Synthesis, mechanical properties of fluorescent carbon dots loaded nanocomposites chitosan film for wound healing and drug delivery

2.1 Materials

Chitosan (Ch), with a molar mass of 375 k Da and degree of de-acetylation of 98% was obtained from Hi Media Chemicals, Mumbai, India and was used as received. The carbon resource material, 1,2,3,4-Butanetetracarboxylic acid (BTCA; molar mass 243.16) was obtained from E. Merck Chemicals, Mumbai, India and was analytical grade. Other chemicals were obtained from S. D. fine Chemicals, Pune, India and were GR grade. The double distilled water was used throughout the investigations.

2.2 Methods

3.1 Synthesis of CNPs from BTCA

BTCA is a water soluble compound with four carboxylic acid groups per molecule. This makes it a suitable resource material to prepare CNPs with their surface covered with negatively charged carboxylate groups. When an aqueous solution of BTCA is exposed to microwaves, the uniform and intense heating leads to formation of carbon dots. This involves evaporation of solvent molecules, followed by cleavage of covalent bonds within the molecules. Finally, the semi-solid brown colored fluid is left in the beaker, which, on addition of water, yields a pale yellow solution. This solution produced greenish fluorescence when exposed to UV radiations.

3.2 Quantum yield

In order to determine QY of carbon dots, Eq. (1) was used. The integrated photoluminescence intensities, obtained at different concentrations of CNPs solutions, were plotted against corresponding absorbance (data not shown). The respective slopes (m values) were put in Eq. (1) and the QY of carbon nanoparticles was calculated, using 54% as the value of QY_std.. The QY of carbon dots, prepared from BTCA, was calculated using Eq. (1) and was found to be 21%. The value obtained is almost the same as obtained by Liu et al. (Wang et al., 2017), who prepared CNPs from poly(acrylamide) and sodium citrate and reported a QY value of 18%. However, the QY value of 21% is fairly high as compared to those obtained in majority of studies. For example, Zhou et al. (Samer et al., 2017) reported a QY value of 7.1% in the case of CNPs obtained from Watermelon.

3.3 Characterization of CNPs and CNP/Ch film

The FTIR spectrum of carbon nanoparticles synthesized is shown in Fig. 1(a). A wide band corresponding to stretching vibrations of the hydroxyl group (band of a free OH group (from water) and hydrogen bonds) occurs around 3434 cm⁻¹. In addition, C—H stretching of alkane appears at 2962 cm⁻¹. A small peak at 1404 cm⁻¹ is due to deformation band of C—H. Moreover, peak corresponding to 1242 cm⁻¹ is due to C—O stretching. In this way it may be inferred that carboxylic groups are present on the surface of carbon dots. In addition, CNPs display the characteristic bands for C⚌C and C—O—C vibrations at ∼1636 cm⁻¹ and 1048 cm⁻¹ respectively, thereby indicating the presence of sp². The above observation entails that the synthesized high fluorescent CNPs made up of sp² hybridized carbon atoms with hydroxyl, carbonyl and carboxylic functional groups.

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Fig. 1 (a) FTIR spectrum of Carbon nanoparticles; X-ray diffraction pattern of (b) plain chitosan Ch/CNP(0) and (c) Ch/CNP(4) film.

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The zeta potential of carbon nanoparticles is a significant property as it reveals the stability of the colloidal dispersion and the nature of charge existing on the surface of carbon dots., which leads to the probable interactions of CNPs with biological entities. In this work, zeta potential of CNPs was found to be −11.6 mV, thus indicating presence of negative charge on their surface (see Fig. 4). This may be due to the presence of carboxylic groups (from BTCA molecules) on the surface of carbon dots. Similar results have also been reported elsewhere. For example, Bayda et al. (Samer et al., 2017) used “black tea”, as a suitable precursor for the synthesis of CNPs by nitric acid (HNO₃) oxidation and reported a zeta potential of −16.6 mV. In another study, Sivasankaran et al. (2017) prepared ethylenediamine passivated Carbon nanoparticles from tissue paper and reported a zeta potential value of −15.7 mV. Indeed, the nature of charge on Carbon nanoparticles depends upon the resource material iused. The positively charged Carbon nanoparticles, prepared from chitosan, were reported to have a zeta potential value of 27.2 mV. Probably due to the presence of protonated amine groups (Chowdhary et al., 2012). The crystalline nature of the Carbon nanoparticles was investigated by the X-ray diffraction analysis. The XRD patterns of plain chitosan film and CNPs-loaded film are shown in Fig. 1(b) and (c) respectively. It can be seen that chitosan film shows a scattered broad diffraction pattern with two peaks at 2θ values of 14.6 and 18.1. This suggests semi-crystalline nature of chitosan film. These values are very close to those reported elsewhere (Yahya et al., 2006; Kaur et al., 2013). The XRD pattern of Ch/CNP composite film, as shown in Fig. 1(c), also shows almost similar pattern with the two peaks, occupying the same positions. Therefore, it is clear that incorporation of Carbon nanoparticles in to chitosan film does not produce any additional peaks and the diffraction pattern remains the same. Hence, CNPs can be assigned to have non-crystalline or amorphous nature.

3.4 Optical characterization

The investigation of optical properties of Carbon nanoparticles plays an important role in exploring the biomedical applications of CNPs in cell imaging. The exposure of UV light on the CNP solution resulted in green colour fluorescence. It was observed that in the presence of visible light the solution appeared pale yellow while it emitted green fluorescence in UV light (see Fig. 2(a) and (b) respectively). The UV spectrum of Carbon nanoparticles solution is shown in Fig. 2(c). It can be observed that there is maximum absorption peak at 225 nm and then absorption continues to decrease with further increase in the wavelength. It is reported that presence of passivating agent plays a key role in UV spectra of carbon dot solutions. For example, So and co-workers (So et al., 2017) synthesized ethylene diamine (EDA)– passivated Carbon nanoparticles from citric acid reported an absorption around band at 353 nm and a peak at 200 nm with corresponding shoulder peak at 237 nm, whereas the Carbon nanoparticles from citric acid sample gave a low absorption peak at 200 nm and no peak at 353 nm.

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Fig. 2 Solution of CNPs in the (a) absence and (b) presence of UV light; (c) UV spectrum and (d) fluorescence spectra of CNPs solution.

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The emission spectra of CNPs solution, at the excitation wave length of 260–340 nm, are shown in Fig. 2(d). It can be noticed that when the excitation wavelength varies from 260 to 300 nm, no sharp emission peak is noticed. However, with the further increase in excitation wavelength, a well defined peak in emission spectra is observed and maximum emission intensity is obtained at 434 nm for the excitation wavelength of 340 nm. In addition it is also noteworthy in the spectra that there is a red shift of emission peak from 390 to 424 nm as the excitation wavelength increases from 310 to 340 nm. This indicates that the emission of fluorescence from Carbon nanoparticles is excitation-dependent, thus suggesting that desired emission intensity can be achieved by variation in the excitation wavelength. Similar reports are also available which claim excitation wavelength dependent emission from carbon dots. For example, Guo et al. (Yongming et al., 2016) reported a red shift of emission peak for highly luminescent Carbon nanoparticles, obtained from hair. Similarly, Bhaisare et al. (2015) synthesized Carbon nanoparticles from citric acid by microwave induced carbonization and reported a maximum emission intensity at 375 nm for the excitation wavelength of 300 nm. It was further reported that as the excitation wavelength increased from 300 to 460 nm, the corresponding emission peak also shifted towards higher wavelength (i.e. red shift). It has been suggested that this phenomenon is attributable to the presence of functional groups on the surface of Carbon nanoparticles while some have suggested it is because of the defects on the surface red shift. Indeed, there are so many reports in which excitation-independent emission has been reported (Chen et al., 2017).

3.5 TEM analysis

The size of the Carbon nanoparticles was determined by TEM analysis as shown in Fig. 3(a) and (b) respectively. It may be noticed from Fig. 3(a) that the particle shown is quite spherical in shape and it possesses a diameter of around 10 nm. The images of some more Carbon dots with bar length of 10 nm are shown in Fig. 3(b). It also supports our observation that the particles are almost spherical with a size range of 80–95 nm. Furthermore, it can be seen in Fig. 3(c) that CNPs exhibit discernible lattice fringes with inter-planar spacing of 0.33 nm which is close to the diffraction facet of graphitic carbon (Liang et al., 2013). Similarly, in a work by Baker and co-worker (Baker and Baker, 2010), a spacing of 0.32 nm has been reported for the Carbon dots obtained from single walled carbon nano tubes.

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Fig. 3 (a) and (b) TEM images and (c) HRTEM image of carbon dots.

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3.6 UV radiations, wettability and water absorption behavior of Ch/CNP nanocomposite films

Carbon nanoparticles absorb ultraviolet radiations and emit fluorescence. This property allows them to be used as UV rays absorbing medium and may be useful in the development of nanocomposite materials that can be employed as a strong UV rays protector. In this work, we recorded absorbance of plain Ch and Ch/CNP(4) films when exposed to UV radiations with the wavelength range of 290–320 nm.

The results, as shown in Fig. 4(a), reveal that the plain Ch film exhibits low absorbance while the Carbon nanoparticles loaded chitosan film, namely the sample Ch/CNP(4), shows strong absorption of UV radiations. This may simply be attributed to the presence of Carbon nanoparticles within the chitosan film matrix. Such films may be used to block the UV radiations where ever required.

The average contact angles, obtained for the poly(styrene) film, plain chitosan film Ch/CNP(0) and CNPs loaded samples CNP/Ch (2), CNP/Ch (4) and CNP/Ch (7) were found to be 52 ± 5.4, 26 ± 3.4 and 42 ± 4.6, 45 ± 4.2 and 48 ± 4.8 respectively. It can be noticed that the poly(styrene) film showed highest contact angle, thus showing minimum wettability. In addition, the plain chitosan film Ch/CNP(0) exhibited lower contact angle out of all the five samples, thus demonstrating fair wettability. However, as the CNPs content in the films increased, the contact angles of the respective films showed an increasing trend thus suggesting a decrease in the wettability of the films with increase in CNPs content.

As observed in contact angle measurement experiment discussed above, the addition of CNPs reduced the hydrophilicity of the chitosan film. In order to further investigate this aspect further, various Ch/CNP nanocomposite films, containing different volume of CNP solution in the feed mixture, were prepared and their equilibrium swelling ratio was determined in distilled water at 37 °C. The results, as shown in Fig. 4(b), revel that there is drastic fall in the ESR of films due to addition of carbon dots. It can be seen that plain chitosan film demonstrates an ESR of nearly 69.5 g/g which decreases to 9.8 as 2 ml of carbon dot solution is added into film forming solution. In other words, a 7 fold decrease in ESR is observed due to addition of Carbon nanoparticles in the plain chitosan film. Moreover, addition of 4 ml of Carbon nanoparticles causes a further decrease in the ESR to 1.1 g/g. However, the film prepared with the addition of 7 ml of CNP solution into the film forming mixture causes no further decrease in the SR of the respective film. The observed findings are very interesting and significant too. The drastic loss in ESR of the hydrogel film due to presence of Carbon nanoparticles may be attributed to the additional physical crosslinks that have been produced between the protonated NH₃ ⁺ groups of chitosan and negatively charged —COO^– present on the surface of the carbon dots. Hence, the electrostatic attraction between the oppositely charged groups acts as additional crosslinks and a tight network is formed within the chitosan film. As a result, the chains become rigid and do not allow water molecules to enter in to the network. This causes a great reduction in the ESR as compared to the plain chitosan film. The electrostatic attractive force is well depicted in Fig. 4(c). These additional crosslinks not only render stiffness to the chitosan chains but they also reduce the free space available for the accommodation of incoming solvent molecules (Ding et al., 2013).

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Fig. 4 (a) UV absorbance of the plain Ch and Carbon nanoparticles loaded Ch/CNP($) films (b) Equilibrium swelling ratios (ESR) of various Ch/CNP)(4) films in distilled water; (c) Attractive forces producing physical crosslinks.

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3.7 Equilibrium moisture uptake(EMU) study

The results of moisture absorption studies, carried out with the plain Ch and Carbon nanoparticles loaded Ch/CNP films are shown in Fig. 5(a) and (b) respectively.

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Fig. 5 Equilibrium moisture uptake of (a) plain Ch and (b) Ch/CNP films.

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It can be noticed that for both of the samples, EMU increase as the relative water humidity or activity increases. This is simply due to the fact that chitosan contains a number of polar —OH groups within its network. Therefore, water vapor molecules have fair tendency to bind with these active sites. The observed sigmoidal shape is attributable to the fact that when relative humidity (RH) is low, number of water vapor molecules is small that are bound to the active sites. However, with increase in RH, more and more vapor molecules are attached to active sites. In addition, the capillary action is also prominent at higher RH environment. One interesting and significant observation is that the plain Ch film shows higher water absorption as compared to the film Ch.CNP for every RH. This is simply attributed to the fact that carbon nanoparticles, present within the Ch/CNP film, act as effective crosslinkers within the Ch/CNP film matrix. This is due to presence of –OH groups on the surface of carbon dots, which bind through H-bonding interactions with chitosan chains. As a result, Ch/CNP film become highly crosslinked and hence possesses lower water vapor absorption.

The EMU data, obtained for both of the films, was analyzed by three parameters based GAB isotherm model (Tiwari and Rana, 2010). This model is given as: $$\text{M}=\frac{{\text{M}}_0\text{Ck}.{\text{a}}_{\text{w}}}{\left(1-\text{k}{\text{a}}_{\text{w}}\right)\left(1-\text{k}{\text{a}}_{\text{w}}+\text{ck}{\text{a}}_{\text{w}}\right)}$$ where, Mo is the monolayer moisture content, C is a constant related to the first layer heat of sorption and K is a factor related to the heat of sorption of the multilayer. In order to determine the parameters of GAB isotherm model, GAB equation is re-arranged into a second degree polynomial equation.

A non-linear regression analysis between a_w/M and a_w resulted in a polynomial as shown in Fig. 6. The GAB parameters, calculated using Eqs. (5), (6) and (7) were obtained as follows: For plain film K = 0.88, C = 20.26 and M_o = 0.11 g per g dry film; For Ch/CNP film K = 0.99, C = 0.39, M_o = 0.006 g/g dry film respectively. A close look at the values obtained reveals some significant facts about the water absorption behavior of the two films. The relatively higher moisture content M_o for the plain film suggests that plain chitosan film has greater water sorption capacity, probably due to un-crosslinked network within the film matrix. On the other hand, M_o value for Ch/CNP film is almost 17 times less, thus suggestive of the fact that film matrix is fairly crosslinked and hence does not encourage absorption of water molecules. Interestingly, the value of C for plain Ch film is fairly high (i.e. 20.26) which suggests that the isotherm for plain film is Type-II sigmoidal, thus showing low moisture content in the initial water activity environment and a drastic increase for the higher a_w values. In this way a point of inflection is noticed in such type of isotherms. The parameter C for the Ch/CNP film shows a very low value of C, i.e. 0.39 which lies between 0 and 2. As per literature available, this indicates a type –III isotherm with no point of inflection (Xu et al., 2007). This can also be noticed in the Fig. 6(b) where no inflection is observed. In this way, the presence of Carbon nanoparticles within the chitosan film affects the nature of sorption isotherm obtained. Finally, the value of K provides a measure of the interactions between the molecules in multilayer with the adsorbent and tends to fall between the energy values of the molecules in the monolayer and that of liquid water. The prescribed range for K values is 0 < K ≤ 1. As can be seen, the values of K, obtained for all the film samples fall within the prescribed range.

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Fig. 6 a_w/M versus a_w polynomial plots for the plain (a) Ch and (b) Ch/CNP films.

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3.8 Moisture permeation study

As mentioned in the previous section, the electrostatic attractive forces produce physical crosslinks and a dense network is formed, thus discouraging the enterance of water molecules. We also studied the permeability of these films with respect to water vapor molecules.

The results, as shown in Fig. 7, reveal that plain Ch film allows faster permeation of water vapor as compared to the film sample Ch/CNP. This is attributable to the presence of highly crosslinked network within the Ch/CNP film as discussed above. The narrow mesh size of the network does not permit much of vapor molecules to pass through the film, while plain chitosan film, having uncrosslinked network, allows vapor molecules to permeate through the film at a much faster rate. The WVTR for the two film samples, namely Ch and Ch/CNP, was found to be 1758 and 956 g/m²/d respectively. This indicates that addition of Carbon nanoparticles into plain chitosan film could alter its WVTR appreciably. The low moisture permeation rate of the Ch/CNP film makes it a suitable candidate for wound healing management, particularly for the injuries with low exudate (Hu et al., 2001; Ovington, 2007).

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Fig. 7 Dynamic moisture loss profiles for the plain Ch and Ch/CNP films.

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3.9 Expansion and Uv-absorption study

As stated in the section “Introduction”, the CNPs loaded chitosan film can be used in a number of biomedical applications, including wound healing dressings. The use of a polymeric film for wound dressing requires fair structural integrity during the absorption of exudate coming out from wound. The reason is that when a film is placed over the wound, it comes in contact with the exudate and begins to undergo expansion in its size. If the film expands appreciably, then it may lose its integrity, become soft and sticky, and ultimately may cause inconvenience to the patient. It may also be torn and leave the wound surface. Hence, it becomes essential to test its expansion limit and structural integrity. For this, the expansion ratio d/d₀ (where d and d₀ are the diameters of the film before and after absorption of gelatin solution) were plotted against time ‘t’. The results of expansion study are shown in Fig. 8(a). It is noticeable that the plain Ch film undergoes 2.8 fold expansion in its diameter in duration of 60 min, while in the same time frame, the Ch/CNP film does not expand appreciably (a little expansion, around 1.3 fold) and it maintains its structural integrity throughout. On the other hand, the plain Ch film gets hydrated, slippery and becomes difficult to handle properly. Thus it may be concluded from this study that addition of pre-calculated quantity of Carbon nanoparticles in to chitosan film can render it enough mechanical strength and control its water absorption capacity as per requirement. The optical images of the plain Ch and the Ch/CNP films in the swollen state are also shown. It is worth noticing that the Ch/CNP film was intact, rigid and unslippery, while the plain chitosan film was fully hydrated with pale yellow appearance, was difficult to hold properly and was slippery. Therefore it may be concluded that addition of CNP sin to chitosan film could cause a great change in its physico-chemical properties. Finally, we also investigated fluorescent behavior of Ch/CNP film in the presence of UV-radiations. It may be seen in Fig. 8(b) and (c) that Ch/CNP film does not show any emission in the presence of sun light while a green fluorescence is observed in the presence of UV light.

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Fig. 8 (a) d/d_o versus time profiles for the plain Ch and Ch./CNP films in 4% gelatin solution, Ch/CNP films in the presence of (b) sun light and (c) UV light.

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3.10 Biocompatibility tests

Blood compatible nature of the films Ch/CNP (0) and Ch/CNP(2) was evaluated in the terms of % hemolysis. The % hemolysis of these films was found to be (2.12 ± 0.02) and (1.13 ± 0.18) respectively. It is clear that both of the samples, namely plain chitosan film Ch/CNP(0) and CNP-loaded film Ch/CNP(2) are non-hemolytic. In addition, the sample Ch/CNP(2) shows excellent biocompatible nature as its % hemolysis is almost 1.0. The low hemolytic index of Ch/CNP (0) film is because of its highly hydrophilic nature, which causes a decreases in polymer-red blood corpuscles (RBC) interactions. This subsequently lowers the disruption of RBC. However, in the case of the sample Ch/CNP(2), the presence of polar carboxylate groups on the surface of CNPs, lower the interaction of film with red blood cells to a greater extent, thus minimizing the degree of disruption of blood cells. This results in minimum percent hemolysis.

The adsorption of protein on a wound dressing film indicates its cell adhesion behavior. Albumin, a multifunctional transporter protein, is the most abundant protein found in the plasma (approx. 50 mg·ml⁻¹). Its adsorption is related to the inhibition of the coagulation cascade and, consequently, platelet adsorption. In the present work, it was found that plain sample Ch/CNP(0), and CNPs-loaded samples Ch/CNP(2), Ch/CNP(4) and Ch/CNP(7) showed BSA adsorption 24.2, 21.5, 18.3, 15.9 and 14.1 mg/m² respectively. The extremely low BSA adsorption may be indicative of the hydrophilic nature of the film surfaces for all the samples studied. It has been reported (Zhao et al., 2018), that BSA is mainly adsorbed on a polymeric surface due to hydrophobic interactions. As the film base material is chitosan and it contains a number of polar hydroxyl groups, the film surface acquires hydrophilic nature and therefore minimum interaction takes place between the protein BSA and film surface. It is also noticeable that as the CNPs content in the film increase, quantity of BSA adsorbed decreases. This is attributable to the fact that since the surface of carbon dots contains polar –COOH groups, they render additional hydrophilicity to the nano-composite films, thus minimizing the protein-polymer interactions.

Finally, the maximum detachment force (F_max) required to detach the film from mucosal surface, was determined for all the film samples namely, Ch/CNP(0), Ch/CNP(2), Ch/CNP(4) and Ch/CNP(7) respectively. The values of F_max were found to be 88.22 ± 11.52, 45.15 ± 8.61, 43.29 ± 7, and 46.22 ± 5.94 mN respectively. It can be noticed that F_max value obtained for the plain sample Ch/CNP(0) is relatively much higher as compared to those obtained for the CNPs-loaded films. The higher F_max value of plain sample may be attributed to the fact that these films are hydrophilic and absorb a fair quantity of water along with relaxation of chitosan chains. As a result of chain relaxation, new sites are exposed to the mucus membrane and cause greater adhesions are not too high as reported elsewhere.