Preparation and characterization of biocomposite films of carrageenan/locust bean gum/montmorrillonite for transdermal delivery of curcumin

Thickness

The digital micrometer (Mitutoyo, Japan) was used to estimate the thickness of the transdermal films from 3 different points. The average and standard deviation (SD) of the 3 readings were recorded.

Drug content

The transdermal films with the specific area were placed in 100 mL solution containing 50 ml phosphate buffer (pH 7.4) and 50 ml ethanol followed by agitation on a mechanical shaker for 24 hours. Solution was then filtered, diluted suitably and analyzed at 422 nm using UV/Visible spectrophotometer (2202, Systronics, India). The experiment was performed in triplicate and the results were reported as mean ± SD.

Weight variation

Composite films of each batch were weighed individually. Each batch result was reported as mean ± SD.

Percent moisture content

The composite films were weighed individually and placed in a desiccator (37°C for 24 hours) containing silica gel as the desiccant. The final weight was measured when there was no further change in the weight of the film. The percent moisture content was calculated by using equation (Eq) 1.

\[\% Moisture\;content = \frac{{InitialWeight - FinalWeight}}{{InitialWeight}}\]

Percentage moisture absorption

The transdermal composite films were accurately weighed and placed (for 72 hours) in a desiccator maintained at 75% RH condition using a saturated solution of sodium chloride. The percent moisture absorption was calculated by Eq. 2:

\[\% Moisture\;absorption = \frac{{FinalWeight - InitialWeight}}{{InitialWeight}}\]

Folding endurance test

Folding endurance of the films was evaluated by repeatedly folding the film at the same place and the number of times the film could be folded without breaking represents its folding endurance value.

Mechanical properties

The mechanical properties of transdermal films were assessed by texture analyzer (TA XT plus, Stable Microsystems, UK) equipped with 5 kg of a load cell. Filmstrip (10 x 50 mm) was held between two clamps positioned at a distance of 10 mm and pulled apart at a rate of 50 mm/min. The tensile strength and extensibility were measured for 3 replicates of each batch of transdermal film. Tensile strength and extensibility were expressed in N/mm² and percentage respectively.

Swelling ratio

The completely dried, pre-weighed films were equilibrated in 250 mL of phosphate buffer (pH 7.4) at 25°C. The swelling ratio (Q) of the film is calculated using Eq. 3:

Q=W _s /W _d Eq. 3

Where, W_s is the weight of the swollen film at different time intervals and W_d is the weight of dry film.

Water vapor transmission rate

The sample film was set on the top of glass polytope (144 mm²) containing 10 ml of phosphate buffer (pH 7.4). This setup was pre-weighed and placed in an oven at 35ºC for 24 hours. WVTR was determined using following equation:

WVTR= W _i -W _t /A × 10 ⁶ g/m ² day^-1Eq. 4

Where, WVTR is expressed in g²h, A=area of the polytop opening (mm²), W_i and W_t = weight of the polytope before and after placing in the oven, respectively.

Scanning electron microscopy

A Nova NanoSEM 200 (Netherlands) instrument was used for SEM analysis. A double adhesive tape was used for mounting the composite films on SEM stubs for observing the surface morphologies of the film at an accelerating voltage from 10 to 15 kV.

X-ray diffraction

Samples were set up by slicing the films to fit the square tiles of the holder, was mounted on the sample cell and scanned between 2 θ of 0-60º with a counting time of 0.1 seconds step size. X-ray patterns of the films were obtained with XPERT-PRO equipment (PANalytical, Netherland).

ATR-FTIR

ATR-FTIR spectra of the transdermal films were carried out ATR-FTIR spectrophotometer (Bruker Alpha, Germany) in the transmittance mode with the wave number region 4000 – 500 cm^-1.

In vitro drug release

Transdermal films containing 25 mg equivalent of curcumin were applied to a glass slide and secured with a stainless-steel mesh screen. The assembly was placed at the bottom of the paddle time dissolution test apparatus (Lab India DS 8000, India). A 500 mL volume of phosphate buffer (pH 7.4) and ethanol (1:1) was employed as dissolution media under 32˚C and 50 rpm test conditions. Aliquots of 5 ml were withdrawn at specified intervals and were analyzed at 422 nm using UV/Visible spectrophotometer (2202, Systronics, India). In vitro drug release data were fitted to Zero order, First order, Higuchi, Hixson-Crowell and Korsmeyer-Peppas models for understanding the mechanism of drug release from film formulations.

In vitro skin permeation

In vitro drug permeation studies were carried out using Franz diffusion cell apparatus having 20 ml volume capacity of the receptor compartment. Full thickness rat skin from the dorsal surface was used for permeation studies. The animal skin was applied over the receptor compartment. Aliquots of 5 mL were withdrawn at specified intervals and were analyzed at 422 nm using UV/Visible spectrophotometer (2202, Systronics, India).

Wound healing study

The animal study was conducted in accordance with the protocol approved by the animal ethics committee of Chitkara College of Pharmacy, Chitkara University, India (118/PO/ab/08/CPCSEA). Healthy male rats weighing 240-300 g were anesthetized using ketamine (80 mg/kg). The specific skin area was shaved for creating wound of approximately 200 mm². Twelve animals were partitioned into 3 groups, Group 1: (Control), Group 2: (κC/L40), Group 3: (κC/L40/M). Transdermal film formulation was applied on the wound and wound area was measured by tracing on a millimeter scale graph paper on predetermined days i.e., 0, 2, 4, 6, 8, 10, 12 and 14 days post wound treatment. Percent of wound contraction was figured using Eq. 5:

\[{\text{\% Wound}}\;{\text{Contraction = }}\frac{{{\text{Initial}}\;{\text{wound}}\;{\text{area}}\;{\text{specific}}\;{\text{day}}\;{\text{wound}}\;{\text{area}}}}{{{\text{Initial}}\;{\text{wound}}\;{\text{area}}}} \times 100\]

Mechanical properties

Mechanical properties are the indication of strength and resistance of films to external stress. Mechanical properties of the transdermal films were assessed in terms of the tensile strength and percent elongation to break (extensibility). Pure κC films showed the tensile strength of 1.39±0.21 N/mm² and extensibility of 14.4±0.95%. The tensile strength and extensibility of κC/L40 and κC/L100 were found to be 11.30±0.92 N/mm²& 17.14±1.54% and 11.98±0.32 N/mm² & 25.42±1.26% respectively (Table 3). The increase in mechanical properties was attributed to the interaction between the H bond forming groups of κC and unbranched segments of D-mannose backbone of LBG. The tensile strength and extensibility of transdermal composite film (κC/L40/M) were found to be 14.22±1.01 N/mm² and 30.12±1.16% respectively. Incorporation of MMT in κC/LBG further increased the mechanical properties. This enhancement in mechanical properties could be attributed to interfacial affinity, interaction and formation of physically crosslinked networks between the MMT clay particles are the κC/LBG polymeric matrix. Similar results were reported by Martin et al² and Mirzataheri et al.¹¹

Swelling ratio

The swelling ratio of κC and κC/L40 was found to be 2.85±0.28 and 4.51±0.54% respectively. Increase in swelling ratio with the addition of LBG could be due to an increase in the hydrophilic groups in the composite films. Moreover, the physical entanglement of LBG and κC polymeric chains leads to the formation of a hydrogel network. Furthermore, the addition of MMT leads to a decrease in the swelling ratio. This could be due to the physical entanglement of clay particles in the polymer matrix leading to closures of pores/channels, thereby, reducing the swelling ratio.

Water vapor transmission rate

The WVTR was found to be increase with an increase (614.86±3.42 to 8342.10±10.68 g/m² per day) in the concentration of LBG in the films. This may be due to an increase in the hydrophilic groups (with an increase in LBG content) providing more affinity for the transmission of water vapors. Further increase in the concentration of LBG led to decrease in the WVTR, which could be due to an excessive gelling property imparted by the LBG which led to narrow or closure of pores/channels responsible for the transmission of water vapors. Furthermore, the addition of MMT particles led to the closure of pores/ channels which led to a drastic reduction in WVTR of transdermal composite films.

Characterization of curcumin loaded transdermal films

Scanning electron microscopy

The SEM images of different batches of transdermal films are shown in Fig. 1. Presence of rough surface topology and small rod-like shapes confirm uniform distribution of curcumin in the polymer matrix of transdermal films. The SEM images further indicate the presence of κC/LBG rendered intercalated lamellar structures due to entrapment of MMT particles within the polymer matrix. Martin et al suggested that Cloisite^TM 30B forms ex-foliated structure within the polymer matrix of κC/LBG, which is responsible for the enhancement of mechanical property water barrier property and antibacterial activity of κC/LBG composite films.²

Fig. 1

SEM images of transdermal films (A) κC; (B) κCL/40; (C), (D) Curcumin loaded κC/L40 composite films; (E), (F) Curcumin loaded κC/LBG/MMT.

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Fig. 1. SEM images of transdermal films (A) κC; (B) κCL/40; (C), (D) Curcumin loaded κC/L40 composite films; (E), (F) Curcumin loaded κC/LBG/MMT.

X-ray diffraction

As per the XRD pattern (Fig. 2) results, transdermal composite films of curcumin prepared using κC/L40 were found to have peaks corresponding to 2θ of 9.11(d spacing 9.7Å) and 2θ of 17.59 (d spacing 5.04 Å). Whereas κC/L40/M films exhibited diffraction peaks corresponding to 2θ of 5.12 (d spacing 17.26 Å) and 2θ of 9.39(d spacing 9.4 Å). The results suggested intercalation and possible bonding between the clay particles and the polymeric chains of κC/LBG. The results are in agreement with those reported by Aouada et al¹² and Lim et al.¹³

Fig. 2

X-ray Diffraction pattern of (A) κC/L40; (B) κC/L40/M batches of transdermal films.

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Fig. 2. X-ray Diffraction pattern of (A) κC/L40; (B) κC/L40/M batches of transdermal films.

ATR-FTIR

FTIR analysis was used to evaluate the chemical interactions in the transdermal composite films of κC/LBG/MMT (Fig. 3). Pure κC showed a characteristic peak at about 3406 cm^-1 of strong O-H stretching representing the involvement of hydroxyl groups in hydrogen bonding. Strong bands at 1216 cm^-1 representing sulfates stretching and at 1121 cm^-1 and 927 cm^-1 for C-O-C glycosidic linkages for pure κC were also observed. Pure LBG showed characteristic peaks at about 3300 cm^-1 (O-H stretching), 2900 cm^-1 (C-H stretching), 1370 cm^-1 (C-H bending) and 1123 cm^-1 (O-H bending). The broad peak at 1000-1100 cm^-1attributed to C-O-H stretching/bending vibrations. Pure MMT shows a peak at 3449 cm^-1 representing interlayer and intralayer H-bonding. However, peaks at 3619 cm^-1 and 1633 cm^-1 indicates O-H stretching and O-H bending respectively. The bands around 520 – 560 cm^-1 are due to Si-O bending vibrations while 1134 cm^-1 band represents Si-O stretching vibrations. In κC/LBG, κC may interact with D-mannose backbone of LBG through hydrogen bonding. The observed shift and marked an increase in the intensity of the peak corresponding to C-O stretching band of C-O-H group band from 1129 cm^-1(κC) to 1156 cm^-1(κC/LBG) could be due to the hydrogen bridging interaction between the OH group of LBG with the κC structure. In κC/LBG/MMT films, the addition of MMT clay to κC/LBG polymer matrix, leads to the formation of H-bonding between hydroxylated edge groups of MMT and OH groups of κC and LBG. Results are in agreement with those reported by Martin et al.²

Fig. 3

ATR-FTIR of various biocomposite transdermal films (A) Curcumin; (B) Pure κC; (C) Pure LBG; (D) Pure MMT; (E) κC/L40; (F) κC/L40/M.

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Fig. 3. ATR-FTIR of various biocomposite transdermal films (A) Curcumin; (B) Pure κC; (C) Pure LBG; (D) Pure MMT; (E) κC/L40; (F) κC/L40/M.

In vitro release study

The results of in vitro drug release study revealed that the cumulative percent drug release decreases with an increase in the concentration of LBG in the transdermal film formulation. The drug release from κC, κC/L40 and κC/L100 was found to be 80.42±4.12%, 69.38±3.28% and 63.94±3.1% respectively after 500 minutes as shown in Fig. 4. Formation of the polymer matrix by LBG is the possible cause of retardation of drug release from the films. The drug release from κC/L40/M was found to be 44.94±1.26% and 56.39±1.74% after 300 to 500 minutes respectively as shown in Fig. 4. The addition of MMT further retards drug release from transdermal films. This may be due to the interpenetration of the clay particles in the polymeric chains of κC and LBG. The interference of the clay particles with polymeric chain relaxation (up to hydration) could also be a cause of the reduction in drug release by MMT. The drug release data obtained after in vitro release study was fitted into various kinetic models. i.e. zero order, first order, Higuchi, Hixon Crowell and Korsmeyer Peppas model. The value of n was found to be ranging from 0.54 to 0.75 for the selected formulations as shown in Table 4, indicating anomalous non-Fickian drug release behavior from the formulation. Further, diffusion and erosion controlled mechanism could be responsible for the release of drug from the transdermal composite films.

Fig. 4

In vitro release of curcumin from transdermal biocomposite films of κC, κC/L40, κC/L100, κC/L40/M.

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Fig. 4. In vitro release of curcumin from transdermal biocomposite films of κC, κC/L40, κC/L100, κC/L40/M.

In vitro skin permeation

The results of in vitro permeation from animal skin were from diffusion cells are in line with the in vitro drug release results of the films (Fig. 5). κC and LBG produced a polymer matrix which has less affinity for water; this results in a decrease in the thermodynamic activity of the drug in the membrane thereby leading to a decrease in the drug release from the film. The addition of MMT further retarded the drug release from the formulation.

Fig. 5

In vitro permeation of curcumin from transdermal biocomposite films of κC, κC/L40, κC/L100, κC/L40/M.

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Fig. 5. In vitro permeation of curcumin from transdermal biocomposite films of κC, κC/L40, κC/L100, κC/L40/M.

Wound healing

Percent degree of wound contraction is an indication of wound healing property. As compared to control κC film, κC/L40 and κC/L40/M exhibited faster wound healing property (Table 5). The accelerated wound contraction from transdermal composite films could be attributed to the controlled release of curcumin from the formulations.