Microwave- assisted and chemically tailored chlorogenic acid- functionalized silver nanoparticles of Citrus sinensis in gel matrix aiding QbD design for the treatment of acne

Background: Numerous studies have shown that various products of Citrus sinensis , for example, crude extracts, essential oil, and purified components, possess anti- acne properties. However, the development of chlorogenic acid- functionalized silver nanoparticles of C. sinensis in gel matrix aiding QbD design has not been evaluated for acne treatment. Aim: In this study, we have developed chlorogenic acid- functionalized silver nanoparticles of C. sinensis in a gel matrix employing a QbD approach for acne treatment. Material and Method: Citrus sinensis extract- loaded silver nanoparticles were


| INTRODUC TI ON
Acne, popularly known as acne vulgaris, is one of the most commonly occurring disorders majorly affecting the youth and adults within the age group of 11 to 30. This is basically an inflammatory disorder that gets initiated by Propionibacterium acnes, under the effect of the usual circulation of dehydroepiandrosterone. Also, females are more vulnerable to acne (50.9%) in comparison with males (42.5%). 1,2 Some factors that are responsible for causing acne are reformed keratinization, changes in the immune system, hormones, inflammation, etc. Apart from being a dermatological condition, this disorder further leads to depression and anxiety. 3 Many conventional formulations have been developed and widely used to treat acne, but these formulation results in causing irritation and allergic reaction. 4 For the treatment of acne, various products of medicinal plants such as Achyranthes aspera, Allium cepa, Azadirachta indica, Rosmarinus officinalis, and Citrus sinensis have been explored. 5,6 Citrus sinensis, a scientific name for sweet orange that belongs to the family of Rutaceae, is an excellent source of various components such as thiamine, niacin, calcium, vitamin C, potassium, magnesium, and folacin. The presence of anthocyanin and flavones in Citrus sinensis is responsible for providing pharmacological benefits such as chelation of metal and antioxidant effects. In addition to this, the seeds of Citrus sinensis are found to own antimicrobial effects. 7 So, to evaluate the antimicrobial effect of Citrus sinensis for the treatment of acne, metallic nanoparticles are used.
Metallic nanoparticle, mainly silver nanoparticle, has come into action by showing their antimicrobial effect due to the presence of varied surface area to volume ratio. 2 Taking into consideration the significance of the Citrus sinensis, our research focuses on the development of the gel of silver nanoparticles.
This study further involves the modification of the silver nanoparticle gel with chlorogenic acid (CA) using microwave due to its pharmacological effect of antibacterial, anti-lipogenesis, antioxidant, and anti-inflammatory. 8 Also, CGA is utilized as a bioreducing agent without the need for any additional harmful compounds that aid in achieving a higher yield of silver nanoparticles. 9 Phytochemicals such as chlorogenic acid may be added to the surface of AgNPs to increase their dermo-compatibility or antibacterial properties. In research, chlorogenic acid-loaded AgNPs were shown to have enhanced antioxidant activity, reduced cytotoxicity, and low skin deposition. Furthermore, QbD approach has been used for the development of chlorogenic acid fabricated gel of silver nanoparticles loaded with Citrus sinensis. Methanol LR grade and Carbopol 940 P NF were obtained from SD fine chemicals (Mumbai, India). Chemicals and solvents used in the investigations were of analytical quality, and HPLC water was used in all of the experiments.

| Preparation of plant extract
The plant extract was obtained by removing the dust from the peel by rinsing it with distilled water. Peel was then sun-dried to eliminate any remaining moisture. Later, using an electric blender, the peel was grounded into a powder. At 80°C, 2 g of powder samples was heated for 10 min in 100 ml of deionized water. The aqueous peel extract was then cooled and filtered using filter paper (Whatman No. 1). The filtered extract was then put in cold storage at a temperature ranging between 0 and 4°C for future experimentation. Because of their significant dielectric losses, alcohol and water are best for microwave heating stabilizers. For example, polar molecules like H 2 O try to get the electric field of the microwave to point in the right direction. When dipolar molecules try to move around in a changing electric field, they lose energy as heat; this and viscosity index of 159.69 g, 634.95 g s, −116.33 g, and −501.80 g s, respectively, indicating that the AgN-CA gel was stable.

Conclusion:
The current study showed that AgN-CA is an effective drug carrier system for the topical administration of C. sinensis extract for the treatment of acne.

K E Y W O R D S
chlorogenic acid, Citrus sinensis, CLSM, silver nanoparticles, TEM may be one of the reasons dipolar molecules contribute to the lowering of Ag+ ions. In addition to its high energy conversion efficiency, time-saving, cleanliness, and ease of use, the microwaveassisted approach may be employed to generate macro-scale production of highly dispersive chlorogenic acid-functionalized AgNPs. The solution combination was then microwave-irradiated (Panasonic, Model no. NN-CT645BFDG) at a set frequency of 2.45 GHz at a temperature of 90°C. The solution went from being clear to being a dark brown color. The generation of silver nanoparticles was first discovered by a change in color (as shown in Figure 1) and later verified by UV-Spectroscopy (Shimadzu UV-1800) that had shown lambda max more than 400 nm ( Figure 2).
After centrifugation, water-soluble particles were removed from the silver nanoparticles by washing them with distilled water and acetone. 10,11 Later on, AgN-CA nanoparticles were then lyophilized and stored for analysis.

| Optimization of AgN-CA
The three factors at three distinct levels of box-behnken design Absorbance (Y 2 ) of silver nanoparticles were explored systematically ( Table 1). To determine the optimal composition, these independent variables were measured at lowest (−), medium (0), and highest (+) amounts. To evaluate the influence of independent variables, the design represented 17 formulation runs (different compositions) with three centers ( Table 2) The best model is quadratic because the variables used had an effect on the dependent variables both on their own and when they were put together.

| Characterization of AgN-CA
3.2.1 | Particle size, polydispersibility index (PDI), and zeta potential (ZP) The particle size, PDI, and ZP of the generated AgN-CA were measured using Malvern Instruments, Worcestershire, UK. To assess the surface charge, the zeta potential was analyzed, and an optimal value lesser than −30 and more than +30 was deemed stable. The electrostatic potential ZP is proportional to the nanoparticles' surface charge. Nanoparticles having a ZP between the range of +10 and −10 mV are generally considered neutral. 12 Prior to the investigation, double-distilled water was used to dilute the samples 100-fold, and 0.45 μm membrane filters were used to remove the impurities. 13

| Transmission electron microscopy (TEM)
The morphological study of the optimized Citrus sinensis-loaded AgN-CA formulation was performed using a TEM-Tecnai, G20, Philips scientific instrument. After drying, a diluted drop of the specimen was tainted with phosphotungstic acid (1% w/v) and seen under a transmission electron microscope. Images of AgN-CA formulation showed that the developed particles were well-identified enclosed constructs with uniform size distribution and were perfectly spherical in shape, according to electron microscopy. 13,15

| Fourier transform infrared (FTIR) spectroscopy
The KBr pellet technique was used to obtain Fourier transform infrared (Perkin Elmer) spectra of optimized AgN-CA nanoformulation and Citrus sinensis extract in the range of 4000-400 cm −1 band in order to check extract and excipient compatibility. 16

| In vitro release study of drug
This study was carried out utilizing a dialysis bag diffusion method

| Confocal laser scanning microscopy (CLSM)
This study was done to compare the prepared AgN-CA formulation depth to that of rhodamine solution. To determine the depth of penetration, a rhodamine red B dye layered AgN-CA formulation was produced. In a similar fashion to the permeation research, this investigation was carried out. Fixed in a diffusion cell, the nasal mucosa was incubated with AgN-CA formulation that has been loaded with rhodamine and a rhodamine solution. As a check, a sample of rhodamine solution was employed, and extra rhodamine was removed and cleaned with double-distilled water. Later, the treated nasal mucosa was sliced into tiny pieces for microscopy after 6 h. The permeation of AgN-CA formulation across the nasal mucosal layers was examined using Leica TCS SPE (UK). Rhodamine B was excited by a 488 nm Argon laser beam, after which fluorescence emission was measured at 532 nm.
By using CLSM, the researchers were able to compare the AgN-CA nanoparticle's penetration depth with that of the control group. 17

| Loading of AgN-CA into carbopol 934P gel matrix
In a way to sustain the Citrus sinensis-loaded optimized AgN-CA formulation on the skin for a longer span of time, a gel was developed. For the development of a gel, a considered quantity of carbopol 934P, that is, 1% w/w, was suspended with double-distilled water to produce gel dispersion. This gel dispersion was kept overnight to allow it to swell.
Later, additional components, including polyethylene glycol 400, that is, 15% w/w, as a plasticizer and chlorocresol (0.1) as a preservative, as well as triethanolamine for pH adjustment, were added to the gel base described above. After this, the optimum AgN-CA formulation was dropwise added to this previously created gel with continuous mixing to produce a homogenous gel formulation referred to as AgN-CA Gel.

| Characterization and evaluation of AgN-CA gel 16-19
3.6.1 | pH The pH of the prepared gels was evaluated using a digital pH meter (Mettler Toledo). In 100 cc of distilled water, 1 g of gel was dissolved that was kept for 2 h at 4°C. Results of each formulation were provided as the average of three measurements.

| Extrudability
The technique used to assess the extrudability of AgN-CA gel was based on the % of gel squeezed out from the tube when a certain load was applied. More the quantity extruded better was extrudability. Filling the one-ounce tube with the study's formulation was done in a clean lacquered aluminum tube with a 5 mm nasal tip. A weight of 200 g to the bottom of the tube was applied in order to release the gel. For this experiment, the tip was used to measure how much gel was extruded through it. It was determined that the greater the gel's extrudability, the easier it is to apply.

| Spreadability
Gels with minimal spreadability but high consistency are preferred.
On a wooden block, a lower slide was attached to a glass slide TA B L E 3 Short term accelerated stability evaluation of CS loaded optimized AgN-CA formulation

| Texture analysis
Utilizing a texture analyzer (TA.XT. plus texture analyzer, USA), the texture of generated BNLCG was examined. The 50 g of optimum formulation (BNLCG) was put in a glass beaker of 100 ml, and the surface was kept as smooth as possible to prevent air bubble entrapment.

| Stability study
Accelerated stability of Citrus sinensis-loaded optimized AgN-CA formulation and gel were performed in accordance with the International

Conference on Harmonization of Technical Requirements for
Registration of Pharmaceuticals for Human Use (ICH-Q1A (R)).
Lyophilized AgN-CA formulation was taken in an Eppendorf, sealed, and kept in a stability chamber with a temperature of 30 ± 2°C and RH 65% ± 5%. The formulation, along with gel, was then evaluated once every month for changes in particle size, PDI, EE%, reconstitution time color appearance phase separation, clarity, homogeneity, pH, and drug content (as shown in Tables 3 and 4).     Table 5). The values of generated predicted R 2 by this design was found to be in adjusted R 2 also; different model graphs were also obtained that are shown in Figure 3, signifying that the data are equivalent to the models selected. However, the power of microwave presented a negative effect on the particle size resulting in decreasing the size. The ratio of surface area to volume increases, which ultimately results in decreasing in the particle size, which would enhance the overall surface area of the nanoparticle, thus, enhancing the absorption of the microwave.

| Optimization of Citrus sinensis loaded AgN-CA formulation by Box-Behnken design
Therefore, the more the power of the microwave, the more will be absorption, but less will be particle size. 23 Particle size (Y1) = +77.64 + 42.20A + 8.58B − 9.08C + 0.6300AB signifying that as the concentration increases, the absorbance value also increases. Correspondingly, the concentration of Citrus sinensis extracts also increases the value of the absorbance as described in the equation. Moreover, the power of microwaves also showed a positive effect on the value of absorbance (Table 6).

| Particle size and PDI
The optimized Citrus sinensis-loaded AgN-CA nanoparticles were found to have a particle size of 71.78 nm utilizing the dynamic light scattering approach with a nanozetasizer. A very low PDI of 0.2972 was found in the particle size distribution (Figure 4).

| Zeta potential
In general, ZP >+30 or <−30 mV is believed to provide sufficient repulsion forces to prevent particle aggregation. 24 Optimized AgN-CA nanoparticles had a ZP of −36.12 mV, assuring the stability of the colloidal dispersion ( Figure 5).

| Estimation of entrapment efficiency
The entrapment efficiency of Citrus sinensis-loaded AgN-CA nanoparticles was found to be 79.42% ± 6.79%.

| Electron microscopy imaging
Citrus sinensis-loaded AgN-CA nanoparticles have definite sealed structures with uniform size distributions, further presenting spherical forms in the TEM image of the optimized formulation ( Figure 6).

| Extract and excipient compatibility study using FTIR
FTIR spectroscopy was used as a tool for compatibility screening. As shown in the ( Figure 7A)

| In vitro release study
The release behavior of the prepared optimized Citrus sinensis loaded AgN-CA formulation and Citrus sinensis suspension was conducted to assess the release of Citrus sinensis, as shown in Figure 8.

| Characterization and evaluation of AgN-CA gel
The optimized AgN-CA gel ( Figure 11) was evaluated for a range of distinguishing characteristics; the findings are shown in

| Stability
For six months, the outcomes of short-term accelerated stability investigations were assessed. Over the course of the investigation, no variations in particle size, ZP, drug content, reconstitution time, color appearance, phase separation, clarity, homogeneity, pH, and drug content were detected (3 and 4).

| DPPH method-evaluation of antioxidant activity
The correlation between the antioxidant capacity of Citrus sinensisloaded AgN-CA formulation and a standard reference solution (ascorbic acid) was performed ( Figure 13).

| Assessment of antimicrobial activity of Citrus sinensis against Propionibacterium acnes
After the preparation of successive dilutions of the drug, MICs in multiples of 2 were generated. The order of MIC so obtained was found in the following fashion: Citrus sinensis (120.84 μg/ml), Citrus sinensis loaded AgN-CA (60.21 μg/ml), and lastly clindamycin phosphate (28.44 μg/ml), as the results suggest that the developed nanoparticle was observed to be moderately effective than clindamycin phosphate. However, Citrus sinensis developed nanoparticle were able to penetrate efficiently across the walls of the bacteria in comparison with clindamycin phosphate.

| CON CLUS ION
In this study, we successfully synthesized silver nanoparticles assisted by microwave using peel extract of Citrus sinensis. The Citrus sinensis-loaded AgN-CA formulation was stable. The outcomes of short-term accelerated stability investigations were assessed that supported the assumption that nanoparticles were stable throughout storage. The outcomes of the present investigation validated that the prepared optimized AgN-CA formulation is a valuable drug carrier system for Citrus sinensis dermal delivery for the management of acne vulgaris.

ACK N OWLED G M ENTS
This study was supported via funding from Prince Sattam bin Abdulaziz University (Project Number-PSAU/2023/R/1444).

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest associated with this research work.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.
F I G U R E 1 3 DPPH assay of Control sample (A), ascorbic acid (B), Citrus sinensis Extract (C), and Citrus sinensis-loaded AgN-CA formulation (D).