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Drug-Loaded Biocompatible Chitosan Polymeric Films with Both Stretchability and Controlled Release for Drug Delivery
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  • 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]
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    Orcidhttps://orcid.org/0000-0002-4456-0128
  • Tomoyuki Tachibana
    Tomoyuki Tachibana
    Chemical Engineering Program, Graduate School of Advanced Science and Engineering, Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima739-8527, Japan
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  • Hijiri Wadamori
    Hijiri Wadamori
    Chemical Engineering Program, Graduate School of Advanced Science and Engineering, Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima739-8527, Japan
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  • Keita Yamana
    Keita Yamana
    Applied Chemistry Program, Graduate School of Advanced Science and Engineering, Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima739-8527, Japan
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  • Riku Kawasaki
    Riku Kawasaki
    Applied Chemistry Program, Graduate School of Advanced Science and Engineering, Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima739-8527, Japan
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  • Shogo Kawamura
    Shogo Kawamura
    Applied Chemistry Program, Graduate School of Advanced Science and Engineering, Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima739-8527, Japan
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  • Hinata Isozaki
    Hinata Isozaki
    Applied Chemistry Program, Graduate School of Advanced Science and Engineering, Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima739-8527, Japan
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  • Mina Sakuragi
    Mina Sakuragi
    Department of Nanoscience, Sojo University, 4-22-1 Ikeda, Nishi-ku, Kumamoto860-0082, Japan
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    Orcidhttps://orcid.org/0000-0002-6362-3673
  • Isamu Akiba
    Isamu Akiba
    Department of Chemistry and Biochemistry, Kitakyushu University, 1-1 Hibikino, Wakamatsu, Kitakyushu808-0135, Japan
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    Orcidhttps://orcid.org/0000-0003-3085-5555
  • 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]
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ACS Omega

Cite this: ACS Omega 2023, 8, 1, 1282–1290
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https://doi.org/10.1021/acsomega.2c06719
Published December 20, 2022

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Abstract

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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(%)=(L1−L0)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.

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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.

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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).

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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%.

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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.

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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).

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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).

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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.

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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).

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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.

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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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  • Corresponding Authors
    • Ji Ha Lee - Chemical Engineering Program, Graduate School of Advanced Science and Engineering, Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima739-8527, Japan;  Orcidhttps://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, Japan;  Orcidhttps://orcid.org/0000-0002-6362-3673
    • Isamu Akiba - Department of Chemistry and Biochemistry, Kitakyushu University, 1-1 Hibikino, Wakamatsu, Kitakyushu808-0135, Japan;  Orcidhttps://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

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This article references 33 other publications.

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    Adsorption of tetracycline (TC) and cefotaxime (CTX) by novel NiFe2O4-COF-chitosan-terephthalaldehyde nanocomposites film (NCCT) was explored. To assess the feasibility of NCCT as a potential adsorbent for TC and CTX removal, a series of adsorption expts. were conducted. These results showed that the chem. crosslink of chitosan with terephthalaldehyde (TPA) restricted NCCT degrdn. of acid. In addn., NCCT as a film adsorbent could be sepd. completely in only 2 s without mass loss, much faster than any other magnetic materials. The abundant functional groups of chitosan, COF and TPA could significantly improve the adsorption capacity of NCCT on antibiotics. The pseudo-second-order kinetics model better illustrated the adsorption of TC and CTX onto NCCT, and the max. adsorption capacity are 388.52 mg g-1 and 309.26 mg g-1, resp. The nanocomposites film NCCT also exhibit excellent reproducibility after six adsorption cycles. In addn., various anal. results implied the adsorption mechanism of TC on NCCT was mainly through complexation, cation exchange, electrostatic attraction, hydrogen bonding and the π-π interaction. For CTX, the mechanism of adsorption included condensation reaction, electrostatic attraction, hydrogen bonding and π-π interaction. This kind of high-efficiency and novel nanocomposites film adsorbents can be ultrafastly sepd. from water, verifying NCCT has huge potential in water treatment for antibiotic contaminants removal.
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    Depending on the modifications proposed, chitosan films present different characteristics, for instance correlated to hydrophilicity, chem. and mech. properties. The aim of this study was to evaluate the influence of glutaraldehyde crosslinking and an alk. post-treatment with NaOH on the characteristics of chitosan based films. Films were obtained by casting and characterized by thickness, swelling degree, mech. and thermal properties and chem. structure. The water vapor permeability (WVP) was also evaluated for food packaging application. It was obsd. that crosslinking and NaOH post-treatment have great influence on the chitosan films characteristics. Crosslinking reduced the swelling degree of films and increased its fragility, whereas NaOH treatment also reduces the swelling degree and changes mech. properties, acting in the same way as a crosslinker. The WVP analyses showed that the basic treatment could substitute the glutaraldehyde crosslinking for film water stability, without greatly compromising the barrier properties of chitosan based films.
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    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, 9145– 9152,  DOI: 10.1021/acssuschemeng.8b01468
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    Wu, Jimin; Su, Chen; Jiang, Lei; Ye, Shan; Liu, Xiufeng; Shao, Wei
    ACS Sustainable Chemistry & Engineering (2018), 6 (7), 9145-9152CODEN: ASCECG; ISSN:2168-0485. (American Chemical Society)
    The aim of this study was to prep. nonleaching chitosan-based wound dressings via chem. modification. A green and facile method was applied to fabricate ampicillin grafted chitosan (CSAP) sponges. The morphol., porosity, and swelling behavior of CSAP sponges were analyzed. Specifically, the antibacterial activity of CSAP sponges toward Staphylococcus aureus, Candida albicans, and Escherichia coli was assessed using a series of assays, including bacterial growth curve, nucleic acids leakage, and live/dead staining. As expected, CSAP sponges exhibit excellent antibacterial activity without any leaching. The cytotoxicity assay was carried out on HEK293 cells in vitro, and the result prove the good biocompatibility of the developed sponges. Moreover, the wound healing ability was evaluated using a wound model in vivo, and the result shows that the sponge could speed up wound healing efficiently. Thus, the chem. modified chitosan sponges exhibit great potential as promising wound dressings.
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    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
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    Vivcharenko, Vladyslav; Wojcik, Michal; Przekora, Agata
    Cells (2020), 9 (5), 1185CODEN: CELLC6; ISSN:2073-4409. (MDPI AG)
    The treatment of chronic wounds is still a meaningful challenge to physicians. The aim of this work was to produce vitamin C-enriched chitosan/agarose (CHN/A) film that could serve as potential artificial skin substitute for chronic wound treatment. The biomaterial was fabricated by a newly developed and simplified method via mixing acidic chitosan soln. with alk. agarose soln. that allowed to obtain slightly acidic pH (5.97) of the resultant material, which is known to support skin regeneration. Vitamin C was immobilized within the matrix of the film by entrapment method during prodn. process. Produced films (CHN/A and CHN/A + vit C) were subjected to comprehensive evaluation of cellular response with the use of human skin fibroblasts, epidermal keratinocytes, and macrophages. It was demonstrated that novel biomaterials support adhesion and growth of human skin fibroblasts and keratinocytes, have ability to slightly reduce transforming growth factor-beta 1 (TGF-β1) (known to be present at augmented levels in the epidermis of chronic wounds), and increase platelet-derived growth factor-BB (PDGF-BB) secretion by the cells. Nevertheless, addn. of vitamin C to the biomaterial formulation does not significantly improve its biol. properties due to burst vitamin release profile. Obtained results clearly demonstrated that produced CHN/A film has great potential to be used as cellular dermal, epidermal, or dermo-epidermal graft pre-seeded with human skin cells for chronic wound treatment.
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    Parvez, Shahed; Rahman, M. Mizanur; Khan, Mubarak A.; Khan, M. Anwar H.; Islam, Jahid M. M.; Ahmed, Mostak; Rahman, M. Fizur; Ahmed, Belal
    Polymer Bulletin (Heidelberg, Germany) (2012), 69 (6), 715-731CODEN: POBUDR; ISSN:0170-0839. (Springer)
    A bioadhesive wound-dressing material based on the combination of gelatin and chitosan with a proper ratio was developed and successfully applied in biomedical fields. The composite films were prepd. with increase in chitosan concn. in a fixed amt. of gelatin and were evaluated for mech. stability (e.g., tensile strength, elongation-at-break), water and buffer uptake capacity, water and buffer aging, mol. structure, morphol., thermal stability, and for biol. properties (e.g., antimicrobial activity, cytotoxicity, in vivo wound-healing performance). It is noteworthy that the 10:3 (gelatin:chitosan) composite films showed the best physico-mech., thermal, and antimicrobial properties among the other ratios blend films. The improved mech. and thermal stability of the 10:3 composite film suggested its promising use as carrier for controlled release drug. The composite film was evaluated using a rat model for in vivo tests to ascertain the applicability of the proper ratio of the chitosan and gelatin in the film for best wound-healing activity. Wound sites dresses with gelatin/chitosan composite films showed excellent rapid healing of the wound surface than those dressed with eco-plaster and gauze. Within a day after dressing with 10:3 composite film, the healing efficiency was found to be 80 %.
  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, 1963– 1969,  DOI: 10.1016/j.cclet.2021.11.047
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    In situ wound sprayable double-network hydrogel: Preparation and characterization
    Cai, Chenglong; Wang, Ting; Han, Xu; Yang, Shaoqiang; Lai, Chengteng; Yuan, Tao; Feng, Zhangqi; He, Nongyue
    Chinese Chemical Letters (2022), 33 (4), 1963-1969CODEN: CCLEE7; ISSN:1001-8417. (Elsevier B.V.)
    In clin. settings the wound-dressing was required easy to use and can match the wound area immediately, at the same time they need to have the properties of hemostats, anti-inflammation and promoting wound healing. To get an ideal wound dressing, we developed a type of gel-like wound adhesive patch from spraying double-network hydrogel, which own the properties of self-antibacterial and can promote wound healing. By spraying, the gel-like wound adhesive patch can match the wound area immediately and form a gel-film in 10 s. Sodium CM-cellulose as pH sensitive materials accelerated the speed to form the gel-film and enhanced ductility of the wound adhesive patch. In vitro expts. show that, this gel-like wound adhesive patch can promote cell proliferation and reduce cell apoptosis. In vivo studies show that, compared with commercialized wound dressings in clinic using, the spraying gel-like wound adhesive patch from our work has a better effect on wound healing. In conclusion, the spraying gel-like wound patch in our work is easy to use and can form a gel-film match on wound area in a short time, also it has the properties of hemostats, anti-inflammation and promoting wound healing. Its feasibility for mass prodn. shows a good potential for com. use.
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    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, 5630– 5642,  DOI: 10.1016/j.actbio.2012.10.032
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    A multilayered scaffold of a chitosan and gelatin hydrogel supported by a PCL core for cardiac tissue engineering
    Pok, Seokwon; Myers, Jackson D.; Madihally, Sundararajan V.; Jacot, Jeffrey G.
    Acta Biomaterialia (2013), 9 (3), 5630-5642CODEN: ABCICB; ISSN:1742-7061. (Elsevier Ltd.)
    A three-dimensional scaffold composed of self-assembled polycaprolactone (PCL) sandwiched in a gelatin-chitosan hydrogel was developed for use as a biodegradable patch with a potential for surgical reconstruction of congenital heart defects. The PCL core provides surgical handling, suturability and high initial tensile strength, while the gelatin-chitosan scaffold allows for cell attachment, with pore size and mech. properties conducive to cardiomyocyte migration and function. The ultimate tensile stress of the PCL core, made from blends of 10, 46 and 80 kDa (Mn) PCL, was controllable in the range of 2-4 MPa, with lower av. mol. wt. PCL blends correlating with lower tensile stress. Blends with lower mol. wt. PCL also had faster degrdn. (controllable from 0% to 7% wt. loss in saline over 30 days) and larger pores. PCL scaffolds supporting a gelatin-chitosan emulsion gel showed no significant alteration in tensile stress, strain or tensile modulus. However, the compressive modulus of the composite tissue was similar to that of native tissue (∼15 kPa for 50% gelatin and 50% chitosan). Electron microscopy revealed that the gelatin-chitosan gel had a three-dimensional porous structure, with a mean pore diam. of ∼80 μm, showed migration of neonatal rat ventricular myocytes (NRVM), maintained NRVM viability for over 7 days, and resulted in spontaneously beating scaffolds. This multi-layered scaffold has sufficient tensile strength and surgical handling for use as a cardiac patch, while allowing migration or pre-loading of cardiac cells in a biomimetic environment to allow for eventual degrdn. of the patch and incorporation into native tissue.
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    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
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    A review of mechanical properties of scaffold in tissue engineering: Aloe Vera composites
    Tran, T. T.; Hamid, Z. A.; Cheong, K. Y.
    Journal of Physics: Conference Series (2018), 1082 (Regional Conference on Materials and ASEAN Microscopy Conference 2017), 012080/1-012080/6CODEN: JPCSDZ; ISSN:1742-6588. (IOP Publishing Ltd.)
    A review. Aloe vera (AV) is a well-known pharmaceutical herb and traditional biomaterial from thousand years ago. AV is also a great potential material in tissue engineering because of its excellent properties such as biocompatible, biodegradable, antioxidant, anti-inflammatory, anti-diabetic, and antimicrobial. In tissue regeneration, scaffold plays an important role to form template for cell growth or tissue repair. There are some requirements in fabricating scaffold to mimic the structure of native parts of the body such as skin, cardiac, nerve, or bone. Here, mech. properties of AV aiming to be used as scaffold are reviewed. Tensile strength as the main mech. property of scaffold made from AV and incorporated with other natural materials (chitosan, alginate, collagen, etc.) and synthetic polymers (PCL, PVA, PLGA, etc.) will be discussed. The methods to measure mech. properties are also being presented in this brief review article. Finally, some of the perspectives will be given for the future development of AV in tissue engineering.
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    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, 801– 812,  DOI: 10.1021/acs.biomac.8b01420
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    Double Cross-Linked Chitosan Composite Films Developed with Oxidized Tannic Acid and Ferric Ions Exhibit High Strength and Excellent Water Resistance
    Yang, Jie; Li, Man; Wang, Yanfei; Wu, Hao; Zhen, Tianyuan; Xiong, Liu; Sun, Qingjie
    Biomacromolecules (2019), 20 (2), 801-812CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
    There is tremendous scientific interest in developing biodegradable films through facile and versatile strategies. Although extensive studies on the prepn. of chitosan films have been conducted, the reported results commonly present low mech. strength and weak water resistance. In the present study, high strength and significantly water resistance single-cross-linked chitosan-oxidized tannic acid (SC-CS/OTA) composite films and double cross-linked chitosan/oxidized tannic acid/FeIII (DC-CS/OTA/FeIII) composite films were created through a Schiff base reaction and metal coordination. As a result, the optimal tensile strength of SC-CS/OTA composite films and DC-CS/OTA/FeIII composite films was 35.92 and 209 MPa, resp. Notably, when compared with other chitosan-based films, the tensile strength of DC-CS/OTA/FeIII composite films was approx. three times stronger. Moreover, the water vapor permeability (WVP) values of the films with FeIII(0.66 ± 0.03 × 10-10 g/m·h·Pa) was lower than that of films without FeIII (1.33 ± 0.01 × 10-10 g/m·h·Pa). More importantly, WVP values of the DC-CS/OTA/FeIII composite films were 3-4 orders of magnitude lower than those of chitosan films previously reported. The SC-CS/OTA composite films (96.69%) and DC-CS/OTA/FeIII composite films (99.06%) also presented high DPPH radical scavenging activity. Furthermore, SC-CS/OTA and DC-CS/OTA/FeIII hydrogels were also prepd. This work can be widely applied in the food, biomedical science, and wastewater treatment fields.
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    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, 194– 203,  DOI: 10.1016/j.ijbiomac.2020.05.061
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    Silver loaded hydroxyethylacryl chitosan/sodium alginate hydrogel films for controlled drug release wound dressings
    Chalitangkoon, Jongjit; Wongkittisin, Marisa; Monvisade, Pathavuth
    International Journal of Biological Macromolecules (2020), 159 (), 194-203CODEN: IJBMDR; ISSN:0141-8130. (Elsevier B.V.)
    Wound dressings composed of hydroxyethylacryl chitosan (HC) and sodium alginate (SA) were developed with antibacterial activity by loading Ag particles. The formation of Ag particle in the HC/SA films was achieved by an immersion method through in situ chem. redn. of AgNO3 soln. and confirmed by FTIR, SEM-EDS, XRD and XRF techniques. The effect of Ag loading in the Ca-crosslinked HC/SA films with different crosslinking d. was studied on swelling behavior, mech. properties, cytotoxicity, antibacterial activity and drug release behavior. The results showed that Ag loading increased swelling degree in phosphate buffer and enhanced mech. properties. The HC/SA films with Ag loading exhibited antibacterial activity against E. coli and S. aureus as well as no toxicity on Vero cell. In vitro drug release profiles of the films were examd. using para-acetylaminophenol, as a sol. model drug. The increase in crosslinking d. and Ag loading prolonged drug releasing rate and almost the films showed linearity profiles. It can be concluded that the HC/SA films with Ag loading have a promising potential in modern wound dressings with antibacterial property and controlled drug release.
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    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, 4964– 4974,  DOI: 10.1021/acs.langmuir.0c00438
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    Diminishing Cohesion of Chitosan Films in Acidic Solution by Multivalent Metal Cations
    Kan, Yajing; Yang, Qiang; Tan, Qiyan; Wei, Zhiyong; Chen, Yunfei
    Langmuir (2020), 36 (18), 4964-4974CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
    Chitosan is a natural polymer with good biocompatibility, biodegradability, and bioactivity that has great potential for biomedical and industrial applications. Like other natural sugar-based polymers, chitosan mols. own versatile adhesion abilities to bind with various surfaces, owing to multiple functional moieties contained in the chain. To develop the promising biomaterials based on the chitosan chem., it is fundamentally important to figure out its adhesion mechanism under a certain condition, which leaves us nos. of open questions. In this work, we characterized the chitosan films adsorbed on a mica substrate in acidic soln. and investigated the effects of multivalent salts on the cohesive behaviors of the films by means of the surface forces app. The results showed that the cohesion capacities of chitosan films were reduced to around 30% of their original states after the addn. of 10-7 M LaCl3 into 150 mM acetic acid, which could be partially recovered by holding the films at the contact position for a longer time. Surprisingly, the cohesion loss in the films exhibited the dependence on the properties of the metal cations including valance and concn. The topog. of the chitosan-coated surface also showed obvious aggregation in the presence of submicromolar of the salts. Here, we attributed these phenomena regarding cohesion loss to the mechanisms involved in the absorption of metal cations by the chitosan chains, which not only consumed the binding sites but also induced conformation change in the polymer network. Our findings may offer a suggestion for the prodn. of chitosan-based materials to notice the potential impacts of ultralow concd. salts that are usually neglected even under acidic conditions.
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    Rivero, S.; García, M. A.; Pinotti, A. Crosslinking Capacity of Tannic Acid in Plasticized Chitosan Films. Carbohydr. Polym. 2010, 82, 270– 276,  DOI: 10.1016/j.carbpol.2010.04.048
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    Crosslinking capacity of tannic acid in plasticized chitosan films
    Rivero, S.; Garcia, M. A.; Pinotti, A.
    Carbohydrate Polymers (2010), 82 (2), 270-276CODEN: CAPOD8; ISSN:0144-8617. (Elsevier Ltd.)
    Plasticized and unplasticized chitosan films with tannic acid addn. were developed. The crosslinking capacity of tannic acid was studied by evaluating both the structural modification produced on chitosan films and its effect on physicochem., barrier and mech. properties. Likewise the changes of these properties during the storage were analyzed; formulations contg. tannic acid increased 29% tensile strength whereas decreased 24% water vapor permeability. Film soly. decreased at 100 °C, confirming that exposure at high temps. facilitates the crosslinking process. The presence of tannic acid and glycerol simultaneously showed a synergic effect on the studied properties, all of which presented intermediate behavior in relation to those detd. with the addn. of either acid or plasticizer. The effect of the storage was also relevant since the films evidenced their tendency to a more stable structure due to the reorganization toward an anhyd. conformation, as was demonstrated by X-ray diffraction and FTIR anal.
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    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, 18354– 18365,  DOI: 10.1021/acssuschemeng.0c04426
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    Phosphorylated Micro- and Nanocellulose-Filled Chitosan Nanocomposites as Fully Sustainable, Biologically Active Bioplastics
    Blilid, Sara; Kedzierska, Marta; Milowska, Katarzyna; Wronska, Natalia; El Achaby, Mounir; Katir, Nadia; Belamie, Emmanuel; Alonso, Bruno; Lisowska, Katarzyna; Lahcini, Mohammed; Bryszewska, Maria; El Kadib, Abdelkrim
    ACS Sustainable Chemistry & Engineering (2020), 8 (50), 18354-18365CODEN: ASCECG; ISSN:2168-0485. (American Chemical Society)
    Controlled cellulose fragmentation and its downsizing to micro- and nanocrystals have recently captured tremendous attention to access sustainable nanomaterials. Hitherto, few functionalized cellulose derivs. have been used as fillers, and addnl. knowledge is needed to establish an accurate structure-performance relationship in the realm of sustainable nanocomposites. Herein, a range of phosphorylated microcellulose (MCC) and nanosized cellulose (CNC) have been prepd. and used as reinforcing fillers to build transparent and flexible chitosan-cellulose nanostructured films. Regardless of their functionalization, all nanocellulose fillers reach good dispersion in the matrix, while those that are microcellulose aggregate slightly inside of the films. Distinctively, improved thermal stability was seen for chitosan films reinforced with cyclotriphosphazene grafted on cellulose nanocrystals (PN-CNC), where only half wt. of the bioplastic was decompd. at 700°C. Moreover, better mech. properties were obtained using nanocellulose instead of microcellulose as fillers, with PN-CNC-filled chitosan reaching the highest value of 1.649 MPa in tensile modulus compared to 1.195 MPa for neat chitosan films. Phosphorylated cellulose fillers (P-CNC and P-MCC) also bring interesting antibacterial and intercellular catalase activities, compared to neat chitosan and unmodified cellulose-filled chitosan. In total, this study sheds light on the pivotal role of cellulose phosphorylation in improving the thermal, mech., and biol. properties of the next generation of rationally designed bioplastics. Fully sustainable bioplastics by merging chitosan as a soft matrix and phosphorylated (microsized and nanosized) cellulose as a filler.
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    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, 35– 40,  DOI: 10.1080/17518253.2010.500621
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    Modification of chitosan films with environmentally benign reagents for increased water resistance
    Cui, Zheng; Beach, Evan S.; Anastas, Paul T.
    Green Chemistry Letters and Reviews (2011), 4 (1), 35-40CODEN: GCLRAI; ISSN:1751-8253. (Taylor & Francis Ltd.)
    Chitosan is a non-toxic, renewable, abundant natural material with excellent film-forming properties. It is shown here that water absorption by chitosan films can be decreased by chem. modification with the bio-based reagents citric acid and glycerol. IR spectroscopy showed that citric acid reacted with chitosan amine groups to form an acyclic amide structure. Glycerol imparted flexibility and water repellency to the films. When soaked in water for six hours, a chitosan-citric acid-glycerol (1:1:9) film absorbed 44% water by wt. after initial exposure and remained unchanged for the duration of the expt. In comparison, a pure chitosan membrane initially absorbed 70% water and gradually increased to 96%. After soaking, the modified film swelled by only 11% compared to 42% for pure chitosan. Similar results were obsd. for water contact angle with the film surfaces. For the modified film, change in contact angle over time could be attributed solely to evapn., whereas the pure chitosan film absorbed the droplets. These results show that the properties of chitosan can be improved using environmentally benign reagents in accordance with green chem. principles.
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    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
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    Structure and properties of chitosan films: Effect of the type of solvent acid
    Qiao, Congde; Ma, Xianguang; Wang, Xujie; Liu, Libin
    LWT--Food Science and Technology (2021), 135 (), 109984CODEN: LSTWB3; ISSN:0023-6438. (Elsevier Ltd.)
    The properties of chitosan films can be modified by changing the solvent type, attributing to the different interaction patterns between chitosan and acids. However, little is known about how these interactions affect the structure and properties of chitosan films. In this work, the influence of acid type on the structure and properties of chitosan films was studied by Fourier transform IR spectroscopy (FTIR), X-ray diffraction (XRD) and differential scanning calorimetry (DSC). FTIR spectra showed that the ionic interactions and hydrogen bonding could occur between chitosan and acid ions. Structural anal. revealed that chitosan was partially cryst. in hydrochloric and acetic acid films, whereas it was amorphous in lactic and citric acid films. DSC result indicated that the glass transition temp. of chitosan was much lower in citric acid film with low water content than in all the other films, suggesting that the citrate ions interacted more strongly with chitosan. A melting transition appeared in hydrochloric and acetic acid films. In addn., the tensile strength of these films decreased with an increase in the vol. of acid. These observations indicate that the choice of a proper solvent for chitosan may be desirable for certain special applications.
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    Yeng, C. M.; Salmah, H.; Ting, S. Corn Cob Filled Chitosan Biocomposite Films. Adv. Mater. Res. 2013, 747, 649– 652,  DOI: 10.4028/www.scientific.net/AMR.747.649
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    Corn cob filled chitosan biocomposite films
    Yeng, Chan Ming; Husseinsyah, Salmah; Ting, Sam Sung
    Advanced Materials Research (Durnten-Zurich, Switzerland) (2013), 747 (Multi-Functional Materials and Structures IV), 649-652CODEN: AMREFI; ISSN:1662-8985. (Trans Tech Publications Ltd.)
    Recently, there has been renews interest in chitosan as materials in producing of biocomposite films. The chitosan (CS)/corn cob (CC) biocomposite films were prepd. by solvent casting method. The effect of CC content on tensile properties of CS/CC biocomposite films was studied. The tensile strength and elongation at break of CS/CC biocomposite films decreased as increasing of CC content. However, the increasing of CC content was increased the tensile modulus of CS/CC biocomposite films. SEM (SEM) was indicated that the deceasing of tensile properties was due to the poor interfacial adhesion between CC filler and CS matrix.
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    Ghosh, A.; Ali, M. A. Studies on Physicochemical Characteristics of Chitosan Derivatives with Dicarboxylic Acids. J. Mater. Sci. 2012, 47, 1196– 1204,  DOI: 10.1007/s10853-011-5885-x
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    Studies on physicochemical characteristics of chitosan derivatives with dicarboxylic acids
    Ghosh, Arun; Ali, M. Azam
    Journal of Materials Science (2012), 47 (3), 1196-1204CODEN: JMTSAS; ISSN:0022-2461. (Springer)
    Chitosan (N-deacetylated deriv. of chitin) was solubilized in different aq. dicarboxylic acid solns., including oxalic acid, malonic acid, adipic acid, azelaic acid and also in monocarboxylic acetic acid. These dicarboxylic acid solns. were used with the objective that they not only act as solvents but also enhance material properties of chitosan gel films through chem. crosslinking. The properties including conformational changes of chitosan, chem. interaction, and mech., morphol. and thermal characteristics of selected chitosan samples studied in this work. The CD study indicated that the intensity of the broad neg. transition of chitosan helical structure in the wavelength region of 190-230 nm decreased with decreasing the chain length of the dicarboxylic acids. The IR spectra revealed the formation of amide linkage between chitosan and carboxylic acids in solid state. The cross-sections of the films produced from malonic acid and acetic acid solns. of chitosan exhibited granular morphologies with different granule sizes and hill-valley-structures under at. force microscope. The chitosan/malonic acid film showed improved water resistance and decreased tensile properties compared with the chitosan/acetic acid and chitosan/adipic acid films. These phys. characteristics of chitosan/malonic acid film are attributed to the dual effects of malonic acid, which acts as a chem. cross-linker and also as a plasticizer. A strong glass transition (Tg) peak at 166 °C in differential scanning calorimetric anal. was obsd., indicating the possible plasticizing effect with malonic acid.
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    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
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    Controlled release of paroxetine from chitosan/montmorillonite composite films
    Altunkaynak, Funda; Okur, Mujgan; Saracoglu, Nurdan
    Journal of Drug Delivery Science and Technology (2022), 68 (), 103099CODEN: JDDSAL; ISSN:1773-2247. (Elsevier B.V.)
    Controlled drug delivery studies popular in pharmaceutical area; polymeric composites prepd. with clay has a great potential importance in drug release studies. The aim of the study is to produce biocoposite material suitable for controlled release of Paroxetine Hydrochloride. In this study, chitosan/clay/paroxetine (CS/MMT/PHH and CS/NaMMT/PHH) composite films were prepd. to investigate drug release properties of paroxetine (PHH). Films contg. various amts. of clay (MMT/NaMMT) (0, 0.1, 0.2, 0.4 g) and glycerol (0.25, 0.50) were prepd. by solvent casting method. The structural properties of drug-contg. and non-drug contg. films were characterized by FT-IR and SEM anal. The release studies of PHH were done in vitro and pH 7.4 and at 37°C temp. The highest percent of drug release was obsd. with CS/PHH film after 170 h (69%). It was obsd. that the drug release profiles of chitosan films contg. clay (MMT or NaMMT) were better than films without clay. In order to investigate the drug release mechanism, Korsmeyer-Peppas, Higuchi, Zero and First order kinetic models were used. It was detd. that release kinetics of the most of films fit the Korsmeyer-Peppas model, and according to this model, drug release occurs through two mechanisms, swelling-controlled and diffusion-controlled. It has been obsd. that all films contg. clay have long-term drug release. Increasing in clay ratio in the composite, caused decrease in drug release rate. PHH loaded CS/MMT/PHH and CS/NaMMT/PHH films showed steady and prolonged drug delivery. Results indicated that prepd. CS/MMT/PHH and CS/NaMMT/PHH films has a potential to act as suitable carrier for drugs.
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    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, 106035– 106045,  DOI: 10.1039/c6ra16746b
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    Physically cross-linked pH-responsive chitosan-based hydrogels with enhanced mechanical performance for controlled drug delivery
    Che, YuJu; Li, Dongping; Liu, Yulong; Ma, Qinglin; Tan, Yebang; Yue, Qinyan; Meng, Fanjun
    RSC Advances (2016), 6 (107), 106035-106045CODEN: RSCACL; ISSN:2046-2069. (Royal Society of Chemistry)
    A novel phys. cross-linked pH-responsive hydrogel with enhanced mech. performance (PCAD) was prepd. from chitosan (CS), acrylic acid (AA) and (2-dimethylamino) Et methacrylate (DMAEMA) via in situ free radical polymn. for controlled drug delivery. The successful fabrication of the hydrogels was verified by Fourier transform IR spectroscopy (FT-IR), X-ray diffraction (XRD), differential scanning calorimetry (DSC) and thermogravimetric anal. (TGA) measurements. SEM (SEM) and mech. analyses demonstrated that the morphol. and mech. behaviors of the resultant hydrogels were strongly affected by the content of AA and DMAEMA. Moreover, the swelling properties of these hydrogels were systematically investigated, and the results indicated that they exhibited strong pH sensitivity. The drug delivery applications of such fabricated hydrogels were further evaluated, from which Bovine serum albumin (BSA) and 5-fluorouracil (5-Fu) were chosen as the model drugs for in vitro release. The results showed that the amt. of 5-Fu and BSA released can be tuned by changing the compn. of the hydrogel and the pH of the medium. Toxicity assays confirmed that the blank hydrogels had negligible toxicity to normal cells, whereas the 5-Fu-loaded hydrogels remained high in cytotoxicity for LO2 and HepG-2 cancer cells. As seen from the results, PCAD hydrogels seem to have a potential application in drug-delivery systems controlled by the external pH value for cancer therapy.
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    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, 88– 103,  DOI: 10.1016/j.ejpb.2018.05.017
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    Poly (vinyl alcohol) microneedles: Fabrication, characterization, and application for transdermal drug delivery of doxorubicin
    Nguyen, Hiep X.; Bozorg, Behnam Dasht; Kim, Yujin; Wieber, Alena; Birk, Gudrun; Lubda, Dieter; Banga, Ajay K.
    European Journal of Pharmaceutics and Biopharmaceutics (2018), 129 (), 88-103CODEN: EJPBEL; ISSN:0939-6411. (Elsevier B.V.)
    Poly (vinyl alc.) microneedles were fabricated, characterized, and applied to enhance in vitro transdermal delivery of doxorubicin. The microneedles were fabricated using the micromolding technique with the drug load in different locations within the needle array. The polymer soln. was assessed for rheol. properties, drug dissoln., and chem. structure studies. Microneedles (unloaded) and drug-loaded microneedles were characterized by optical microscopy, fluorescent microscopy, SEM, and drug release kinetics. Successful microporation of dermatomed human cadaver skin was demonstrated by dye binding, pore uniformity, histol., confocal laser microscopy, and skin integrity studies. The microneedles-mediated transdermal delivery of doxorubicin was investigated using vertical Franz diffusion cells. The fabricated microneedles were sharp, strong, and uniform. In vitro permeation studies showed that the microneedle-treated skin (4351.55 ± 560.87 ng/sq.cm) provided a significantly greater drug permeability than the untreated group (0.00 ± 0.00 ng/sq.cm, n = 4, p < 0.01). The drug location within the needle array was found to affect the drug release profile as well as its permeation into and across human skin. Skin microporation achieved by poly (vinyl alc.) microneedles was found to enhance transdermal delivery of doxorubicin in vitro.
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    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, 83– 94,  DOI: 10.5138/ijdd.2010.0975.0215.03057
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    Effect of penetration enhancer DMSO on in-vitro skin permeation of Acyclovir transdermal microemulsion formulation
    Kumar, Brajesh; Jain, S. K.; Prajapati, S. K.
    International Journal of Drug Delivery (2011), 3 (), 83-94CODEN: IJDDC9; ISSN:0975-0215. (Advanced Research Journals)
    The aim of this research was to enhance the flux of transdermal drug delivery by using penetration enhancers DMSO. Skin penetration enhancers have been used to improve bioavailability and increase the range of drugs for which topical and transdermal delivery is a viable option which penetrate into skin to reversibly decrease the barrier resistance. Penetration enhancing activity of dimethylsulfoxide (DMSO) at 5% wt./wt. and 10% wt./wt. concn. were detd. in aq. soln. of ACV and in microemulsion formulations though calcn. of transdermal flux of ACV with Keshary Chein Frenz Diffusion cell by using wistar albino rat skin. The transdermal flux of formulations PD, PD5D, PD10D, ME1 and ME10D was found to be 2.47, 50.7529, 119.7691, 238.1432 and 266.6721μg/cm2/h. The flux of microemulsion formulation ME10D was found 266.6721 ± 8.49 μg/cm2/h. Which showed highest value and skin flux of the drug could be enhanced up to 107 fold compared to its aq. soln. by prepg. microemulsion ME10D. DMSO in microemulsion formulation is safe to the skin at 10% DMSO wt./wt.

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  • Abstract

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    Figure 1

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

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    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.

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    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).

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    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%.

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    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.

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    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).

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    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).

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    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.

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    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).

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    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.

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      We have developed polysaccharide composite films made of anionic polysaccharides and chitosan (CHI) by utilizing hot press techniques. In order to demonstrate the versatility of these films as cell scaffolds, the present study investigated the adhesion and proliferation of fibroblasts on composite films prepd. by using various kinds of anionic polysaccharides and that were modified with proteins. Cells were spread on heparin/CHI and alginic acid/CHI films and grew well, whereas those on chondroitin sulfate C (CS)/CHI and hyaluronic acid/CHI films were round in shape. The differences in adhesion and proliferation behaviors of cells could be explained by the differences in the biochem. function of the anionic polysaccharides and the phys. properties of the films such as morphol., storage modulus, ζ-potentials, and swelling ratios. Among them, the no. of cells on CS/CHI films remained almost unchanged. The mechanisms underlying growth suppression on CS/CHI films were investigated by using an integrin stimulator, the TNIIIA2 peptide, and platelet-derived growth factor-B. It was indicated that the growth suppression was due to the lack of fibronectin-integrin growth signaling. The surface modification of CS/CHI films with fibronectin promoted the adhesion and proliferation of cells. These results show that the chem. and phys. properties of the polysaccharide composite films, which resulted from the chem. species of anionic polysaccharides or surface modifications of the films, can modulate cell adhesion and proliferation properties thereon.
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      In this study, a stimuli-responsive, biodegradable and bioactive film was produced by blending cashew gum polysaccharide (CGP) and polyvinyl alc. (PVA). The film presented malleability and mech. properties enabling an easy handling. Wetting the film changed the optical property from opacity to levels of transparency higher than 70% and resulted in up to 2-fold increase in its superficial area. Different swelling indexes were obtained varying the pH of solvent, which allows classifying the CGP/PVA film as pH sensitive stimuli-responsive material. The bioactivity was achieved through covalent immobilization of papain, which remained active after storage of CGP/PVA-papain film for 24 h in the presence of buffer or in a dry form. These results evidenced that CGP/PVA-papain film is a very promising material for biomedical applications.
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      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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      Physicochemical features assessment of acemannan-based ternary blended films for biomedical purposes
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      Carbohydrate Polymers (2021), 257 (), 117601CODEN: CAPOD8; ISSN:0144-8617. (Elsevier Ltd.)
      The exploitation of natural origin macromols., as complex phys. mixts. or drugs, increases in biomedical or tissue engineering (TE) solns. Aloe Vera is a highly explored medicinal plant, from which the main polysaccharide is acemannan (ACE). The ACE combination with chitosan and alginate results in interactions that lead to mixed junction zones formation, predicting membrane functionality improvement. This work proposes the development and characterization of ACE-based blended films as a promising strategy to design a nature-derived bioactive platform. The results confirmed that stable complex polyelectrolyte structures were formed through different intermol. interactions. The films present good dimensional stability, flexibility, an adequate swelling ability with mostly radial water uptake, and a sustainable ACE release to the medium. Pos. biol. performance of the ACE-based blended films with L929 cells also suggested that they can be applied in TE solns., with the potential to act as bioactive topical platforms.
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      To develop a new potential scaffold, biocomposite nanofibrous membranes composed of poly(vinyl acetate) (PVAc)/polysaccharide were prepd. using an electrospinning process. The resulting nanofibrous membranes exhibited a fully interconnected pore structure and their av. diam. was influenced by a change of cond. and viscosity of the electrospinning solns. due to the addn. of polysaccharides. According to the X-ray diffraction (XRD) results, the cryst. phase of PVAc was hardly affected by the addn. of polysaccharides. However, the mech. properties were markedly changed by the addn. of polysaccharides. In addn., the water contact angle and zeta potential were slightly affected by the use of polysaccharides. Cytocompatibility results showed that cell attachment improved by the addn. of chitosan, while cell proliferation enormously increased by blending pullulan. Therefore, PVAc/polysaccharide biocomposite nanofibrous membranes appear to be promising biomaterials for tissue engineering.
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      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, 339– 354,  DOI: 10.1016/j.ijbiomac.2021.03.034
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      Newer guar gum ester/chicken feather keratin interact films for tissue engineering
      Das, Aatrayee; Das, Ankita; Basu, Aalok; Datta, Pallab; Gupta, Mradu; Mukherjee, Arup
      International Journal of Biological Macromolecules (2021), 180 (), 339-354CODEN: IJBMDR; ISSN:0141-8130. (Elsevier B.V.)
      This work intends to synthesis newer guar gum indole acetate ester and design film scaffolds based on protein-polysaccharide interactions for tissue engineering applications. Guar gum indole acetate(GGIA) was synthesized for the first time from guar gum in presence of aprotic solvent activated hofmeister ions. The newer biopolymer was fully characterized in FT-IR, 13C NMR, XRD and TGA anal. High DS (Degree of Substitution, DS = 0.61) GGIA was crosslinked with hydrolyzed keratin, extd. from chicken feather wastes. Films were synthesized from different biopolymer ratios and the surface chem. appeared interesting. Physicochem. properties for GGIA-keratin assocn. were notable. Fully bio-based films were non-cytotoxic and exhibited excellent biocompatibility for human dermal fibroblast cell cultivations. The film scaffold showed 63% porosity and the recorded tensile strength at break was 6.4 MPa. Furthermore, the standardised film exerted superior antimicrobial activity against both the Gram-pos. and Gram-neg. bacteria. MICs were recorded at 130 Μg/mL and 212 Μg/mL for E. coli and S. aureus resp. In summary, GGIA-keratin film scaffolds represented promising platforms for skin tissue engineering applications.
    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
      11
      Chitosan/polydopamine layer by layer self-assembled silk fibroin nanofibers for biomedical applications
      Ma, Xiao; Wu, Guomin; Dai, Fangfang; Li, Dan; Li, Hao; Zhang, Li; Deng, Hongbing
      Carbohydrate Polymers (2021), 251 (), 117058CODEN: CAPOD8; ISSN:0144-8617. (Elsevier Ltd.)
      Silk fibroin (SF) is increasingly needed in tissue engineering for its superior biocompatibility. However, the practical applications of pure SF biomaterials confront bacterial infection problems. In this study, chitosan (CS) and polydopamine (PDA) were introduced into electrospun nanofibrous SF mats through layer-by-layer self-assembly (LBL) to obtain enhanced antibacterial ability and cytocompatibility. The surface morphol. and compn. anal. confirmed the successful deposition. After depositing 15 bilayers, the tensile modulus of the mats in wet condition increased from 2.16 MPa (pristine SF mats) to 4.89 MPa. A trend towards better hydrophilicity performance was also recorded with more bilayers coating on the mats. Besides, LBL structured mats showed improved antibacterial ability of more than 98% against E. coli and S. aureus. In addn., advancement in biocompatibility was obsd. during the proliferation expt. of L929 cells. Overall, the deposition of CS and PDA may further expand the use of SF in biomedical field.
    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, 166– 172,  DOI: 10.1016/j.carbpol.2006.07.024
      12
      In vitro biocompatibility evaluations of hexanoylchitosan film
      Neamnark, Artphop; Sanchavanakit, Neeracha; Pavasant, Prasit; Bunaprasert, Tanom; Supaphol, Pitt; Rujiravanit, Ratana
      Carbohydrate Polymers (2007), 68 (1), 166-172CODEN: CAPOD8; ISSN:0144-8617. (Elsevier B.V.)
      The present contribution reports for the first time some in vitro biocompatibility evaluations of hexanoylchitosan (H-chitosan) for possible utilization in biomedical applications. The evaluations comprised the cytotoxicity testing and the attachment, proliferation, and spreading of L929, mouse connective tissue, fibroblast-like cells that were cultured on the surface of H-chitosan film in comparison with those on chitosan film. These films were fabricated by soln.-casting technique. Some thermal, physico-chem., and morphol. characteristics of H-chitosan film were also investigated. H-chitosan film exhibited two steps in the loss of its mass at 242 and 299°, resp., while chitosan film exhibited only one at 297°. The water contact angle on the surface of H-chitosan film was 76°, while that on the surface of chitosan counterpart was 71°, a result indicating the more hydrophobicity of H-chitosan film in comparison with the chitosan counterpart. Indirect cytotoxicity evaluation of H-chitosan film using L929 revealed non-toxicity of the film to the cells. Lastly, both the attachment and the proliferation of L929 cells on H-chitosan film were inferior to those on tissue-culture polystyrene plate (TCPS). The attachment of the cells on H-chitosan film was better than that on the chitosan counterpart at a short seeding time (i.e., <5 h), while the proliferation of the cells on H-chitosan film was better than that on the chitosan counterpart after 2 and 3 days in culture.
    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
      13
      Removal and adsorption mechanism of tetracycline and cefotaxime contaminants in water by NiFe2O4-COF-chitosan-terephthalaldehyde nanocomposites film
      Li, Zehao; Liu, Yinyin; Zou, Shuiyang; Lu, Chunbo; Bai, Hu; Mu, Haibo; Duan, Jinyou
      Chemical Engineering Journal (Amsterdam, Netherlands) (2020), 382 (), 123008CODEN: CMEJAJ; ISSN:1385-8947. (Elsevier B.V.)
      Adsorption of tetracycline (TC) and cefotaxime (CTX) by novel NiFe2O4-COF-chitosan-terephthalaldehyde nanocomposites film (NCCT) was explored. To assess the feasibility of NCCT as a potential adsorbent for TC and CTX removal, a series of adsorption expts. were conducted. These results showed that the chem. crosslink of chitosan with terephthalaldehyde (TPA) restricted NCCT degrdn. of acid. In addn., NCCT as a film adsorbent could be sepd. completely in only 2 s without mass loss, much faster than any other magnetic materials. The abundant functional groups of chitosan, COF and TPA could significantly improve the adsorption capacity of NCCT on antibiotics. The pseudo-second-order kinetics model better illustrated the adsorption of TC and CTX onto NCCT, and the max. adsorption capacity are 388.52 mg g-1 and 309.26 mg g-1, resp. The nanocomposites film NCCT also exhibit excellent reproducibility after six adsorption cycles. In addn., various anal. results implied the adsorption mechanism of TC on NCCT was mainly through complexation, cation exchange, electrostatic attraction, hydrogen bonding and the π-π interaction. For CTX, the mechanism of adsorption included condensation reaction, electrostatic attraction, hydrogen bonding and π-π interaction. This kind of high-efficiency and novel nanocomposites film adsorbents can be ultrafastly sepd. from water, verifying NCCT has huge potential in water treatment for antibiotic contaminants removal.
    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, 2748– 2757,  DOI: 10.1007/s10924-017-1166-3
      14
      Influence of Glutaraldehyde Crosslinking and Alkaline Post-treatment on the Properties of Chitosan-Based Films
      Frick, Julia Menegotto; Ambrosi, Alan; Pollo, Liliane Damaris; Tessaro, Isabel Cristina
      Journal of Polymers and the Environment (2018), 26 (7), 2748-2757CODEN: JPENFW; ISSN:1566-2543. (Springer)
      Depending on the modifications proposed, chitosan films present different characteristics, for instance correlated to hydrophilicity, chem. and mech. properties. The aim of this study was to evaluate the influence of glutaraldehyde crosslinking and an alk. post-treatment with NaOH on the characteristics of chitosan based films. Films were obtained by casting and characterized by thickness, swelling degree, mech. and thermal properties and chem. structure. The water vapor permeability (WVP) was also evaluated for food packaging application. It was obsd. that crosslinking and NaOH post-treatment have great influence on the chitosan films characteristics. Crosslinking reduced the swelling degree of films and increased its fragility, whereas NaOH treatment also reduces the swelling degree and changes mech. properties, acting in the same way as a crosslinker. The WVP analyses showed that the basic treatment could substitute the glutaraldehyde crosslinking for film water stability, without greatly compromising the barrier properties of chitosan based films.
    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, 9145– 9152,  DOI: 10.1021/acssuschemeng.8b01468
      15
      Green and Facile Preparation of Chitosan Sponges as Potential Wound Dressings
      Wu, Jimin; Su, Chen; Jiang, Lei; Ye, Shan; Liu, Xiufeng; Shao, Wei
      ACS Sustainable Chemistry & Engineering (2018), 6 (7), 9145-9152CODEN: ASCECG; ISSN:2168-0485. (American Chemical Society)
      The aim of this study was to prep. nonleaching chitosan-based wound dressings via chem. modification. A green and facile method was applied to fabricate ampicillin grafted chitosan (CSAP) sponges. The morphol., porosity, and swelling behavior of CSAP sponges were analyzed. Specifically, the antibacterial activity of CSAP sponges toward Staphylococcus aureus, Candida albicans, and Escherichia coli was assessed using a series of assays, including bacterial growth curve, nucleic acids leakage, and live/dead staining. As expected, CSAP sponges exhibit excellent antibacterial activity without any leaching. The cytotoxicity assay was carried out on HEK293 cells in vitro, and the result prove the good biocompatibility of the developed sponges. Moreover, the wound healing ability was evaluated using a wound model in vivo, and the result shows that the sponge could speed up wound healing efficiently. Thus, the chem. modified chitosan sponges exhibit great potential as promising wound dressings.
    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
      16
      Cellular response to vitamin C-enriched chitosan/agarose film with potential application as artificial skin substitute for chronic wound treatment
      Vivcharenko, Vladyslav; Wojcik, Michal; Przekora, Agata
      Cells (2020), 9 (5), 1185CODEN: CELLC6; ISSN:2073-4409. (MDPI AG)
      The treatment of chronic wounds is still a meaningful challenge to physicians. The aim of this work was to produce vitamin C-enriched chitosan/agarose (CHN/A) film that could serve as potential artificial skin substitute for chronic wound treatment. The biomaterial was fabricated by a newly developed and simplified method via mixing acidic chitosan soln. with alk. agarose soln. that allowed to obtain slightly acidic pH (5.97) of the resultant material, which is known to support skin regeneration. Vitamin C was immobilized within the matrix of the film by entrapment method during prodn. process. Produced films (CHN/A and CHN/A + vit C) were subjected to comprehensive evaluation of cellular response with the use of human skin fibroblasts, epidermal keratinocytes, and macrophages. It was demonstrated that novel biomaterials support adhesion and growth of human skin fibroblasts and keratinocytes, have ability to slightly reduce transforming growth factor-beta 1 (TGF-β1) (known to be present at augmented levels in the epidermis of chronic wounds), and increase platelet-derived growth factor-BB (PDGF-BB) secretion by the cells. Nevertheless, addn. of vitamin C to the biomaterial formulation does not significantly improve its biol. properties due to burst vitamin release profile. Obtained results clearly demonstrated that produced CHN/A film has great potential to be used as cellular dermal, epidermal, or dermo-epidermal graft pre-seeded with human skin cells for chronic wound treatment.
    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, 715– 731,  DOI: 10.1007/s00289-012-0761-7
      17
      Preparation and characterization of artificial skin using chitosan and gelatin composites for potential biomedical application
      Parvez, Shahed; Rahman, M. Mizanur; Khan, Mubarak A.; Khan, M. Anwar H.; Islam, Jahid M. M.; Ahmed, Mostak; Rahman, M. Fizur; Ahmed, Belal
      Polymer Bulletin (Heidelberg, Germany) (2012), 69 (6), 715-731CODEN: POBUDR; ISSN:0170-0839. (Springer)
      A bioadhesive wound-dressing material based on the combination of gelatin and chitosan with a proper ratio was developed and successfully applied in biomedical fields. The composite films were prepd. with increase in chitosan concn. in a fixed amt. of gelatin and were evaluated for mech. stability (e.g., tensile strength, elongation-at-break), water and buffer uptake capacity, water and buffer aging, mol. structure, morphol., thermal stability, and for biol. properties (e.g., antimicrobial activity, cytotoxicity, in vivo wound-healing performance). It is noteworthy that the 10:3 (gelatin:chitosan) composite films showed the best physico-mech., thermal, and antimicrobial properties among the other ratios blend films. The improved mech. and thermal stability of the 10:3 composite film suggested its promising use as carrier for controlled release drug. The composite film was evaluated using a rat model for in vivo tests to ascertain the applicability of the proper ratio of the chitosan and gelatin in the film for best wound-healing activity. Wound sites dresses with gelatin/chitosan composite films showed excellent rapid healing of the wound surface than those dressed with eco-plaster and gauze. Within a day after dressing with 10:3 composite film, the healing efficiency was found to be 80 %.
    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, 1963– 1969,  DOI: 10.1016/j.cclet.2021.11.047
      18
      In situ wound sprayable double-network hydrogel: Preparation and characterization
      Cai, Chenglong; Wang, Ting; Han, Xu; Yang, Shaoqiang; Lai, Chengteng; Yuan, Tao; Feng, Zhangqi; He, Nongyue
      Chinese Chemical Letters (2022), 33 (4), 1963-1969CODEN: CCLEE7; ISSN:1001-8417. (Elsevier B.V.)
      In clin. settings the wound-dressing was required easy to use and can match the wound area immediately, at the same time they need to have the properties of hemostats, anti-inflammation and promoting wound healing. To get an ideal wound dressing, we developed a type of gel-like wound adhesive patch from spraying double-network hydrogel, which own the properties of self-antibacterial and can promote wound healing. By spraying, the gel-like wound adhesive patch can match the wound area immediately and form a gel-film in 10 s. Sodium CM-cellulose as pH sensitive materials accelerated the speed to form the gel-film and enhanced ductility of the wound adhesive patch. In vitro expts. show that, this gel-like wound adhesive patch can promote cell proliferation and reduce cell apoptosis. In vivo studies show that, compared with commercialized wound dressings in clinic using, the spraying gel-like wound adhesive patch from our work has a better effect on wound healing. In conclusion, the spraying gel-like wound patch in our work is easy to use and can form a gel-film match on wound area in a short time, also it has the properties of hemostats, anti-inflammation and promoting wound healing. Its feasibility for mass prodn. shows a good potential for com. use.
    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, 5630– 5642,  DOI: 10.1016/j.actbio.2012.10.032
      19
      A multilayered scaffold of a chitosan and gelatin hydrogel supported by a PCL core for cardiac tissue engineering
      Pok, Seokwon; Myers, Jackson D.; Madihally, Sundararajan V.; Jacot, Jeffrey G.
      Acta Biomaterialia (2013), 9 (3), 5630-5642CODEN: ABCICB; ISSN:1742-7061. (Elsevier Ltd.)
      A three-dimensional scaffold composed of self-assembled polycaprolactone (PCL) sandwiched in a gelatin-chitosan hydrogel was developed for use as a biodegradable patch with a potential for surgical reconstruction of congenital heart defects. The PCL core provides surgical handling, suturability and high initial tensile strength, while the gelatin-chitosan scaffold allows for cell attachment, with pore size and mech. properties conducive to cardiomyocyte migration and function. The ultimate tensile stress of the PCL core, made from blends of 10, 46 and 80 kDa (Mn) PCL, was controllable in the range of 2-4 MPa, with lower av. mol. wt. PCL blends correlating with lower tensile stress. Blends with lower mol. wt. PCL also had faster degrdn. (controllable from 0% to 7% wt. loss in saline over 30 days) and larger pores. PCL scaffolds supporting a gelatin-chitosan emulsion gel showed no significant alteration in tensile stress, strain or tensile modulus. However, the compressive modulus of the composite tissue was similar to that of native tissue (∼15 kPa for 50% gelatin and 50% chitosan). Electron microscopy revealed that the gelatin-chitosan gel had a three-dimensional porous structure, with a mean pore diam. of ∼80 μm, showed migration of neonatal rat ventricular myocytes (NRVM), maintained NRVM viability for over 7 days, and resulted in spontaneously beating scaffolds. This multi-layered scaffold has sufficient tensile strength and surgical handling for use as a cardiac patch, while allowing migration or pre-loading of cardiac cells in a biomimetic environment to allow for eventual degrdn. of the patch and incorporation into native tissue.
    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
      20
      A review of mechanical properties of scaffold in tissue engineering: Aloe Vera composites
      Tran, T. T.; Hamid, Z. A.; Cheong, K. Y.
      Journal of Physics: Conference Series (2018), 1082 (Regional Conference on Materials and ASEAN Microscopy Conference 2017), 012080/1-012080/6CODEN: JPCSDZ; ISSN:1742-6588. (IOP Publishing Ltd.)
      A review. Aloe vera (AV) is a well-known pharmaceutical herb and traditional biomaterial from thousand years ago. AV is also a great potential material in tissue engineering because of its excellent properties such as biocompatible, biodegradable, antioxidant, anti-inflammatory, anti-diabetic, and antimicrobial. In tissue regeneration, scaffold plays an important role to form template for cell growth or tissue repair. There are some requirements in fabricating scaffold to mimic the structure of native parts of the body such as skin, cardiac, nerve, or bone. Here, mech. properties of AV aiming to be used as scaffold are reviewed. Tensile strength as the main mech. property of scaffold made from AV and incorporated with other natural materials (chitosan, alginate, collagen, etc.) and synthetic polymers (PCL, PVA, PLGA, etc.) will be discussed. The methods to measure mech. properties are also being presented in this brief review article. Finally, some of the perspectives will be given for the future development of AV in tissue engineering.
    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, 801– 812,  DOI: 10.1021/acs.biomac.8b01420
      21
      Double Cross-Linked Chitosan Composite Films Developed with Oxidized Tannic Acid and Ferric Ions Exhibit High Strength and Excellent Water Resistance
      Yang, Jie; Li, Man; Wang, Yanfei; Wu, Hao; Zhen, Tianyuan; Xiong, Liu; Sun, Qingjie
      Biomacromolecules (2019), 20 (2), 801-812CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
      There is tremendous scientific interest in developing biodegradable films through facile and versatile strategies. Although extensive studies on the prepn. of chitosan films have been conducted, the reported results commonly present low mech. strength and weak water resistance. In the present study, high strength and significantly water resistance single-cross-linked chitosan-oxidized tannic acid (SC-CS/OTA) composite films and double cross-linked chitosan/oxidized tannic acid/FeIII (DC-CS/OTA/FeIII) composite films were created through a Schiff base reaction and metal coordination. As a result, the optimal tensile strength of SC-CS/OTA composite films and DC-CS/OTA/FeIII composite films was 35.92 and 209 MPa, resp. Notably, when compared with other chitosan-based films, the tensile strength of DC-CS/OTA/FeIII composite films was approx. three times stronger. Moreover, the water vapor permeability (WVP) values of the films with FeIII(0.66 ± 0.03 × 10-10 g/m·h·Pa) was lower than that of films without FeIII (1.33 ± 0.01 × 10-10 g/m·h·Pa). More importantly, WVP values of the DC-CS/OTA/FeIII composite films were 3-4 orders of magnitude lower than those of chitosan films previously reported. The SC-CS/OTA composite films (96.69%) and DC-CS/OTA/FeIII composite films (99.06%) also presented high DPPH radical scavenging activity. Furthermore, SC-CS/OTA and DC-CS/OTA/FeIII hydrogels were also prepd. This work can be widely applied in the food, biomedical science, and wastewater treatment fields.
    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, 194– 203,  DOI: 10.1016/j.ijbiomac.2020.05.061
      22
      Silver loaded hydroxyethylacryl chitosan/sodium alginate hydrogel films for controlled drug release wound dressings
      Chalitangkoon, Jongjit; Wongkittisin, Marisa; Monvisade, Pathavuth
      International Journal of Biological Macromolecules (2020), 159 (), 194-203CODEN: IJBMDR; ISSN:0141-8130. (Elsevier B.V.)
      Wound dressings composed of hydroxyethylacryl chitosan (HC) and sodium alginate (SA) were developed with antibacterial activity by loading Ag particles. The formation of Ag particle in the HC/SA films was achieved by an immersion method through in situ chem. redn. of AgNO3 soln. and confirmed by FTIR, SEM-EDS, XRD and XRF techniques. The effect of Ag loading in the Ca-crosslinked HC/SA films with different crosslinking d. was studied on swelling behavior, mech. properties, cytotoxicity, antibacterial activity and drug release behavior. The results showed that Ag loading increased swelling degree in phosphate buffer and enhanced mech. properties. The HC/SA films with Ag loading exhibited antibacterial activity against E. coli and S. aureus as well as no toxicity on Vero cell. In vitro drug release profiles of the films were examd. using para-acetylaminophenol, as a sol. model drug. The increase in crosslinking d. and Ag loading prolonged drug releasing rate and almost the films showed linearity profiles. It can be concluded that the HC/SA films with Ag loading have a promising potential in modern wound dressings with antibacterial property and controlled drug release.
    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, 4964– 4974,  DOI: 10.1021/acs.langmuir.0c00438
      23
      Diminishing Cohesion of Chitosan Films in Acidic Solution by Multivalent Metal Cations
      Kan, Yajing; Yang, Qiang; Tan, Qiyan; Wei, Zhiyong; Chen, Yunfei
      Langmuir (2020), 36 (18), 4964-4974CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
      Chitosan is a natural polymer with good biocompatibility, biodegradability, and bioactivity that has great potential for biomedical and industrial applications. Like other natural sugar-based polymers, chitosan mols. own versatile adhesion abilities to bind with various surfaces, owing to multiple functional moieties contained in the chain. To develop the promising biomaterials based on the chitosan chem., it is fundamentally important to figure out its adhesion mechanism under a certain condition, which leaves us nos. of open questions. In this work, we characterized the chitosan films adsorbed on a mica substrate in acidic soln. and investigated the effects of multivalent salts on the cohesive behaviors of the films by means of the surface forces app. The results showed that the cohesion capacities of chitosan films were reduced to around 30% of their original states after the addn. of 10-7 M LaCl3 into 150 mM acetic acid, which could be partially recovered by holding the films at the contact position for a longer time. Surprisingly, the cohesion loss in the films exhibited the dependence on the properties of the metal cations including valance and concn. The topog. of the chitosan-coated surface also showed obvious aggregation in the presence of submicromolar of the salts. Here, we attributed these phenomena regarding cohesion loss to the mechanisms involved in the absorption of metal cations by the chitosan chains, which not only consumed the binding sites but also induced conformation change in the polymer network. Our findings may offer a suggestion for the prodn. of chitosan-based materials to notice the potential impacts of ultralow concd. salts that are usually neglected even under acidic conditions.
    24. 24
      Rivero, S.; García, M. A.; Pinotti, A. Crosslinking Capacity of Tannic Acid in Plasticized Chitosan Films. Carbohydr. Polym. 2010, 82, 270– 276,  DOI: 10.1016/j.carbpol.2010.04.048
      24
      Crosslinking capacity of tannic acid in plasticized chitosan films
      Rivero, S.; Garcia, M. A.; Pinotti, A.
      Carbohydrate Polymers (2010), 82 (2), 270-276CODEN: CAPOD8; ISSN:0144-8617. (Elsevier Ltd.)
      Plasticized and unplasticized chitosan films with tannic acid addn. were developed. The crosslinking capacity of tannic acid was studied by evaluating both the structural modification produced on chitosan films and its effect on physicochem., barrier and mech. properties. Likewise the changes of these properties during the storage were analyzed; formulations contg. tannic acid increased 29% tensile strength whereas decreased 24% water vapor permeability. Film soly. decreased at 100 °C, confirming that exposure at high temps. facilitates the crosslinking process. The presence of tannic acid and glycerol simultaneously showed a synergic effect on the studied properties, all of which presented intermediate behavior in relation to those detd. with the addn. of either acid or plasticizer. The effect of the storage was also relevant since the films evidenced their tendency to a more stable structure due to the reorganization toward an anhyd. conformation, as was demonstrated by X-ray diffraction and FTIR anal.
    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, 18354– 18365,  DOI: 10.1021/acssuschemeng.0c04426
      25
      Phosphorylated Micro- and Nanocellulose-Filled Chitosan Nanocomposites as Fully Sustainable, Biologically Active Bioplastics
      Blilid, Sara; Kedzierska, Marta; Milowska, Katarzyna; Wronska, Natalia; El Achaby, Mounir; Katir, Nadia; Belamie, Emmanuel; Alonso, Bruno; Lisowska, Katarzyna; Lahcini, Mohammed; Bryszewska, Maria; El Kadib, Abdelkrim
      ACS Sustainable Chemistry & Engineering (2020), 8 (50), 18354-18365CODEN: ASCECG; ISSN:2168-0485. (American Chemical Society)
      Controlled cellulose fragmentation and its downsizing to micro- and nanocrystals have recently captured tremendous attention to access sustainable nanomaterials. Hitherto, few functionalized cellulose derivs. have been used as fillers, and addnl. knowledge is needed to establish an accurate structure-performance relationship in the realm of sustainable nanocomposites. Herein, a range of phosphorylated microcellulose (MCC) and nanosized cellulose (CNC) have been prepd. and used as reinforcing fillers to build transparent and flexible chitosan-cellulose nanostructured films. Regardless of their functionalization, all nanocellulose fillers reach good dispersion in the matrix, while those that are microcellulose aggregate slightly inside of the films. Distinctively, improved thermal stability was seen for chitosan films reinforced with cyclotriphosphazene grafted on cellulose nanocrystals (PN-CNC), where only half wt. of the bioplastic was decompd. at 700°C. Moreover, better mech. properties were obtained using nanocellulose instead of microcellulose as fillers, with PN-CNC-filled chitosan reaching the highest value of 1.649 MPa in tensile modulus compared to 1.195 MPa for neat chitosan films. Phosphorylated cellulose fillers (P-CNC and P-MCC) also bring interesting antibacterial and intercellular catalase activities, compared to neat chitosan and unmodified cellulose-filled chitosan. In total, this study sheds light on the pivotal role of cellulose phosphorylation in improving the thermal, mech., and biol. properties of the next generation of rationally designed bioplastics. Fully sustainable bioplastics by merging chitosan as a soft matrix and phosphorylated (microsized and nanosized) cellulose as a filler.
    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, 35– 40,  DOI: 10.1080/17518253.2010.500621
      26
      Modification of chitosan films with environmentally benign reagents for increased water resistance
      Cui, Zheng; Beach, Evan S.; Anastas, Paul T.
      Green Chemistry Letters and Reviews (2011), 4 (1), 35-40CODEN: GCLRAI; ISSN:1751-8253. (Taylor & Francis Ltd.)
      Chitosan is a non-toxic, renewable, abundant natural material with excellent film-forming properties. It is shown here that water absorption by chitosan films can be decreased by chem. modification with the bio-based reagents citric acid and glycerol. IR spectroscopy showed that citric acid reacted with chitosan amine groups to form an acyclic amide structure. Glycerol imparted flexibility and water repellency to the films. When soaked in water for six hours, a chitosan-citric acid-glycerol (1:1:9) film absorbed 44% water by wt. after initial exposure and remained unchanged for the duration of the expt. In comparison, a pure chitosan membrane initially absorbed 70% water and gradually increased to 96%. After soaking, the modified film swelled by only 11% compared to 42% for pure chitosan. Similar results were obsd. for water contact angle with the film surfaces. For the modified film, change in contact angle over time could be attributed solely to evapn., whereas the pure chitosan film absorbed the droplets. These results show that the properties of chitosan can be improved using environmentally benign reagents in accordance with green chem. principles.
    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
      27
      Structure and properties of chitosan films: Effect of the type of solvent acid
      Qiao, Congde; Ma, Xianguang; Wang, Xujie; Liu, Libin
      LWT--Food Science and Technology (2021), 135 (), 109984CODEN: LSTWB3; ISSN:0023-6438. (Elsevier Ltd.)
      The properties of chitosan films can be modified by changing the solvent type, attributing to the different interaction patterns between chitosan and acids. However, little is known about how these interactions affect the structure and properties of chitosan films. In this work, the influence of acid type on the structure and properties of chitosan films was studied by Fourier transform IR spectroscopy (FTIR), X-ray diffraction (XRD) and differential scanning calorimetry (DSC). FTIR spectra showed that the ionic interactions and hydrogen bonding could occur between chitosan and acid ions. Structural anal. revealed that chitosan was partially cryst. in hydrochloric and acetic acid films, whereas it was amorphous in lactic and citric acid films. DSC result indicated that the glass transition temp. of chitosan was much lower in citric acid film with low water content than in all the other films, suggesting that the citrate ions interacted more strongly with chitosan. A melting transition appeared in hydrochloric and acetic acid films. In addn., the tensile strength of these films decreased with an increase in the vol. of acid. These observations indicate that the choice of a proper solvent for chitosan may be desirable for certain special applications.
    28. 28
      Yeng, C. M.; Salmah, H.; Ting, S. Corn Cob Filled Chitosan Biocomposite Films. Adv. Mater. Res. 2013, 747, 649– 652,  DOI: 10.4028/www.scientific.net/AMR.747.649
      28
      Corn cob filled chitosan biocomposite films
      Yeng, Chan Ming; Husseinsyah, Salmah; Ting, Sam Sung
      Advanced Materials Research (Durnten-Zurich, Switzerland) (2013), 747 (Multi-Functional Materials and Structures IV), 649-652CODEN: AMREFI; ISSN:1662-8985. (Trans Tech Publications Ltd.)
      Recently, there has been renews interest in chitosan as materials in producing of biocomposite films. The chitosan (CS)/corn cob (CC) biocomposite films were prepd. by solvent casting method. The effect of CC content on tensile properties of CS/CC biocomposite films was studied. The tensile strength and elongation at break of CS/CC biocomposite films decreased as increasing of CC content. However, the increasing of CC content was increased the tensile modulus of CS/CC biocomposite films. SEM (SEM) was indicated that the deceasing of tensile properties was due to the poor interfacial adhesion between CC filler and CS matrix.
    29. 29
      Ghosh, A.; Ali, M. A. Studies on Physicochemical Characteristics of Chitosan Derivatives with Dicarboxylic Acids. J. Mater. Sci. 2012, 47, 1196– 1204,  DOI: 10.1007/s10853-011-5885-x
      29
      Studies on physicochemical characteristics of chitosan derivatives with dicarboxylic acids
      Ghosh, Arun; Ali, M. Azam
      Journal of Materials Science (2012), 47 (3), 1196-1204CODEN: JMTSAS; ISSN:0022-2461. (Springer)
      Chitosan (N-deacetylated deriv. of chitin) was solubilized in different aq. dicarboxylic acid solns., including oxalic acid, malonic acid, adipic acid, azelaic acid and also in monocarboxylic acetic acid. These dicarboxylic acid solns. were used with the objective that they not only act as solvents but also enhance material properties of chitosan gel films through chem. crosslinking. The properties including conformational changes of chitosan, chem. interaction, and mech., morphol. and thermal characteristics of selected chitosan samples studied in this work. The CD study indicated that the intensity of the broad neg. transition of chitosan helical structure in the wavelength region of 190-230 nm decreased with decreasing the chain length of the dicarboxylic acids. The IR spectra revealed the formation of amide linkage between chitosan and carboxylic acids in solid state. The cross-sections of the films produced from malonic acid and acetic acid solns. of chitosan exhibited granular morphologies with different granule sizes and hill-valley-structures under at. force microscope. The chitosan/malonic acid film showed improved water resistance and decreased tensile properties compared with the chitosan/acetic acid and chitosan/adipic acid films. These phys. characteristics of chitosan/malonic acid film are attributed to the dual effects of malonic acid, which acts as a chem. cross-linker and also as a plasticizer. A strong glass transition (Tg) peak at 166 °C in differential scanning calorimetric anal. was obsd., indicating the possible plasticizing effect with malonic acid.
    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
      30
      Controlled release of paroxetine from chitosan/montmorillonite composite films
      Altunkaynak, Funda; Okur, Mujgan; Saracoglu, Nurdan
      Journal of Drug Delivery Science and Technology (2022), 68 (), 103099CODEN: JDDSAL; ISSN:1773-2247. (Elsevier B.V.)
      Controlled drug delivery studies popular in pharmaceutical area; polymeric composites prepd. with clay has a great potential importance in drug release studies. The aim of the study is to produce biocoposite material suitable for controlled release of Paroxetine Hydrochloride. In this study, chitosan/clay/paroxetine (CS/MMT/PHH and CS/NaMMT/PHH) composite films were prepd. to investigate drug release properties of paroxetine (PHH). Films contg. various amts. of clay (MMT/NaMMT) (0, 0.1, 0.2, 0.4 g) and glycerol (0.25, 0.50) were prepd. by solvent casting method. The structural properties of drug-contg. and non-drug contg. films were characterized by FT-IR and SEM anal. The release studies of PHH were done in vitro and pH 7.4 and at 37°C temp. The highest percent of drug release was obsd. with CS/PHH film after 170 h (69%). It was obsd. that the drug release profiles of chitosan films contg. clay (MMT or NaMMT) were better than films without clay. In order to investigate the drug release mechanism, Korsmeyer-Peppas, Higuchi, Zero and First order kinetic models were used. It was detd. that release kinetics of the most of films fit the Korsmeyer-Peppas model, and according to this model, drug release occurs through two mechanisms, swelling-controlled and diffusion-controlled. It has been obsd. that all films contg. clay have long-term drug release. Increasing in clay ratio in the composite, caused decrease in drug release rate. PHH loaded CS/MMT/PHH and CS/NaMMT/PHH films showed steady and prolonged drug delivery. Results indicated that prepd. CS/MMT/PHH and CS/NaMMT/PHH films has a potential to act as suitable carrier for drugs.
    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, 106035– 106045,  DOI: 10.1039/c6ra16746b
      31
      Physically cross-linked pH-responsive chitosan-based hydrogels with enhanced mechanical performance for controlled drug delivery
      Che, YuJu; Li, Dongping; Liu, Yulong; Ma, Qinglin; Tan, Yebang; Yue, Qinyan; Meng, Fanjun
      RSC Advances (2016), 6 (107), 106035-106045CODEN: RSCACL; ISSN:2046-2069. (Royal Society of Chemistry)
      A novel phys. cross-linked pH-responsive hydrogel with enhanced mech. performance (PCAD) was prepd. from chitosan (CS), acrylic acid (AA) and (2-dimethylamino) Et methacrylate (DMAEMA) via in situ free radical polymn. for controlled drug delivery. The successful fabrication of the hydrogels was verified by Fourier transform IR spectroscopy (FT-IR), X-ray diffraction (XRD), differential scanning calorimetry (DSC) and thermogravimetric anal. (TGA) measurements. SEM (SEM) and mech. analyses demonstrated that the morphol. and mech. behaviors of the resultant hydrogels were strongly affected by the content of AA and DMAEMA. Moreover, the swelling properties of these hydrogels were systematically investigated, and the results indicated that they exhibited strong pH sensitivity. The drug delivery applications of such fabricated hydrogels were further evaluated, from which Bovine serum albumin (BSA) and 5-fluorouracil (5-Fu) were chosen as the model drugs for in vitro release. The results showed that the amt. of 5-Fu and BSA released can be tuned by changing the compn. of the hydrogel and the pH of the medium. Toxicity assays confirmed that the blank hydrogels had negligible toxicity to normal cells, whereas the 5-Fu-loaded hydrogels remained high in cytotoxicity for LO2 and HepG-2 cancer cells. As seen from the results, PCAD hydrogels seem to have a potential application in drug-delivery systems controlled by the external pH value for cancer therapy.
    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, 88– 103,  DOI: 10.1016/j.ejpb.2018.05.017
      32
      Poly (vinyl alcohol) microneedles: Fabrication, characterization, and application for transdermal drug delivery of doxorubicin
      Nguyen, Hiep X.; Bozorg, Behnam Dasht; Kim, Yujin; Wieber, Alena; Birk, Gudrun; Lubda, Dieter; Banga, Ajay K.
      European Journal of Pharmaceutics and Biopharmaceutics (2018), 129 (), 88-103CODEN: EJPBEL; ISSN:0939-6411. (Elsevier B.V.)
      Poly (vinyl alc.) microneedles were fabricated, characterized, and applied to enhance in vitro transdermal delivery of doxorubicin. The microneedles were fabricated using the micromolding technique with the drug load in different locations within the needle array. The polymer soln. was assessed for rheol. properties, drug dissoln., and chem. structure studies. Microneedles (unloaded) and drug-loaded microneedles were characterized by optical microscopy, fluorescent microscopy, SEM, and drug release kinetics. Successful microporation of dermatomed human cadaver skin was demonstrated by dye binding, pore uniformity, histol., confocal laser microscopy, and skin integrity studies. The microneedles-mediated transdermal delivery of doxorubicin was investigated using vertical Franz diffusion cells. The fabricated microneedles were sharp, strong, and uniform. In vitro permeation studies showed that the microneedle-treated skin (4351.55 ± 560.87 ng/sq.cm) provided a significantly greater drug permeability than the untreated group (0.00 ± 0.00 ng/sq.cm, n = 4, p < 0.01). The drug location within the needle array was found to affect the drug release profile as well as its permeation into and across human skin. Skin microporation achieved by poly (vinyl alc.) microneedles was found to enhance transdermal delivery of doxorubicin in vitro.
    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, 83– 94,  DOI: 10.5138/ijdd.2010.0975.0215.03057
      33
      Effect of penetration enhancer DMSO on in-vitro skin permeation of Acyclovir transdermal microemulsion formulation
      Kumar, Brajesh; Jain, S. K.; Prajapati, S. K.
      International Journal of Drug Delivery (2011), 3 (), 83-94CODEN: IJDDC9; ISSN:0975-0215. (Advanced Research Journals)
      The aim of this research was to enhance the flux of transdermal drug delivery by using penetration enhancers DMSO. Skin penetration enhancers have been used to improve bioavailability and increase the range of drugs for which topical and transdermal delivery is a viable option which penetrate into skin to reversibly decrease the barrier resistance. Penetration enhancing activity of dimethylsulfoxide (DMSO) at 5% wt./wt. and 10% wt./wt. concn. were detd. in aq. soln. of ACV and in microemulsion formulations though calcn. of transdermal flux of ACV with Keshary Chein Frenz Diffusion cell by using wistar albino rat skin. The transdermal flux of formulations PD, PD5D, PD10D, ME1 and ME10D was found to be 2.47, 50.7529, 119.7691, 238.1432 and 266.6721μg/cm2/h. The flux of microemulsion formulation ME10D was found 266.6721 ± 8.49 μg/cm2/h. Which showed highest value and skin flux of the drug could be enhanced up to 107 fold compared to its aq. soln. by prepg. microemulsion ME10D. DMSO in microemulsion formulation is safe to the skin at 10% DMSO wt./wt.

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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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