectrophotometer within a range of 200–700 nm. The DOX-loaded films were immersed in 7 mL of pure water with the pH adjusted using (pH = 7.0, 5.0) aqueous HCl. A 1 mL sample was gathered from the solution at a specified time and was then returned to the original solvent each time after measurement. The release ratio was calculated from the release amount and the initial amount in the film using eq 3. A quartz cell with a path length of 10 mm was used. A calibration curve of DOX was determined at a wavelength of 520 nm (Figure S1).
drugrelease(%)=releaseamountofDOXinwaterloadingamountofDOXinfilm×100
(3)

Transdermal Drug Penetration

A transdermal penetration test was performed using Franz diffusion cells (PermeGear, Inc.) with a diameter of 5.0 mm. A 20 mM phosphate buffer solution of pH 7.4 was placed in the receptor chamber and was maintained at 37 °C with continuous stirring at 600 rpm. After the subcutaneous fat was removed from full-thickness skin, the skin was placed on the receptor chamber facing upward toward the donor cell. The DOX-loaded film was placed onto the skin after soaking in water for 2 min. Then, a 10% DMSO aqueous solution was added to the surface of the film. The concentration of DOX in the receptor chamber was determined using a fluorescence spectrometer (LS-55, PerkinElmer) after 18, 24, and 48 h. DOX was excited at λex = 485 nm, and the emission was recorded at λem = 596 nm.

Anticancer Effects of Drugs Released from Hydrogels

Murine colon carcinoma cells (Colon26) were seeded onto the 12-well plate at a density of 1.0 × 105 cells per well and incubated for 18 h. The cells were coincubated with DOX-loaded hydrogels (CS of 350 μg and DOX of 400 μg) for 24 h. After an exchange of fresh medium, the cells were treated with a Cell Counting Kit-8 (Dojindo Laboratories) for 2 h. The absorbance at 450 and 650 nm was measured using a microplate reader to determine cell viability.

Subcellular Distribution of Drugs

Colon26 cells were seeded onto a glass-bottom dish at a density of 1.0 × 105 cells per well and incubated for 18 h. The cells were coincubated with DOX-loaded hydrogels (CS of 350 μg and DOX of 400 μg) for 24 h. After an exchange with fresh medium, the nuclei were stained with Hoechst33342, and the cells were observed using a confocal laser scanning microscope (LSM700, Zeiss, Germany).

Anticancer Effects toward Cancer Spheroid

Colon26 spheroids were prepared by seeding Colon26 cells to an EZ sphere at 200 cells per well. The cells were incubated for 4 days. The cancer spheroids were exposed to DOX-loaded hydrogels. The sizes of the spheroids were monitored via a microscope at specified points of time (0, 1, 3, 5, and 7 days).

Results and Discussion

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Drug-Loaded Chitosan Hydrogels

A reaction scheme for chitosan (CS) and acetic acid (AA) is illustrated in Figure 1A. CS has a high molecular weight, and it has two amino groups per molecule. These were reacted by adding the AA and the −NH2 groups of CS in the acidic media, which became NH3+COO because of the protonation of the −NH2 groups. (28) The details and discussion of the reaction of CS and AA will be described in a later section. Figure 1B shows the formation procedure of DOX-loaded film with different amounts of AA added. First, 0–1 mL of AA was added and mixed with 1 mL of CS. As shown in Figure 1A, when the −NH2 groups of CS reacted with the AA, they were changed to NH3+CH3COO. The designation number of −NH2 was converted to NH3+CH3COO, depending on the amount of added AA. Furthermore, 50 μL of DOX cancer drug was dissolved in DMSO to reach a concentration of 8 mg/mL, then added to the mixture of CS and AA, and it was well mixed. DOX is encapsulated in the hydrogel in two ways at different concentrations of AA. In the case of a small amount of AA, the −NH2 remained as a factor of the hydrogen bond. When the amount of AA was sufficient to react, it affected the CS as a solvent in the hydrogel. The reaction ratio of the −NH2 depended on the amount of AA as well. Therefore, the NH3+CH3COO number in the mixture seemed to contribute to the drug-release ratio.

Figure 1

Figure 1. (A) Reaction scheme of CS and AA and (B) formation procedure for DOX-loaded film.

Figure 2A shows a photograph of DOX-loaded hydrogel prepared at ratios of AA/CS that ranged from 0 to 67.5. The transparency of the DOX-loaded gel increased as the amount of AA increased. This seems to be related to the amount of AA present in the hydrogel, as mentioned previously. A sufficient amount of AA was expected to replace the NH2 groups of CS with CH3COO, and the remaining amount of AA served as a solvent for the hydrogel and affected the pH. The viscosities were different with each addition of AA. Increasing the AA amount resulted in high viscosity.

Figure 2

Figure 2. (A) Photograph of DOX-loaded hydrogels prepared at AA/CS ratios = 0 (a), 5.4 (b), 27 (c), and 67.5 (d). (B) Ratio of NH3+CH3COO/NH2 vs the ratio of AA/CS.

Figure 2B shows how the ratio of AA/CS affected the development of the ratio of NH3+CH3COO/NH2. The calculation method was as follows: the degree of polymerization (n) for the purchased CS is 480, and the molecular weight per unit is 159 g/mol. Therefore, the total molecular weight of CS is calculated to be 76 320 g/mol (=480 × 159). The volume of CS in this experiment is regarded as 0.02 g because the density of CS is considered to be 1 g/mL. A mole of CS is determined to be 2.62 × 10–7 mol (=0.02 g/76 320 g/mol). A theoretical mole of AA is 1.26 × 10–4 mol (=480 × 2.62 × 10–7 mol) because the reaction between CS and AA should completely replace the NH2 with CH3COO. As a result, the theoretical mass of AA would be 7.57 × 10–3 g (=1.26 × 10–4 mol × 60.05 g/mol; AA molecular weight). In the case of the DOX-loaded hydrogel of AA/CS (b), the practical mass of AA would be 1.08 × 10–3 g (1.8 M AA of 0.01 mL is used). As a consequence, the ratio of NH3+CH3COO/NH2 would be 14.27 (=1.08 × 10–3 g/7.57 × 10–3 g × 100). The other conditions are calculated in the same manner. The black dotted line in Figure 2B shows the experimental values, and the red line shows the theoretical values. In this calculation, the optimal ratio of AA/CS is 37.7, which means all of the NH2 of CS was converted to CH3COO.
Figure 3 shows the FT-IR spectra of DOX-loaded hydrogel to confirm the driving force of gel formation. When the amount of AA was increased, the most notable peak shift was 1575 cm–1 from the primary amide of CS, which shifted to the right. The cause of the peak shift is assumed to be the replacement of the amide bond of CS by AA. Ghosh et al. attributed the red shifts of FT-IR to the amide formation between chitosan and acetic acids. (29) This reaction mechanism between chitosan and acetic acids is consistent with that shown in Figure 1B. In addition, we confirmed that the peak at 1425 cm–1 was increased by the addition of AA. The peak at 2915 cm–1 became sharp for the same reason.

Figure 3

Figure 3. FT-IR spectra of DOX-loaded films prepared at AA/CS ratios = 0 (a), 5.4 (b), 27 (c), and 67.5 (d).

Rheological Properties of Drug-Loaded Hydrogels

The respective storage moduli G′, and loss moduli G″, are shown for DOX-loaded hydrogel prepared at various ratios of AA/CS as functions of the angular frequency at a fixed strain, γ = 0.01% (Figure 4). The G′ values have a substantial elastic response and are always larger than the G″ values over the entire range of frequencies. Among the four films prepared at various ratios of AA/CS, the DOX-loaded hydrogel without AA showed the greatest G′ value, which suggests the effect of gelation in the hydrogen bonding of NH2. When the concentrations of AA were increased, G′ and G″ were decreased. As previously discussed, concerning the effect of AA, the ionic bond between the NH3+ of CS and the CH3COO of AA increases the effect of hydrogen bonding, which lessens gel formation and weakens the gel interaction. As shown in Figure 4d, when the ratio of AA/CS was 67.5, the AA was theoretically in a saturated state. The state of the DOX-loaded hydrogel approximates that of the liquid state, which is G′/G″ < 1.

Figure 4

Figure 4. Frequency sweep tests of DOX-loaded hydrogels prepared at AA/CS ratios of 0 (a), 5.4 (b), 27 (c), and 67.5 (d) at a strain of 0.01%.

Strain-sweep testing of the DOX-loaded hydrogel demonstrated an elastic response that is typical of hydrogels (Figure 5a,b). When the ratio of AA/CS was increased to 27.5, G′ and G″ were closer (Figure 5c). The inversion point of G′/G″ was accelerated from 100 to 40% with the addition of AA. At a rich AA/CS ratio of 67.5, G′ and G″ were inverted (Figure 5d). This means that the prepared hydrogel was no longer a typical gel, but rather, it was a hydrogel that showed a more liquid-like behavior.

Figure 5

Figure 5. Strain-sweep tests of DOX-loaded hydrogels prepared at AA/CS ratios of 0 (a), 5.4 (b), 27 (c), and 67.5 (d) at a frequency of 1 Hz.

Mechanical Properties of Drug-Loaded Films

The tensile stress–strain curves of DOX-loaded film prepared at various ratios of AA/CS are shown in Figure 6. The tension strengths of DOX-loaded film with AA/CS ratios of 5.4 and 27 were lower than that of DOX-loaded film without AA (AA/CS = 0). However, DOX-loaded film with an AA/CS ratio of 67.5 had almost the same tension strength as that of DOX-loaded film without AA (AA/CS = 0). On the other hand, the elongation at the point of breakage for DOX-loaded film that was used to evaluate the stretchability of the film at AA/CS ratios of 27 and 67.5 showed higher values than the DOX-loaded film without AA (AA/CS = 0). That result showed both the high strength and stretchability of the DOX-loaded film with an AA/CS ratio of 67.5. The improvement in stretchability by the addition of AA was due to the reaction of NH3+CH3COO with the amine groups of CS and AA. This induced a cross-linked structure via mutual ionic interactions. (29) These interactions seem to affect the stretchability of the chitosan polymeric film. It was obvious that the DOX-loaded CS film was changed as the NH2 groups of CS were all converted to CH3COO. When immersed in water, the film prepared in a converted CH3COO state seems to expand more because it is more hydrophilic, which should lead to an increase in the amount of drug release.

Figure 6

Figure 6. Tensile stress–strain curves of DOX-loaded films prepared at AA/CS ratios of 0 (a), 5.4 (b), 27 (c), and 67.5 (d).

Drug-Release Testing of DOX-Loaded Films

A drug-release test of DOX-loaded films was conducted. The appearance of DOX-loaded films and the degree of drug release according to time changes are shown in Figure 7. We confirmed the drug release by immersing the DOX-loaded films prepared at various AA/CS ratios (0, 5.4, 27, 67.5). When the DOX-loaded films were immersed in water (pH = 7), their swelling correlated positively with increases in the AA/CS ratio. When the AA/CS ratio was 67.5, the width increased almost 4 times compared with the initial film. At that point, the release ratio of the drug was 70%. Consequently, the release ratio of the drug could be controlled 30–70% by fine control of the amount of AA added during the preparation of DOX-loaded films (Figure 7A). Kinetic models of the release were used to analyze the drug-release mechanism to establish why the release stopped after 3 h for all films. Korsmeyer–Peppas modeling equation (eq 4) was used to analyze the drug-release behavior.
MtM=KKPtn
(4)
In this equation, Mt is the amount of drug released at each time, t; KKP is the Korsmeyer–Peppas rate constant; Mt/M is the percentage of drug released at each time, t; and n is the diffusion exponent. The n value shows the release mechanism of the drug. Values of n between 0.5 and 1.0 indicate non-Fickian diffusion (anomalous transport kinetics). If the n value is approximately equal to 0.5, the drug-release mechanism is defined as a diffusion-controlled mechanism (Fickian diffusion). Values of n < 0.5 may be due to drug diffusion partially through swelling behavior. (30) As a result, the n value was decreased from 0.236 to 0.196 as the addition of AA increased. All n values were less than 0.5, which means those drug releases were caused by swelling behaviors of the film in PBS. Therefore, the burst release of DOX was attributed to swelling behavior, and the differences in the drug-release ratio were caused by the amount of added AA. After 3 h, each film swelling almost ended based on observation, and the additional drug release was attributed to Fickian diffusion. Even though the swelling behavior did not seem to have much influence on the drug release, it is considered the reason for the release stop after 3 h for all films. However, the acidic conditions (pH = 5) caused all DOX-loaded CS polymeric films prepared at various AA/CS ratios (0, 5.4, 27, 67.5) to dissolve in the solvent. This is because the amino groups (NH2 and NH3+CH3COO) of CS repeat units and AA are positively charged, which leads to strong cation–cation repulsive forces. (31) These forces are considered to result in structures that destroy DOX-loaded CS polymeric films.

Figure 7

Figure 7. (A) Photograph of prepared DOX-loaded films at AA/CS ratios = 0 (a), 5.4 (b), 27 (c), and 67.5 (d), and a photograph after each film was soaked in water for 1 day: AA/CS ratios = 0 (e), 5.4 (f), 27 (g), and 67.5 (h); (B) cumulative release ratios of DOX from DOX-loaded films prepared at AA/CS ratios = 0 (a), 5.4 (b), 27 (c), and 67.5 (d).

Cellular Cytotoxicity of Drug-Loaded Hydrogels

The applicability of DOX-loaded hydrogels as drug delivery platforms for anticancer therapy was demonstrated. Murine colon carcinoma cells (Colon26) were coincubated with the hydrogels (DOX of 587–657 μM), and the cell viability was quantified via a modified MTT assay. When the AA/CS ratio in the hydrogel was increased, cell destruction to Colon26 cells was induced more efficiently (Figures 8 and S2). The results are comparable to those from in vitro drug-release studies, which suggests that the cellular uptake amount of released DOX is critical in causing cell death. To address the deliverability of DOX using prepared hydrogels, the subcellular distribution of delivered DOX within cells by confocal laser scanning microscopy was observed. DOX functions as an anticancer agent within nuclei by inhibiting the activity of DNA polymerase and RNA polymerase, and the nuclei in living cells were visualized using the commercially available nuclei staining reagent Hoechst33342. As shown in Figure 8, fluorescence signals from DOX were detected within cells treated with DOX-loaded hydrogels, which showed an increase in fluorescence intensity with increases in the ratio of AA/CS. In the case shown in Figure 8C, the delivered DOX was partially located in the nuclei, which suggests this anticancer agent had intercalated the genomic DNA. These tendencies in accumulation support our hypothesis of cell death.

Figure 8

Figure 8. (A) Colon26 cells were seeded onto 12-well plates (1.0 × 105 cells) and incubated for 24 h. Dox-loaded hydrogels were placed in the medium. After an additional 24 h, a Cell Counting Kit-8 (CCK8) solution was added to each sample, and the absorbance at 450 nm was measured using a microplate reader to quantify cell viability. The control is shown as (a), and each of the Dox-loaded hydrogels is shown at its respective AA/CS ratio: 5.4 (b), 27 (c), and 67.5 (d). Panels (B–D) are the subcellular distributions of delivered DOX in Colon26 cells. Colon26 cells were exposed to the DOX-loaded hydrogels (AA/CS ratios: 5.4 (B); 27 (C); and 67.5 (D)). The scale bar represents 20 μm.

To address the usability of the hydrogel system, anticancer effects against cancer cell spheroids were further examined because these are considered to be a more practical cancer model. After 5 days of incubation, fluorescence from DOX was detected in the spheroids (Figures 9 and S3). Next, the growth of spheroids after exposure to the DOX-loaded hydrogels was evaluated. In the absence of the hydrogel, the spheroids grew rapidly (Figure 9A(a)). By contrast, the spheroidal growth was significantly suppressed by applying DOX-loaded chitosan hydrogels. Finally, the cancer spheroids completely collapsed at 7 days. Thus, the proposed system is effective as an anticancer therapy in complex biological settings.

Figure 9

Figure 9. (A) Relative spheroid volume after exposure of prepared hydrogels (a) and DOX-loaded hydrogel prepared at AA/CS ratio = 27 (b). Optical image after 1 day of exposure to a constant phase (B), DOX (C).

Skin Permeation by Drugs

DOX is administrated mainly by nontargeted delivery, such as venous or arterial injection. However, nontargeted delivery of DOX causes various toxic adverse effects. Topical chemotherapy of doxorubicin, such as a transdermal administration, has attracted attention due to the need to reduce systemic toxicity. (32) On the other hand, the skin penetration of DOX is difficult due to its hydrophilicity, charge, and high molecular weight properties. To overcome these problems, DOX-loaded chitosan film may improve the skin permeability of DOX. Subsequently, a 10% DMSO aqueous solution was added to the film instead of water to prevent drying during the skin penetration test. An aqueous solution with a low concentration of DMSO has been widely studied as a skin penetration enhancer because DMSO induces the protein denaturation of skin and interacts with the hydrophilic region of lipid layers. (33) The DOX released from the DOX-loaded chitosan DMSO solution penetrated the skin (Figure 10). The skin penetration amounts of DOX at 18 and 24 h were almost the same, 3.1 and 3.3 μg/cm2, respectively. However, the amount of DOX after 48 h was increased to 4.5 μg/cm2. The films seemed to start partially collapsing after 48 h. The skin penetration amount of DOX seems comparable to values referencing treatment with a microneedle. (29) This result suggests that the present film shows promise as a candidate for an easy chemical method for the transdermal delivery of DOX without the need for physical methods such as a microneedle or iontophoresis.

Figure 10

Figure 10. Skin permeation experiment of DOX-loaded film prepared at an AA/CS ratio of 5.4. (A) Experimental equipment and (B) skin penetration amount of DOX released from the film after 18 h, 24 h, and 48 h.

Conclusions

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In this work, we described the use of a stretchable DOX-loaded chitosan film prepared using different AA concentrations. Under rich AA conditions, the rheological properties of DOX-loaded chitosan showed weak values, such as a liquid state. Tensile stress–strain curves of DOX-loaded chitosan film were measured, and the stretchability of the film was evaluated via elongation at the point of breakage. The release testing of DOX from the film was conducted in water. When DOX-loaded films were prepared at a AA/CS ratio = 67.5, the elongation at the point of breakage reached a stretchability high of 27%, and the DOX release ratio from chitosan film reached a high of 70%. The difference in the mechanical properties of the chitosan film affected the drug release. The cytotoxicity of DOX-loaded chitosan film was measured, and the cancer spheroids that were tested had completely collapsed at 7 days. As a result of the skin permeability test, the DOX-loaded chitosan film showed practical utility as a drug seal. We confirmed that the DOX was permeable to the skin and that the amount of permeation increased in proportion to time.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.2c06719.

  • UV-measurement of free DOX (Figure S1), cell viability test of hydrogels at different concentration AA (Figure S2), and relative spheroid volume after exposure (Figure S3)(PDF)

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Author Information

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  • Corresponding Authors
    • Ji Ha Lee - Chemical Engineering Program, Graduate School of Advanced Science and Engineering, Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima739-8527, JapanOrcidhttps://orcid.org/0000-0002-4456-0128 Email: [email protected]
    • Akihiro Yabuki - Chemical Engineering Program, Graduate School of Advanced Science and Engineering, Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima739-8527, Japan Email: [email protected]
  • Authors
    • Tomoyuki Tachibana - Chemical Engineering Program, Graduate School of Advanced Science and Engineering, Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima739-8527, Japan
    • Hijiri Wadamori - Chemical Engineering Program, Graduate School of Advanced Science and Engineering, Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima739-8527, Japan
    • Keita Yamana - Applied Chemistry Program, Graduate School of Advanced Science and Engineering, Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima739-8527, Japan
    • Riku Kawasaki - Applied Chemistry Program, Graduate School of Advanced Science and Engineering, Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima739-8527, Japan
    • Shogo Kawamura - Applied Chemistry Program, Graduate School of Advanced Science and Engineering, Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima739-8527, Japan
    • Hinata Isozaki - Applied Chemistry Program, Graduate School of Advanced Science and Engineering, Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima739-8527, Japan
    • Mina Sakuragi - Department of Nanoscience, Sojo University, 4-22-1 Ikeda, Nishi-ku, Kumamoto860-0082, JapanOrcidhttps://orcid.org/0000-0002-6362-3673
    • Isamu Akiba - Department of Chemistry and Biochemistry, Kitakyushu University, 1-1 Hibikino, Wakamatsu, Kitakyushu808-0135, JapanOrcidhttps://orcid.org/0000-0003-3085-5555
  • Author Contributions

    The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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This work was supported by MEXT Promotion of Distinctive Joint Research Center Program (Grant Number JPMXP 0621467946).

References

ARTICLE SECTIONS
Jump To

This article references 33 other publications.

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    Mohamed, S. A. A.; El-Sakhawy, M.; El-Sakhawy, M. A. M. Polysaccharides, Protein and Lipid -Based Natural Edible Films in Food Packaging: A Review. Carbohydr. Polym. 2020, 238, 116178  DOI: 10.1016/j.carbpol.2020.116178
  2. 2
    Luo, Y.; Wang, Q. Recent Development of Chitosan-Based Polyelectrolyte Complexes with Natural Polysaccharides for Drug Delivery. Int. J. Biol. Macromol. 2014, 64, 353367,  DOI: 10.1016/j.ijbiomac.2013.12.017
  3. 3
    Azeredo, H. M. C.; Waldron, K. W. Crosslinking in Polysaccharide and Protein Films and Coatings for Food Contact - A Review. Trends Food Sci. Technol. 2016, 52, 109122,  DOI: 10.1016/j.tifs.2016.04.008
  4. 4
    Iijima, K.; Tsuji, Y.; Kuriki, I.; Kakimoto, A.; Nikaido, Y.; Ninomiya, R.; Iyoda, T.; Fukai, F.; Hashizume, M. Control of Cell Adhesion and Proliferation Utilizing Polysaccharide Composite Film Scaffolds. Colloids Surf., B 2017, 160, 228237,  DOI: 10.1016/j.colsurfb.2017.09.025
  5. 5
    Cazón, P.; Velazquez, G.; Ramírez, J. A.; Vázquez, M. Polysaccharide-Based Films and Coatings for Food Packaging: A Review. Food Hydrocolloids 2017, 68, 136148,  DOI: 10.1016/j.foodhyd.2016.09.009
  6. 6
    Liu, C.; Huang, J.; Zheng, X.; Liu, S.; Lu, K.; Tang, K.; Liu, J. Heat Sealable Soluble Soybean Polysaccharide/Gelatin Blend Edible Films for Food Packaging Applications. Food Packag. Shelf Life 2020, 24, 100485  DOI: 10.1016/j.fpsl.2020.100485
  7. 7
    Silva, F. E. F.; Batista, K. A.; Di-Medeiros, M. C. B.; Silva, C. N. S.; Moreira, B. R.; Fernandes, K. F. A Stimuli-Responsive and Bioactive Film Based on Blended Polyvinyl Alcohol and Cashew Gum Polysaccharide. Mater. Sci. Eng. C 2016, 58, 927934,  DOI: 10.1016/j.msec.2015.09.064
  8. 8
    Rodrigues, L. C.; Fernandes, E. M.; Ribeiro, A. R.; Ribeiro, A. P.; Silva, S. S.; Reis, R. L. Physicochemical Features Assessment of Acemannan-Based Ternary Blended Films for Biomedical Purposes. Carbohydr. Polym. 2021, 257, 117601  DOI: 10.1016/j.carbpol.2020.117601
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    Jeong, H. G.; Kim, Y. E.; Kim, Y. J. Fabrication of Poly(Vinyl Acetate)/Polysaccharide Biocomposite Nanofibrous Membranes for Tissue Engineering. Macromol. Res. 2013, 21, 12331240,  DOI: 10.1007/s13233-013-1155-x
  10. 10
    Das, A.; Das, A.; Basu, A.; Datta, P.; Gupta, M.; Mukherjee, A. Newer Guar Gum Ester/Chicken Feather Keratin Interact Films for Tissue Engineering. Int. J. Biol. Macromol. 2021, 180, 339354,  DOI: 10.1016/j.ijbiomac.2021.03.034
  11. 11
    Ma, X.; Wu, G.; Dai, F.; Li, D.; Li, H.; Zhang, L.; Deng, H. Chitosan/Polydopamine Layer by Layer Self-Assembled Silk Fibroin Nanofibers for Biomedical Applications. Carbohydr. Polym. 2021, 251, 117058  DOI: 10.1016/j.carbpol.2020.117058
  12. 12
    Neamnark, A.; Sanchavanakit, N.; Pavasant, P.; Bunaprasert, T.; Supaphol, P.; Rujiravanit, R. In Vitro Biocompatibility Evaluations of Hexanoyl Chitosan Film. Carbohydr. Polym. 2007, 68, 166172,  DOI: 10.1016/j.carbpol.2006.07.024
  13. 13
    Li, Z.; Liu, Y.; Zou, S.; Lu, C.; Bai, H.; Mu, H.; Duan, J. Removal and Adsorption Mechanism of Tetracycline and Cefotaxime Contaminants in Water by NiFe2O4-COF-Chitosan-Terephthalaldehyde Nanocomposites Film. Chem. Eng. J. 2020, 382, 123008  DOI: 10.1016/j.cej.2019.123008
  14. 14
    Frick, J. M.; Ambrosi, A.; Pollo, L. D.; Tessaro, I. C. Influence of Glutaraldehyde Crosslinking and Alkaline Post-Treatment on the Properties of Chitosan-Based Films. J. Polym. Environ. 2018, 26, 27482757,  DOI: 10.1007/s10924-017-1166-3
  15. 15
    Wu, J.; Su, C.; Jiang, L.; Ye, S.; Liu, X.; Shao, W. Green and Facile Preparation of Chitosan Sponges as Potential Wound Dressings. ACS Sustainable Chem. Eng. 2018, 6, 91459152,  DOI: 10.1021/acssuschemeng.8b01468
  16. 16
    Vivcharenko, V.; Wojcik, M.; Przekora, A. Cellular Response to Vitamin C-Enriched Chitosan/Agarose Film with Potential Application as Artificial Skin Substitute for Chronic Wound Treatment. Cells 2020, 9, 1185  DOI: 10.3390/cells9051185
  17. 17
    Parvez, S.; Rahman, M. M.; Khan, M. A.; Khan, M. A. H.; Islam, J. M. M.; Ahmed, M.; Rahman, M. F.; Ahmed, B. Preparation and Characterization of Artificial Skin Using Chitosan and Gelatin Composites for Potential Biomedical Application. Polym. Bull. 2012, 69, 715731,  DOI: 10.1007/s00289-012-0761-7
  18. 18
    Cai, C.; Wang, T.; Han, X.; Yang, S.; Lai, C.; Yuan, T.; Feng, Z.; He, N. In Situ Wound Sprayable Double-Network Hydrogel: Preparation and Characterization. Chin. Chem. Lett. 2022, 33, 19631969,  DOI: 10.1016/j.cclet.2021.11.047
  19. 19
    Pok, S.; Myers, J. D.; Madihally, S. V.; Jacot, J. G. A Multilayered Scaffold of a Chitosan and Gelatin Hydrogel Supported by a PCL Core for Cardiac Tissue Engineering. Acta Biomater. 2013, 9, 56305642,  DOI: 10.1016/j.actbio.2012.10.032
  20. 20
    Tran, T. T.; Hamid, Z. A.; Cheong, K. Y. A Review of Mechanical Properties of Scaffold in Tissue Engineering: Aloe Vera Composites. J. Phys.: Conf. Ser. 2018, 1082, 012080,  DOI: 10.1088/1742-6596/1082/1/012080
  21. 21
    Yang, J.; Li, M.; Wang, Y.; Wu, H.; Zhen, T.; Xiong, L.; Sun, Q. Double Cross-Linked Chitosan Composite Films Developed with Oxidized Tannic Acid and Ferric Ions Exhibit High Strength and Excellent Water Resistance. Biomacromolecules 2019, 20, 801812,  DOI: 10.1021/acs.biomac.8b01420
  22. 22
    Chalitangkoon, J.; Wongkittisin, M.; Monvisade, P. Silver Loaded Hydroxyethylacryl Chitosan/Sodium Alginate Hydrogel Films for Controlled Drug Release Wound Dressings. Int. J. Biol. Macromol. 2020, 159, 194203,  DOI: 10.1016/j.ijbiomac.2020.05.061
  23. 23
    Kan, Y.; Yang, Q.; Tan, Q.; Wei, Z.; Chen, Y. Diminishing Cohesion of Chitosan Films in Acidic Solution by Multivalent Metal Cations. Langmuir 2020, 36, 49644974,  DOI: 10.1021/acs.langmuir.0c00438
  24. 24
    Rivero, S.; García, M. A.; Pinotti, A. Crosslinking Capacity of Tannic Acid in Plasticized Chitosan Films. Carbohydr. Polym. 2010, 82, 270276,  DOI: 10.1016/j.carbpol.2010.04.048
  25. 25
    Blilid, S.; Kȩdzierska, M.; Miłowska, K.; Wrońska, N.; El Achaby, M.; Katir, N.; Belamie, E.; Alonso, B.; Lisowska, K.; Lahcini, M.; Bryszewska, M.; El Kadib, A. Phosphorylated Micro- A Nd Nanocellulose-Filled Chitosan Nanocomposites as Fully Sustainable, Biologically Active Bioplastics. ACS Sustainable Chem. Eng. 2020, 8, 1835418365,  DOI: 10.1021/acssuschemeng.0c04426
  26. 26
    Cui, Z.; Beach, E. S.; Anastas, P. T. Modification of Chitosan Films with Environmentally Benign Reagents for Increased Water Resistance. Green Chem. Lett. Rev. 2011, 4, 3540,  DOI: 10.1080/17518253.2010.500621
  27. 27
    Qiao, C.; Ma, X.; Wang, X.; Liu, L. Structure and Properties of Chitosan Films: Effect of the Type of Solvent Acid. LWT 2021, 135, 109984  DOI: 10.1016/j.lwt.2020.109984
  28. 28
    Yeng, C. M.; Salmah, H.; Ting, S. Corn Cob Filled Chitosan Biocomposite Films. Adv. Mater. Res. 2013, 747, 649652,  DOI: 10.4028/www.scientific.net/AMR.747.649
  29. 29
    Ghosh, A.; Ali, M. A. Studies on Physicochemical Characteristics of Chitosan Derivatives with Dicarboxylic Acids. J. Mater. Sci. 2012, 47, 11961204,  DOI: 10.1007/s10853-011-5885-x
  30. 30
    Altunkaynak, F.; Okur, M.; Saracoglu, N. Controlled Release of Paroxetine from Chitosan/Montmorillonite Composite Films. J. Drug Delivery Sci. Technol. 2022, 68, 103099  DOI: 10.1016/j.jddst.2022.103099
  31. 31
    Che, Y.; Li, D.; Liu, Y.; Ma, Q.; Tan, Y.; Yue, Q.; Meng, F. Physically Cross-Linked PH-Responsive Chitosan-Based Hydrogels with Enhanced Mechanical Performance for Controlled Drug Delivery. RSC Adv. 2016, 6, 106035106045,  DOI: 10.1039/c6ra16746b
  32. 32
    Nguyen, H. X.; Bozorg, B. D.; Kim, Y.; Wieber, A.; Birk, G.; Lubda, D.; Banga, A. K. Poly (Vinyl Alcohol) Microneedles: Fabrication, Characterization, and Application for Transdermal Drug Delivery of Doxorubicin. Eur. J. Pharm. Biopharm. 2018, 129, 88103,  DOI: 10.1016/j.ejpb.2018.05.017
  33. 33
    Kumar, B.; Jain, S. K.; Prajapati, S. K. Effect of Penetration Enhancer DMSO on In-Vitro Skin Permeation of Acyclovir Transdermal Microemulsion Formulation. Int. J. Drug Delivery 2011, 3, 8394,  DOI: 10.5138/ijdd.2010.0975.0215.03057

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  • Abstract

    Figure 1

    Figure 1. (A) Reaction scheme of CS and AA and (B) formation procedure for DOX-loaded film.

    Figure 2

    Figure 2. (A) Photograph of DOX-loaded hydrogels prepared at AA/CS ratios = 0 (a), 5.4 (b), 27 (c), and 67.5 (d). (B) Ratio of NH3+CH3COO/NH2 vs the ratio of AA/CS.

    Figure 3

    Figure 3. FT-IR spectra of DOX-loaded films prepared at AA/CS ratios = 0 (a), 5.4 (b), 27 (c), and 67.5 (d).

    Figure 4

    Figure 4. Frequency sweep tests of DOX-loaded hydrogels prepared at AA/CS ratios of 0 (a), 5.4 (b), 27 (c), and 67.5 (d) at a strain of 0.01%.

    Figure 5

    Figure 5. Strain-sweep tests of DOX-loaded hydrogels prepared at AA/CS ratios of 0 (a), 5.4 (b), 27 (c), and 67.5 (d) at a frequency of 1 Hz.

    Figure 6

    Figure 6. Tensile stress–strain curves of DOX-loaded films prepared at AA/CS ratios of 0 (a), 5.4 (b), 27 (c), and 67.5 (d).

    Figure 7

    Figure 7. (A) Photograph of prepared DOX-loaded films at AA/CS ratios = 0 (a), 5.4 (b), 27 (c), and 67.5 (d), and a photograph after each film was soaked in water for 1 day: AA/CS ratios = 0 (e), 5.4 (f), 27 (g), and 67.5 (h); (B) cumulative release ratios of DOX from DOX-loaded films prepared at AA/CS ratios = 0 (a), 5.4 (b), 27 (c), and 67.5 (d).

    Figure 8

    Figure 8. (A) Colon26 cells were seeded onto 12-well plates (1.0 × 105 cells) and incubated for 24 h. Dox-loaded hydrogels were placed in the medium. After an additional 24 h, a Cell Counting Kit-8 (CCK8) solution was added to each sample, and the absorbance at 450 nm was measured using a microplate reader to quantify cell viability. The control is shown as (a), and each of the Dox-loaded hydrogels is shown at its respective AA/CS ratio: 5.4 (b), 27 (c), and 67.5 (d). Panels (B–D) are the subcellular distributions of delivered DOX in Colon26 cells. Colon26 cells were exposed to the DOX-loaded hydrogels (AA/CS ratios: 5.4 (B); 27 (C); and 67.5 (D)). The scale bar represents 20 μm.

    Figure 9

    Figure 9. (A) Relative spheroid volume after exposure of prepared hydrogels (a) and DOX-loaded hydrogel prepared at AA/CS ratio = 27 (b). Optical image after 1 day of exposure to a constant phase (B), DOX (C).

    Figure 10

    Figure 10. Skin permeation experiment of DOX-loaded film prepared at an AA/CS ratio of 5.4. (A) Experimental equipment and (B) skin penetration amount of DOX released from the film after 18 h, 24 h, and 48 h.

  • References

    ARTICLE SECTIONS
    Jump To

    This article references 33 other publications.

    1. 1
      Mohamed, S. A. A.; El-Sakhawy, M.; El-Sakhawy, M. A. M. Polysaccharides, Protein and Lipid -Based Natural Edible Films in Food Packaging: A Review. Carbohydr. Polym. 2020, 238, 116178  DOI: 10.1016/j.carbpol.2020.116178
    2. 2
      Luo, Y.; Wang, Q. Recent Development of Chitosan-Based Polyelectrolyte Complexes with Natural Polysaccharides for Drug Delivery. Int. J. Biol. Macromol. 2014, 64, 353367,  DOI: 10.1016/j.ijbiomac.2013.12.017
    3. 3
      Azeredo, H. M. C.; Waldron, K. W. Crosslinking in Polysaccharide and Protein Films and Coatings for Food Contact - A Review. Trends Food Sci. Technol. 2016, 52, 109122,  DOI: 10.1016/j.tifs.2016.04.008
    4. 4
      Iijima, K.; Tsuji, Y.; Kuriki, I.; Kakimoto, A.; Nikaido, Y.; Ninomiya, R.; Iyoda, T.; Fukai, F.; Hashizume, M. Control of Cell Adhesion and Proliferation Utilizing Polysaccharide Composite Film Scaffolds. Colloids Surf., B 2017, 160, 228237,  DOI: 10.1016/j.colsurfb.2017.09.025
    5. 5
      Cazón, P.; Velazquez, G.; Ramírez, J. A.; Vázquez, M. Polysaccharide-Based Films and Coatings for Food Packaging: A Review. Food Hydrocolloids 2017, 68, 136148,  DOI: 10.1016/j.foodhyd.2016.09.009
    6. 6
      Liu, C.; Huang, J.; Zheng, X.; Liu, S.; Lu, K.; Tang, K.; Liu, J. Heat Sealable Soluble Soybean Polysaccharide/Gelatin Blend Edible Films for Food Packaging Applications. Food Packag. Shelf Life 2020, 24, 100485  DOI: 10.1016/j.fpsl.2020.100485
    7. 7
      Silva, F. E. F.; Batista, K. A.; Di-Medeiros, M. C. B.; Silva, C. N. S.; Moreira, B. R.; Fernandes, K. F. A Stimuli-Responsive and Bioactive Film Based on Blended Polyvinyl Alcohol and Cashew Gum Polysaccharide. Mater. Sci. Eng. C 2016, 58, 927934,  DOI: 10.1016/j.msec.2015.09.064
    8. 8
      Rodrigues, L. C.; Fernandes, E. M.; Ribeiro, A. R.; Ribeiro, A. P.; Silva, S. S.; Reis, R. L. Physicochemical Features Assessment of Acemannan-Based Ternary Blended Films for Biomedical Purposes. Carbohydr. Polym. 2021, 257, 117601  DOI: 10.1016/j.carbpol.2020.117601
    9. 9
      Jeong, H. G.; Kim, Y. E.; Kim, Y. J. Fabrication of Poly(Vinyl Acetate)/Polysaccharide Biocomposite Nanofibrous Membranes for Tissue Engineering. Macromol. Res. 2013, 21, 12331240,  DOI: 10.1007/s13233-013-1155-x
    10. 10
      Das, A.; Das, A.; Basu, A.; Datta, P.; Gupta, M.; Mukherjee, A. Newer Guar Gum Ester/Chicken Feather Keratin Interact Films for Tissue Engineering. Int. J. Biol. Macromol. 2021, 180, 339354,  DOI: 10.1016/j.ijbiomac.2021.03.034
    11. 11
      Ma, X.; Wu, G.; Dai, F.; Li, D.; Li, H.; Zhang, L.; Deng, H. Chitosan/Polydopamine Layer by Layer Self-Assembled Silk Fibroin Nanofibers for Biomedical Applications. Carbohydr. Polym. 2021, 251, 117058  DOI: 10.1016/j.carbpol.2020.117058
    12. 12
      Neamnark, A.; Sanchavanakit, N.; Pavasant, P.; Bunaprasert, T.; Supaphol, P.; Rujiravanit, R. In Vitro Biocompatibility Evaluations of Hexanoyl Chitosan Film. Carbohydr. Polym. 2007, 68, 166172,  DOI: 10.1016/j.carbpol.2006.07.024
    13. 13
      Li, Z.; Liu, Y.; Zou, S.; Lu, C.; Bai, H.; Mu, H.; Duan, J. Removal and Adsorption Mechanism of Tetracycline and Cefotaxime Contaminants in Water by NiFe2O4-COF-Chitosan-Terephthalaldehyde Nanocomposites Film. Chem. Eng. J. 2020, 382, 123008  DOI: 10.1016/j.cej.2019.123008
    14. 14
      Frick, J. M.; Ambrosi, A.; Pollo, L. D.; Tessaro, I. C. Influence of Glutaraldehyde Crosslinking and Alkaline Post-Treatment on the Properties of Chitosan-Based Films. J. Polym. Environ. 2018, 26, 27482757,  DOI: 10.1007/s10924-017-1166-3
    15. 15
      Wu, J.; Su, C.; Jiang, L.; Ye, S.; Liu, X.; Shao, W. Green and Facile Preparation of Chitosan Sponges as Potential Wound Dressings. ACS Sustainable Chem. Eng. 2018, 6, 91459152,  DOI: 10.1021/acssuschemeng.8b01468
    16. 16
      Vivcharenko, V.; Wojcik, M.; Przekora, A. Cellular Response to Vitamin C-Enriched Chitosan/Agarose Film with Potential Application as Artificial Skin Substitute for Chronic Wound Treatment. Cells 2020, 9, 1185  DOI: 10.3390/cells9051185
    17. 17
      Parvez, S.; Rahman, M. M.; Khan, M. A.; Khan, M. A. H.; Islam, J. M. M.; Ahmed, M.; Rahman, M. F.; Ahmed, B. Preparation and Characterization of Artificial Skin Using Chitosan and Gelatin Composites for Potential Biomedical Application. Polym. Bull. 2012, 69, 715731,  DOI: 10.1007/s00289-012-0761-7
    18. 18
      Cai, C.; Wang, T.; Han, X.; Yang, S.; Lai, C.; Yuan, T.; Feng, Z.; He, N. In Situ Wound Sprayable Double-Network Hydrogel: Preparation and Characterization. Chin. Chem. Lett. 2022, 33, 19631969,  DOI: 10.1016/j.cclet.2021.11.047
    19. 19
      Pok, S.; Myers, J. D.; Madihally, S. V.; Jacot, J. G. A Multilayered Scaffold of a Chitosan and Gelatin Hydrogel Supported by a PCL Core for Cardiac Tissue Engineering. Acta Biomater. 2013, 9, 56305642,  DOI: 10.1016/j.actbio.2012.10.032
    20. 20
      Tran, T. T.; Hamid, Z. A.; Cheong, K. Y. A Review of Mechanical Properties of Scaffold in Tissue Engineering: Aloe Vera Composites. J. Phys.: Conf. Ser. 2018, 1082, 012080,  DOI: 10.1088/1742-6596/1082/1/012080
    21. 21
      Yang, J.; Li, M.; Wang, Y.; Wu, H.; Zhen, T.; Xiong, L.; Sun, Q. Double Cross-Linked Chitosan Composite Films Developed with Oxidized Tannic Acid and Ferric Ions Exhibit High Strength and Excellent Water Resistance. Biomacromolecules 2019, 20, 801812,  DOI: 10.1021/acs.biomac.8b01420
    22. 22
      Chalitangkoon, J.; Wongkittisin, M.; Monvisade, P. Silver Loaded Hydroxyethylacryl Chitosan/Sodium Alginate Hydrogel Films for Controlled Drug Release Wound Dressings. Int. J. Biol. Macromol. 2020, 159, 194203,  DOI: 10.1016/j.ijbiomac.2020.05.061
    23. 23
      Kan, Y.; Yang, Q.; Tan, Q.; Wei, Z.; Chen, Y. Diminishing Cohesion of Chitosan Films in Acidic Solution by Multivalent Metal Cations. Langmuir 2020, 36, 49644974,  DOI: 10.1021/acs.langmuir.0c00438
    24. 24
      Rivero, S.; García, M. A.; Pinotti, A. Crosslinking Capacity of Tannic Acid in Plasticized Chitosan Films. Carbohydr. Polym. 2010, 82, 270276,  DOI: 10.1016/j.carbpol.2010.04.048
    25. 25
      Blilid, S.; Kȩdzierska, M.; Miłowska, K.; Wrońska, N.; El Achaby, M.; Katir, N.; Belamie, E.; Alonso, B.; Lisowska, K.; Lahcini, M.; Bryszewska, M.; El Kadib, A. Phosphorylated Micro- A Nd Nanocellulose-Filled Chitosan Nanocomposites as Fully Sustainable, Biologically Active Bioplastics. ACS Sustainable Chem. Eng. 2020, 8, 1835418365,  DOI: 10.1021/acssuschemeng.0c04426
    26. 26
      Cui, Z.; Beach, E. S.; Anastas, P. T. Modification of Chitosan Films with Environmentally Benign Reagents for Increased Water Resistance. Green Chem. Lett. Rev. 2011, 4, 3540,  DOI: 10.1080/17518253.2010.500621
    27. 27
      Qiao, C.; Ma, X.; Wang, X.; Liu, L. Structure and Properties of Chitosan Films: Effect of the Type of Solvent Acid. LWT 2021, 135, 109984  DOI: 10.1016/j.lwt.2020.109984
    28. 28
      Yeng, C. M.; Salmah, H.; Ting, S. Corn Cob Filled Chitosan Biocomposite Films. Adv. Mater. Res. 2013, 747, 649652,  DOI: 10.4028/www.scientific.net/AMR.747.649
    29. 29
      Ghosh, A.; Ali, M. A. Studies on Physicochemical Characteristics of Chitosan Derivatives with Dicarboxylic Acids. J. Mater. Sci. 2012, 47, 11961204,  DOI: 10.1007/s10853-011-5885-x
    30. 30
      Altunkaynak, F.; Okur, M.; Saracoglu, N. Controlled Release of Paroxetine from Chitosan/Montmorillonite Composite Films. J. Drug Delivery Sci. Technol. 2022, 68, 103099  DOI: 10.1016/j.jddst.2022.103099
    31. 31
      Che, Y.; Li, D.; Liu, Y.; Ma, Q.; Tan, Y.; Yue, Q.; Meng, F. Physically Cross-Linked PH-Responsive Chitosan-Based Hydrogels with Enhanced Mechanical Performance for Controlled Drug Delivery. RSC Adv. 2016, 6, 106035106045,  DOI: 10.1039/c6ra16746b
    32. 32
      Nguyen, H. X.; Bozorg, B. D.; Kim, Y.; Wieber, A.; Birk, G.; Lubda, D.; Banga, A. K. Poly (Vinyl Alcohol) Microneedles: Fabrication, Characterization, and Application for Transdermal Drug Delivery of Doxorubicin. Eur. J. Pharm. Biopharm. 2018, 129, 88103,  DOI: 10.1016/j.ejpb.2018.05.017
    33. 33
      Kumar, B.; Jain, S. K.; Prajapati, S. K. Effect of Penetration Enhancer DMSO on In-Vitro Skin Permeation of Acyclovir Transdermal Microemulsion Formulation. Int. J. Drug Delivery 2011, 3, 8394,  DOI: 10.5138/ijdd.2010.0975.0215.03057
  • Supporting Information

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.2c06719.

    • UV-measurement of free DOX (Figure S1), cell viability test of hydrogels at different concentration AA (Figure S2), and relative spheroid volume after exposure (Figure S3)(PDF)


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STEP 1:
Click to create an ACS ID

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

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