INTRODUCTION:

Colorectal cancer is still the most common type of cancer in men and women worldwide. After lung and breast, colon is the third most common cancer worldwide. In India, colorectal cancer is the third most common cancer¹. There are many therapeutic strategies to fight colon cancer, such as chemotherapy, radiation, surgery and hormonal therapy or immunotherapy².

Chemotherapy has not been very successful in clinical application because current small molecule drugs have low selectivity and efficacy and cause systemic toxicity ^3,4. Therefore, there is an urgent need for safe and less toxic delivery systems that are also highly effective and non-invasive.

Amygdalin (AMY) is a traditional natural remedy extracted from bitter almonds and has been used for many years to treat cancer, particularly for its anti-cancer effects. However, the effectiveness of amygdalin in the treatment of cancer is still controversial in the healthcare sector⁵. Nevertheless, it continues to be used by some cancer patients in various countries. AMY is a cyanogenic glycoside used in the treatment of various cancers such as breast, prostate, lung, skin, and colon cancer. Amygdalin can prevent tumor growth in various ways. It interrupts the cell cycle, triggers cell apoptosis, and impairs the ability of cancer cells to adhere and invade⁶. Amygdalin was found to effectively inhibit the proliferation and invasion of castration-sensitive and castration-resistant prostate cancer cells⁷. The main problem with amygdalin is its elimination half-life of about 120 minutes. The aqueous solubility of amygdalin is almost 82g/mol and the permeability is low (logP 0.82). However, the clinical applications of amygdalin are limited due to its relatively low inhibitory effect and the possibility of systemic toxicity at high doses¹.

Recently, some research investigations have already done by researchers in the field of cancer treatment using amygdalin as a delivery system. To overcome this problem, Zhou et al.,⁸ have successfully developed starch coated MNPs coupled with β-Glu and PEG for the effectively treating the prostate cancers. However, PEG-β-Glu-MNP/amygdalin significantly inhibits tumor growth. Askar et al., developed folic acid-nanoparticles loaded with amygdalin to inhibit the proliferation of breast cancer MDA-MB-231 and MCF-7 cells. Their findings showed that Amy-F had a significant impact on inhibiting the expansion of cancer cells and improving the effectiveness of radiotherapy. This was achieved by causing cell cycle arrest and promoting cell apoptosis⁹. In other investigations, some delivery systems for amygdalin including chitosan nanoparticles¹⁰, noisome ¹¹, cyclodextrin nanoparticle¹², nanofibrous patches¹³, nanofibers¹⁴, and hydrogel¹⁵ have been reported for cancer treatments.

Nanosponges (NSs) are tiny material-like structures with a diameter of approximately 0–500 nm and a porous surface that encapsulate the drug in their polymeric structure and release the drug in a more predictable and controlled manner¹⁶. NSs have many beneficial properties that can improve solubility and dissolution rate and ensure sustained release of natural and synthetic drugs. The porous nature of NSs can absorb more drugs and the diffusion of the solvent is facilitated. NS carriers are used to treat various medical problems. Preliminary research suggests that this nanotechnology is five times more successful than conventional methods of drug delivery, particularly in the treatment of malignant tumors^17,18.

In the present work, amygdalin-loaded nanosponges were successfully prepared using the ultrasound-assisted emulsion solvent evaporation method and subsequently optimized by implementing CCFD for the effective treatment of colon cancer. The effect of independent variables such as Eudragit RS-100 and PVA were examined on the formulation response variables including Particle size (Y1), %EE (Y2), and %CDR_24h (Y3). The optimization process was carried out with the objective to develop a stable formulation. The optimized formulation was physically characterized for the its ATR-FTIR, DSC, XRD, and SEM. Particle size, PDI, zeta potential, %EE, and %CDR_24h for optimum formula was also determined. Finally, the cytotoxicity study of pure amygdalin was also examined in HT-29 colon cancer cell line.

MATERIALS AND METHODS:

Materials:

Amygdalin (Assay 97%) and Dimethyl Sulfoxide (DMSO) were purchased from Sisco Research Laboratory (SRL), Mumbai, India. Polyvinyl alcohol (PVA), Eudragit RS-100, disodium dihydrogen phosphate dihydrate, potassium dihydrogen phosphate, sodium hydroxide, sodium chloride, and pepsin were purchased from Central Drug House (CDH) (P) Ltd., New Delhi, India. Fetal Bovine Serum (FBS) was purchased from HIMEDIA Maharashtra, India. All other chemicals and reagents were used at analytical grade.

Experimental design using CCFD:

In the present work, we used a CCFD approach to optimize Amy-loaded NS by considering two factors at three levels each. Thirteen experimental runs were generated using Design-expert® software to examine the influence of ERS-100 and PVA as independent variables. Amy-loaded nanosponges were evaluated for particle size (Y1), % EE (Y2), and %CDR_24h(Y3). In this design, 5 central points and 8 non-central points were used to obtain a robust model. Other substances used in the formulation of the nanosponges were kept constant throughout the study. In addition, the response surface methodology was used for data analysis. Regression analysis was used to examine the mathematical model. The correlation coefficient, P-value and lack of fit analysis were also performed to analyze the fit of the model. The 3D response surface and 2D contour plot were used to examine the effects of the independent variables on the formulation response variables ¹⁹.

Table 1: Independent variables with their coded value and response variables with their targets.

Independent variables				Fixed value
Code d values		ERS-100 mg	PVA %	Drug	Ethanol	Water
	Low (-1)	75	0.5	10 mg	10 ml	100 ml
	Middle (0)	100	1.0
	High (+1)	125	1.5
Responses variables with goals
Particle size (Y1)		Minimum
% EE (Y2)		Maximum
%CDR_24h (Y3)		Maximum

Preparation of nanosponges:

Nanosponges was fabricated by utilizing of various quantity of Eudragit RS-100 by ultrasonication assisted-emulsion solvent evaporation method²⁰. Briefly, drug and polymer were dissolved in ethanol by means of ultrasonicate for 5min. Separately, an aqueous solution of PVA was prepared by probe sonication at 60% amplitude for 15min. The organic phase was then incorporated dropwise to the aqueous phase under magnetic stirrer 2000rpm for 2h at room temperature to allow complete evaporation of ethanol from the mixture. The formed nanosponges were vacuum-filtered, and dried in a hot air oven at 40ºC for 30min and placed in a desiccator for 24h.

Particle size, polydispersity indexed, and zeta potential:

Particle size, polydispersity index and zeta potential of Amy-loaded NS were measured using Malvern Zetasizer Nano ZS. To obtain the required scattering intensity, the formulation was diluted with HPLC-grade water during sample preparation. The samples were then analyzed in triplicate at 25ºC±1ºC. The results were expressed as mean diameter ±SD^21,22.

Percentage entrapment efficacy (% EE):

The nanosponges were evaluated for the amount of AMY using a previously reported method. The prepared dispersion of nanosponges (15ml) was centrifuged at 2500rpm for 30min. The supernatant was separated, diluted, and analyzed spectrophotometrically (UV spectrophotometer, Shimadzu) at 210nm to determine drug entrapment^23,24. Entrapment efficacy was analyzed using the formula given below.

Total drug-free drug

% Entrapment efficacy = ----------------------------- ×100

Total drug

In-vitro drug release:

Drug release studies for Amy-loaded NS were carried out using dialysis bag method in various SGF fluids at pH 1.2, 6.8 and 7.4. In this study, Amy-loaded NS formulation was sealed in a dialysis bag and suspended in a 100ml beaker containing SGF fluid with pH 1.2 and rotation speed of 100rpm at a temperature of 37±0.5ºC to maintain the sinking condition. Before the experiment began, the dialysis bag was spread out in a pH 1.2 SGF liquid. SGF fluid was prepared by dissolving pepsin (1.6 g), sodium chloride (1g) and hydrochloric acid (3.5ml) in 500ml distilled water and maintained pH with 0.1N HCl solution. After completing a 2h study, the exact weight amounts of potassium dihydrogen phosphate (0.17g) and disodium dihydrogen phosphate dihydrate (0.22g) were then added to the media and the pH was controlled with 0.1M sodium hydroxide for the next 2h. Subsequently, the pH was adjusted to 7.4 using 1M NaOH solution for the next 20h. At a predetermined time interval, 2ml of sample was extracted and replaced with an equal volume of fresh medium. Before analysing with a UV spectrophotometer at 210nm, the samples were filtered with syringe filter (0.45µm). The experiment was carried out in triplicate and measured mean±SD was determined^19,25

Optimization of Amygdalin-loaded NS:

The optimized formulation of Amy-loaded NS was selected using Design of Expert^® software based on particle size, %EE, and %CDR_24h constraints. Using numerical optimization and a desirability function, target values for the responses were defined to achieve the desired goals, resulting in the optimized formulation. Graphical optimization was also performed to find the optimized formulation in the design space. After formulation, the resulting optimized formulation of Amy-loaded NS was formulated and examined to confirm the authenticity of the predicted variables and formulation response variables. Furthermore, the optimized formulation of Amy-loaded NS was characterized in terms of its particle size, %EE, and %CDR_24h.

Characterization of the optimized formulation of Amy-loaded NS:

PS analysis, EE%, and %CDR_24h

The optimized formulation of Amy-loaded NS was determined for PS analysis, EE%, and %CDR_24has previously described.

Compatibility assessment using Attenuated Total Reflectance-Fourier Transformer Infrared:

The ATR-FTIR spectrums were performed using Shimadzu model No. IRSPIRT-T, spectrophotometer to carry out the interaction in amygdalin, ERS-100, PVA, physical mixture and to ensure the optimized formulation formation. The spectra were recorded from 4000 to 400 cm^-1 ^26,27.

Thermal analysis behaviour using Differential scanning calorimetry:

Thermal analysis behaviour was investigated using DSC-60Plus, Shimadzu, Japan, and the amygdalin, ERS-100, PVA, physical mixture and optimized formulation were hermetically sealed in aluminum pans with the assistance of a tight-fitting aluminum cover. The samples were sealed and heated at a rate of 10 °C/min from 5°C to 300°C while being subjected to a nitrogen flow (10mL/min) for purging purposes. The thermograms were recorded and the firmly sealed empty pan was retained as the reference^28,29.

X-ray diffraction (XRD) analysis:

The XRD analysis was conducted using Rigaku modal no Mini Flex 600, Tokyo, Japan, and the spectrum was captured for amygdalin, ERS-100, PVA, physical mixture and optimized formulation at a 15mA fixed tube current and voltage 40kV^30,31.

Morphological evaluation using scanning electron microscopy:

Morphology of the optimized formulation of Amy-loaded NS was examined using SEM (JEOL, JSM-IT200). The samples were positioned on a metal stand and carefully covered with sputtered Au under vacuum (0.25 Torr) before analysis. The images were taken at an acceleration voltage of 15 kV³².

Kinetic release studies:

The kinetic release data were performed using different kinetic models (zero-order kinetics, first-order kinetics, Higuchi diffusion, and Korsmeyer-Peppas models) for the optimized formulation Amy-loaded NS²⁸.

Cell line studies:

The cytotoxicity study of amygdalin was performed by Deshmukh et al. with minor modifications³³ on HT-29 human colon cancer cells purchased from NCCS, Pune, India. The cells were cultured at 37°C±2°C with 5% CO₂ in DMEM medium supplemented with 10% FBS and 1% antibiotic solution. Cells were treated with different concentrations of amygdalin (0-1000µm) to examine the effect of pure amygdalin on the HT-29 cell line. In 96-well plates, 10000cells/well cells were cultured for 24h. Untreated cells were considered as the control group, whereas cells without MTT were considered as the blank group. After 24h of incubation, MTT solution was added to the cell culture, and it was then incubated for a further 2 hours. Following the experiment, an ELISA plate reader (iMark, Biorad, USA) was used to measure the cell layer matrix at 540 nm after it had been dissolved in 100µl dimethyl sulfoxide. Next, the culture supernatant was collected. To calculate the IC₅₀, Graph Pad Prism-6 was the software used. Photographs were taken under an Olympus ek2 inverted microscope using a camera (AmScope Digital Camera 10 MP Aptima CMOS). Results were presented as the mean±SEM.

% Viable cells = (A_test/ A_Control) *100

(A _{test =}Absorbance of test sample)

(A _{Control =}Absorbance of Control)

RESULTS:

AMY-loaded nanosponges were successfully prepared using ultrasonication assisted-emulsion solvent evaporation method to target the colon area for the successful treatment of CC. In this design, we employed CCFD design for the successful development of Amy-loaded nanosponges. Independent variables with their coded value and response variables with their targets are given in Table 1.

Effect on particles size (Y1):

The particle sizes of AMY-loaded NS were found to be in the range of 120±10.5nm to 210±9.19nm, as shown in Table 2. The quadratic model for particle size was proposed with R² coefficient of 0.9686. The difference between the predicted R² (0.9467) and adjusted R² (7464) was reasonable, Adeq precision (21.124) indicates adequate accuracy of the adopted model to navigate the design space. The positive sign shows the direct relationship and negative sign represent the indirect relationship. The polynomial equation for particle size with coded value was given below.

Particle size = +137.10 + 25.67 A - 2.67 B -7.75 AB + 15.14 A² + 27.14 B² ………………………….. (1)

One-way ANOVA test indicates that the amount of Eudragit RS-100 and PVA had a significant effect on the particle size, with a P-value of >0.0001. Figure 1 (a and d) represent that Eudragit RS-100 has significant positive effect, which means an increasing the concentration of Eudragit RS-100 resulted an increased the particle size. On the other hand, PVA shows significant negative influence on particle size as decrease the concentration of PVA enhance the formation of nanosponges in increased size particles.

Effect on % Encapsulation efficacy (Y2):

The % EE of AMY-loaded NSs ranged from 40±1.1% to 78±2.1%, is displayed in Table 2. For the %EE, the quadratic model was proposed with an R² coefficient of 0.9763. The difference between the predicted R² (0.9470) and adjusted R² (0.9593) was reasonable, Adeq precision (24.126) indicating the adequate signal of adopted model to navigate the design space. The polynomial equation for %EE with coded value was given below.

% EE = +74.379 + 5.5 A + 2.66 B - 6.75 AB - 11.32 A² - 8.82 B²……………………………………………... (2)

The ANOVA findings demonstrated that the quantity of ERS-100 and PVA had a significant effect on the % EE, with a P-value of >0.0001. The 3D surface and contour plots represent that Eudragit RS-100 and PVA have significant positive effect, meaning that increasing the concentration of Eudragit RS-100 and PVA results in a higher %EE (Figure 1 (b and e)).

Effect on % CDR_24h (Y3):

The in-vitro drug release of AMY-loaded NSs ranged from 71.01±0.7 to 87.66±1.05%, as displayed in Table 2. ANOVA findings from statistical analysis indicated that the linear model was the best-fitting model for %CDR_24h with model R² coefficient of 0.9756. The 3D surface and contour plot of the effect of Eudragit RS-100 and PVA on the %CDR_24his depicted in Figure 1 (c and f). The polynomial equation for %CDR with coded value was given below.

%CDR_24h = +78.58 - 6.49 A - 1.83 B………………. (3)

The abovementioned equation shows the linear model, suggesting that the interaction between Eudragit RS-100 and PVA on the formulation response variables %CDR_24hwas insignificant. The predicted versus actual plot for % CDR_24h was qualitatively evaluated to compare the formulation response value to the predicted value from the developed model.

Statistical analysis and Optimization of nanosponges using CCFD design:

CCFD were used to investigate the effect of dependent variables on the formulation response variables. CCFD were used for the least number of experimental runs, to confirm and evaluate the nanosponges formulation by putting the response value obtained from the experiments by the DoE. The optimization process was done using graphical and numerical method to achieving the minimum Particle size, while maximum % EE and %CDR_24h. The predicted versus actual plot for particle size, % EE and %CDR_24h were qualitatively evaluated to compare the formulation response value to the predicted value from the developed model. The predicted vs. actual plot in Figure 2 (a-c) shows maximum correlation and agreement. Figure 2(d-f) shows the normal plot of residuals for this model which is very close to a straight line, indicating a normal distribution of residuals. Figure 2(g) represented the desirability of 0.731, indicating the optimum value for optimized formulation. The overlay plot shows the design space in Figure 2(h). The predicted and experimental value are shown in Table 3.

Figure 1. 3D-response surface and 2D-contour plots for Particle size (a and d), %EE (b and e), % CDR_24h (c and f).

Figure 2. Predicted vs. actual plot (a-c), normal plot of residuals (d-f), Desirability plot (g), and Overlay plot (h).

Table 2. AMY-loaded NSs formulation using Central composite faced design.

Runs	Eudragit RS-100 (X1)	PVA (X2)	Particle size nm (Y1)	Encapsulation efficacy (%) (Y2)	% CDR_24h (Y3)	PDI	Zeta potential
AF1	75	1.5	165 ± 5.14	58.30 ± 2.5	83.14 ± 0.15	0.563 ± 0.25	-3.80 ± 3.32
AF2	125	1.5	191 ± 7.41	55.43 ± 1.3	71.01 ± 0.71	0.608 ± 0.10	-2.50 ± 4.16
AF3	125	1	183 ± 3.04	69.01 ± 2.0	72.40 ± 1.12	0.493 ± 0.09	1.34 ± 3.06
AF4	100	0.5	172 ± 1.55	62.62 ± 0.8	79.84 ± 0.90	0.379 ± 0.21	1.14 ± 2.83
AF5	75	0.5	149 ± 5.03	40.32 ± 1.1	87.66 ± 1.11	0.423 ± 0.11	4.15 ± 3.68
AF6	100	1	133 ± 7.55	76.29 ± 1.6	79.08 ± 0.65	0.573 ± 0.01	1.78 ± 3.07
AF7	100	1	135 ± 6.07	74.13 ± 3.7	78.99 ± 1.63	0.783 ± 0.02	-0.02 ± 3.81
AF8	100	1.5	155 ± 1.75	69.46 ± 3.2	76.62 ± 1.50	0.878 ± 0.07	0.44 ± 3.80
AF9	75	1	120 ± 10.5	57.23 ± 2.8	85.87 ± 1.33	0.601 ± 0.05	2.38 ± 3.15
AF10	100	1	142 ± 14.2	71.25 ± 1.7	77.09 ± 0.92	0.703 ± 0.04	1.1 ± 3.715
AF11	100	1	136 ± 5.96	78.45 ± 2.1	77.26 ± 1.37	0.761 ± 0.09	-1.34 ± 2.08
AF12	100	1	141 ± 7.33	73.41 ± 2.7	78.30 ± 0.78	0.728 ± 0.06	-4.03 ± 3.02
AF13	125	0.5	210 ± 9.19	64.42 ± 1.8	74.29 ± 0.32	0.417 ± 0.03	-2.07 ± 1.95

Table 3. Predicted vs. experimental value on formulation response variables.

Formulation code	ERS-100 mg	PVA %	Particle size (nm)	% EE	% CDR_24h
Predicted value	86.468	1.015	127.714	68.516	82.042
Experimental value	86.468	1.015	154.4 ± 11.46	72.96 ± 1.06	83.191 ± 0.93

Morphological evaluation using Scanning electron microscope:

The morphology of the Amy-loaded NS (optimized formulation) was observed by SEM and depicted in Figure 3 (b). The SEM image showed that the surface of the nanosponges has a porous, uniform and roughly spherical.

ATR-FTIR analysis:

The FTIR spectra of optimized formulation, physical mixture, PVA, Eudragit RS-100, and pure amygdalin were represented in Figure 4 (a). The characteristic peaks of pure amygdalin were obtained at 3420 cm^-1, 2938 cm^-1, 2210 cm^-1, 1596.54 cm^-1, 1501.85 cm^-1and 1122.38 cm^-1, assigned to 0-H Stretch, C-H aromatic stretch, nitrile stretch, C-C aromatic stretch, C=C, and C-O-C aromatic stretch, respectively ⁹. The characteristic peak of ERS-100 was found at 2896.13 cm^-1, 1739.12 cm^-1, 1136.46 cm^-1 assigned to -CH3 band, C=O stretch, C-O stretch ³⁴. PVA peaks were detected at 3246.26 cm^-1 (O-H stretching), 2959.23 cm^-1 (asymmetric stretching vibration), 1716.13 cm^-1 (C=O stretching), 1485.17 cm^-1 (C-H bending), 1108.23 cm^-1 (C-O stretching), 804.32 cm^-1 (C-C stretching) ³⁵. There were no major changes in the characteristic peaks of the physical mixture in the spectra of amygdalin, Eudragit RS-100 and PVA. This confirms that there was no chemical interaction between the drug and other substances. Moreover, the IR spectrum of the optimized formulation confirms the presence of amygdalin with its original functional properties, suggesting that there are no chemical interactions between amygdalin and polymer.

XRD Analysis:

The XRD analysis were used to confirm the crystallinity of amygdalin in nanosponges. The XRD spectra of optimized formulation, physical mixture, PVA, Eudragit RS-100, and pure amygdalin shown in Figure 4 (b). The sharp diffraction peaks with higher intensity at 2θ of 15.04, 18.42, 20.78, 22.18, 25.05 and 37.05º indicating the crystalline nature of amygdalin ⁹. Eudragit RS-100 showed the characteristic crystalline intense peaks at 2θ of 14.28 and 20.72º ³⁴. The PVA (polyvinyl alcohol) show a single sharp peak at 2θ of 16.85°, which shows less crystalline nature ³⁵. The diffractogram of physical mixture showed sharp peak at 2θ of 21.19°, indicating the decrease in the crystallinity of amygdalin. The crystallinity of the physical mixture and the optimized formulation were similar, indicating the amorphous nature of the nanosponges formulation.

DSC Analysis:

DSC thermograms of pure amygdalin, Eudragit RS-100, PVA, physical mixture and optimized formulation are shown in Figure 4(c). Amygdalin showed endothermic peaks at 112.65°C and 223.63°C, the first peak indicates the slight change in thermal behaviour of amygdalin ³⁶. The second peak represents the melting point of amygdalin. ERS-100 showed two endothermic peaks at 81.38°C and 233.03°C ³⁴, and PVA showed endothermic peaks at 194.52°C ³⁵. The amygdalin peaks in the physical mixture were displaced at 192.09°C and 263.09 °C. The optimized formulation showed sharp endothermic peaks at 258ºC, closer to the peak of Eudragit RS-100, and suggesting that inclusion of amygdalin in the amorphous nanosponges core.

Kinetic release studies:

The kinetic release study for an optimized formulation was successfully investigated. The optimized formulation showed zero order kinetic release mechanism compared to other models. Zero order means drug was release from the nanosponges system in constant manner over the period of time. The R² of the optimized formulation was found to be 0.9726.

Figure 3. (a) Drug release studies of AMY-loaded NS at different pH 1.2, 6.8, and 7.4 (b) SEM image of nanosponges with magnification x950.

Figure 4. (a) ATR-FTIR spectrum of pure AMY (black), ERS-100 (red), PVA (blue), physical mixture (pink) and Optimized formulation (green). (b) XRD of pure AMY (black), ERS-100(red), PVA (light green), and physical mixture (blue). (c) DSC of pure AMY (black), ERS-100(red), PVA (light green), and physical mixture (blue).

Cell Viability assay:

The cytotoxicity effect of pure amygdalin on human colon cancer HT-29 cells line proliferation by MTT assay. The results of obtained from MTT assay showed that significant reduction was achieved in HT-29 cells by different dose of pure amygdalin, as shown in Figure 5. It was observed that pure amygdalin exhibited 52% inhibitory concentration at 1000 µM dose of amygdalin.

Figure 5. (a) MTT assay of different concentration of pure amygdalin against HT-29 (0-1000 µM). (b) Microscopic image at different concentration (0-1000 µM) treatment against HT-29 colon cancer cell line.

DISCUSSION:

In this investigation, Amy-loaded nanosponges were formulated for oral sustained release of amygdalin to the colon area. The nanosponges were successfully prepared prepared by exploring ultrasound-assisted emulsion solvent evaporation method followed by optimization through CCFD. The optimized formulation has a particle size of 154.4±11.46, a %EE of 72.96±1.06% and a %CDR_24h of 83.191±0.93%. The results from 3D-surface plot indicates that the Eudragit RS-100 and PVA had significant impact on particle size (Y1), %EE (Y2), and % CDR_24h(Y3). Figure 1 shows that Eudragit RS-100 has a significant positive effect, which means that an increase in the concentration of Eudragit RS-100 leads to an increase in particle size. Pandya et al.,³⁷ proved that high amount of Eudragit RS-100 might enhance the clustering of nanosponges and formation of larger size aggregation. Also, Kumar et al.,³⁸ had similar findings during the formulation of urea loaded microsponges. On the other hand, PVA shows a significant negative effect on particle size, as a decrease in PVA concentration leads to an of nano-sponges increased formation with larger particle size. This can be explained by increased viscosity of the internal phase, which leads to the formation of larger particles. The higher amounts of PVA make the system more stable, lower the surface tension and improve the steric stability of the resulting emulsion, leading to the formation of small particles. As shown in Equation 2, increasing the concentration of Eudragit RS-100 and PVA results in a higher % EE. This can be explained by the fact that increased concentration of Eudragit RS-100 in the formulation increased the % EE, which may be due to the high amount of polymer molecules to coat the maximum AMY molecules, resulting in a higher entrapment efficiency (%) lead. The positive effect of PVA leads to an increase in drug entrapment. This could be occurred by increasing the viscosity of PVA, making it more difficult for the drug to distribute at the interface, leading to retention of the drug in the NSs. As displayed in Equation 3, Eudragit RS-100 and PVA demonstrate the linear relationship at %CDR_24h.

ATR-FTIR results confirm the physical compatibility of drug and other substances utilized the formation of nanosponges. The spectra of the physical mixture and the optimized formulation were observed to be very close to the IR behavior of amygdalin, as shown in Figure 4 (a). Only some peaks were slightly sifted. The crystallinity of the drug and optimized formulation were not similar, indicating the amorphous nature of the nanosponges formulation. The thermal behaviour of the optimized formulation was examined through DSC instrument. The endothermic peak of drug was observed at 223°C. Because of the endothermic peak of ERS-100 occurred at 233.03°C, and the endothermic peak of optimized formulation occurred at 258°C, confirming the proper encapsulation of drug within the polymer and showed their amorphous core comprised amygdalin. The Nanosponges formulations were found to range from particle size of 120±10.5nm to 210±9.19nm, %EE of 40 ±1.1% to 78±2.1%, %CDR_24hof71.01±0.7 to 87.66± 1.05%, PDI of 0.417±0.11 to 0.878±0.75, and zeta potential of -4.03±3.02 to 4.15±3.68. SEM image confirmed the porous nature of nanosponges with spherical shape. The stability study confirms the optimized formulation was stable at 4 and 25°C. The cytotoxicity activity of pure amygdalin confirm that the significant reduction was observed in the cell viability of HT-29 colon cancer cell line.

CONCLUSION:

The present study demonstrated the successful preparation and optimization of AMY-loaded nanosponges using an ultrasound-assisted emulsion solvent evaporation method followed by optimization by CCFD. The optimized formulation showed a particle size of 154.4 ± 11.46, a %EE of 72.96 ± 1.06% and a %CDR_24h of 83.191 ± 0.93%. In addition, the compatibility studies were characterized using ATR-FTIR, DSC and XRD. The results showed that the drug and polymer are compatible with each other. The optimized formulation was stable. Furthermore, the cytotoxicity study of pure amygdalin showed higher cell viability over the HT-29 cell line. Overall, the present study provides a perspective for the development of nanosponges formulation as an effective delivery system for the effective treatment of colorectal cancer.

ABBREVIATIONS:

SEM = Scanning electron microscopy

ATR-FTIR= Attenuated Total Reflectance-Fourier Transformer Infrared

DSC = Differential Scanning Calorimetry

XRD = X-ray Diffraction

CCFD = Central composite face-centered design

DMSO = Dimethyl sulfoxide

AMY = Amygdalin

NS = Nanosponge

AUTHOR CONTRIBUTIONS:

Mahendra Prajapati contributed to the conceptualization, and execution of the experiment and wrote the whole manuscript, data curation, formal analysis, methodology, and investigation. Rohitas Deshmukh and Ranjit K Harwansh participated in conceptualization, supervision, reviewing, and editing.

DISCLOSURE STATEMENT:

The authors declare no conflict of interest.

ACKNOWLEDGMENT:

The authors acknowledge the organizations for immense support in providing SEM analysis of optimized formulations by Sophisticated Instruments Facility National Institute of Pharmaceutical Education and Research (NIPER-Raebareli) and detailed observations of DSC and P-XRD by Central Instrumentation Facility of Jiwaji University, Gwalior, M.P., India, particle size analysis conducted by Department of Chemistry, GLA University, Mathura, and other facility conducted by Institute of Pharmaceutical Research GLA University, Mathura, U.P., India.

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Received on 05.10.2024 Revised on 22.02.2025

Accepted on 10.05.2025 Published on 12.06.2025

Available online from June 14, 2025

Research J. Pharmacy and Technology. 2025;18(6):2746-2755.

DOI: 10.52711/0974-360X.2025.00394

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