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Article

Immobilization of Bromelain on Gold Nanoparticles for Comprehensive Detection of Their Antioxidant, Anti-Angiogenic, and Wound-Healing Potentials

by
Amal Ahmed Ausaj
1,
Hanady S. Al-Shmgani
1,*,
Wijdan Basheer Abid
1,
Abdelalim A. Gadallah
2,3,
Abadi M. Mashlawi
2,
Mohsen A. Khormi
2,
Abdullah Ali Alamri
4,5 and
Emad Abada
2,6,*
1
Department of Biology, College of Education for Pure Sciences/Ibn Al-Haitham, University of Baghdad, Baghdad 10053, Iraq
2
Department of Biology, College of Science, Jazan University, P.O. Box 114, Jazan 45142, Saudi Arabia
3
Zoology Department, Faculty of Science, Mansoura University, Mansoura 35516, Egypt
4
Department of Physical Sciences, Chemistry Division, College of Science, Jazan University, P.O. Box 114, Jazan 45142, Saudi Arabia
5
Nanotechnology Research Unit, College of Science, Jazan University, P.O. Box 114, Jazan 45142, Saudi Arabia
6
Environment and Nature Research Centre, Jazan University, P.O. Box 114, Jazan 45142, Saudi Arabia
*
Authors to whom correspondence should be addressed.
Inorganics 2024, 12(12), 325; https://doi.org/10.3390/inorganics12120325
Submission received: 5 October 2024 / Revised: 6 December 2024 / Accepted: 9 December 2024 / Published: 13 December 2024
(This article belongs to the Section Bioinorganic Chemistry)
Browse Figures
Versions Notes

Abstract

:
Bromelain (Br) is a proteolytic enzyme with various pharmacological properties, such as antibacterial, antioxidant, anti-inflammatory, anticancer, and anti-angiogenic properties. However, due to its low solubility and bioavailability, its absorption is low, so a delivery mechanism is needed to achieve the desired therapeutic effects. Bromelain was chemically synthesized and loaded onto gold nanoparticles (AuNPs). Different methods and techniques were used for detection and characterization, including color-change detection, UV spectroscopy, XRD, SEM, TEM, and FTIR. The in vitro antioxidant activity was detected using DPPH assays, and the wound-healing activity was investigated in mice. The current study revealed that the formulated AuNPs-Br showed effective antioxidant activity and the strongest wound-healing properties, as demonstrated by histopathological and in vivo studies, and showed anti-angiogenic effects.

1. Introduction

Every cell within our intricate and remarkable bodies has an inherent and crucial need for proteins. These proteins not only aid in the growth and development of our bodily structures but also play an important role in ongoing maintenance and repair processes [1]. Nevertheless, it is crucial to note that excess protein within the body can give rise to a host of complications and health issues [2]. For example, the accumulation of superfluous protein within our precious body tissues can lead to inflammation and unwarranted swelling. This excess protein can manifest a variety of severe conditions, such as heart disease, arthritis, and, most alarmingly, cancer [2,3]. To address this, the medical world has diligently sought and discovered invaluable remedies, among which proteolytic enzymes have been brought to the fore.
Proteolytic enzymes or proteases exhibit several pharmacological properties with possible clinical applications, including the ability to prevent and modulate various infections [4]. Proteolytic enzymes from multiple biological sources, including bacteria, fungi, marine organisms, venoms, viruses, and algae, have been tested regarding their anti-infective activities [5,6,7]. Among plant-derived proteases, bromelain (Br) is one of the most characterized for clinical use, mainly due to its combination of low toxicity and good pharmacological value. Br proffers immense therapeutic benefits by effectively breaking down and digesting excess protein molecules. Its remarkable ability to alleviate and counteract inflammation, diminish swelling, and address a host of health concerns is truly overwhelming. The discovery of Br and its extraordinary properties represent a significant milestone in medical research and offer hope for individuals grappling with the deleterious impact of excessive protein accumulation [8,9]. With rapid developments in technology, the medical applications of Br are expected to expand rapidly in the future; Br is considered a new alternative in patient management for some critical diseases.
Nanotechnology, an expanding field with new and exciting applications, plays a vital role in the biomedical industry, specifically in the development of new therapeutic interventions. Its focus is the specific delivery of therapeutics, the driving force of contemporary cancer therapy [10]. Nanoparticles (NPs) are useful for drug delivery partly due to their multimodality, magnetic resonance imaging capability, surface plasmon resonance (SPR), and ability to generate heat and facilitate imaging. The general medical issues surrounding drug-delivering nanomaterials have been widely addressed in the literature, with studies on reducing their costs and toxicity and increasing their efficiency and effectiveness [11].
Given their high surface-area-to-volume ratios, AuNPs can carry significant loads of various therapeutic compounds. This makes them useful in cancer therapy, as they can improve drug delivery efficacy, which is essential for minimizing side effects and enabling lower doses to be used [12]. In addition, their photothermal properties can enable selective cell ablation if the therapeutic agent is activated by temperature [13]. This is employed in light-triggered drug delivery systems, cancer plasmon photosensitizer carriers, and photo-induced hyperthermic therapies. Of particular relevance is their use for photosensitizer drugs, which are suitable for the treatment of deep-seated hypoxic tumors in photodynamic therapy due to the ability of AuNPs to load and deliver high payloads of photosensitizing agents to tumor sites [14,15]. Medication molecules may become more stable and bioavailable when encapsulated into NPs, which could also overcome other problems with medication delivery and therapy.
Angiogenesis can be divided into different processes, such as vasculogenesis, arteriogenesis, and lymphangiogenesis [16]. Vasculogenesis describes the differentiation of endothelial cells from mesodermal clump cells and the subsequent association of the clusters that they form into small channels or sheets. Arteriogenesis starts from pre-existing collateral vessels, becoming functional arterial conduits redirecting blood flow after ischemia. Lymphangiogenesis describes the formation of lymphatic vessels, structures essential for tissue drainage and maintaining appropriate fluid balance in the body. Classic angiogenesis by endothelial cells involves vascular sprouting and the formation of new capillary networks from pre-existing blood vessels under the control of multiple angiogenic regulators and pro-angiogenic factors. Also, controllers of angiogenic processes such as growth factors, growth-inhibiting factors, pro- and anti-angiogenic factors, and invasive proteolytic enzymes mediate extracellular matrix degradation and allow endothelial cells to produce the necessary changes to form new microvessels [17].
Different factors can influence the wound environment and the wound fluid during re-epithelialization [18]. It has been suggested that a lack of growth-promoting cytokines is the cause of chronic wounds. Furthermore, there might also be a net excess of growth-restricting cytokines. The possibility of using cytokines in therapeutic settings to speed up wound healing is being investigated due to the capacity to manufacture pharmacologically large amounts of pure cytokines. The exudate from wounds, or wound fluid, is a mixture of serum and proteins generated from the tissue. Although bacterial action may alter its composition, wound fluid is believed to represent the microenvironment of the wound from which it is collected. It was once believed that the extracellular matrix of the dermis was an inert framework with cells residing in its interstices. It is becoming clear that fibroblasts and the extracellular matrix interact dynamically and that the matrix can change how growth factors act, potentially influencing the fibroblasts’ activity during the wound-healing process.
Bromelain (Br) is a proteolytic enzyme that has demonstrated anti-inflammatory, anti-edematous, antithrombotic, and fibrinolytic activities. Bromelain’s characteristics reflect its ability to hydrolyze protein by cleaving peptide bonds at specific sites. When an externally applied protease does not produce the desired results, factors such as the enzyme’s ability to reach the depths of the wound must be considered. However, many studies have also shown that Br may inhibit cancer-related angiogenesis and invasion. Therefore, Br, as an anti-angiogenic molecule, should be considered a promising agent for preventing unwanted angiogenesis or promoting desirable angiogenesis in wound healing.

2. Results and Discussion

2.1. Preparation of AuNPs and AuNPs-Br

As gold salts were reduced with citrate to prepare AuNPs, the color of the boiled trisodium citrate dehydrate gradually changed from pale yellow to purplish and red with the dropwise addition of tetrachloroauric acid trihydrate solution (Figure 1). After that, Br was added to the synthesized AuNPs and the color gradually turned red to dark wine red. The produced AuNPs conjugated with Br were validated using UV spectrophotometry, based on the absorption spectra of the AuNPs at 520 nm and AuNPs-Br at ~534 nm (Figure 2). Also, the % of Br conjugated with AuNPs was calculated to be 83% using the standard curve. The shift in the maximum absorbance indicated the conjugation of Br molecules on the surfaces of the AuNPs. In the visible spectrum, AuNPs exhibit an SPR band at about 520 nm; the particle size impacts the SPR band. The wavelength is re-shifted, and the particle size increases. AuNPs smaller than 10 nm are mostly damped due to the phase changes induced by the higher rate of electron–surface collisions compared to that observed with larger particles. The dominant contributions from higher-order electron oscillations cause the band broadening to be evident for particles larger than 100 nm [19]. The average particle size of AuNPs decreases with an increasing Na3Ct to HAuCl4 molar ratio (MR), resulting in spherical AuNPs with varying diameters. The absorbance of AuNPs loaded with proteolytic enzymes, particularly at 534 nm, is influenced by several factors including the size of the NPs and the nature of the enzyme [20]. Studies indicate that AuNPs can enhance enzyme stability and activity through immobilization, which is particularly effective for enzymes like horseradish peroxidase (HRP) when modified with thiol groups [21]. The size of the AuNPs, typically around 95–100 nm, plays a crucial role in modulating enzymatic activity and catalytic behaviors [22]. Also, as the size of AuNPs increases, the SPR peak shifts toward longer wavelengths, influenced by quantum size effects, electromagnetic coupling changes, geometric effects, and plasmon damping. AuNPs exhibiting varying dimensions (16, 25, and 40 nm) were synthesized utilizing the citrate reduction technique. The dimensions of the particles were meticulously regulated by manipulating the precursor MR, with diminished ratios resulting in enlarged particles and a red shift in the SPR peak (520, 524, and 528 nm). The NPs were subjected to interactions with progressively elevated concentrations of histamine (spanning from 1 to 100 ppm), and the resultant alterations in the absorbance spectra and the chromatic characteristics of the solution were systematically observed [23]. UV–vis spectroscopy monitors changes in the SPR peak of AuNPs, which is sensitive to changes in size, shape, and the surrounding medium. Upon modification with Br, the SPR peak might shift to a longer wavelength (red-shift) due to an increase in particle size (if Br forms a coating or induces aggregation) [24]. Changes in the dielectric constant around the particles due to Br binding. The magnitude of the shift is an indirect measure of size change. Also, it indicates a larger size due to the formation of a protein corona (Br coating) or aggregation. Red shifts in the SPR peak suggest an apparent increase in size or changes in optical properties [25]. The notable size increase in AuNPs after modification with Br could result from several factors associated with the modification process. Bromelain, being a protein, binds to the surface of AuNPs through physical or chemical interactions, such as electrostatic interactions between charged residues in Br and the AuNP surface and covalent bonding (e.g., thiol groups from cysteine residues forming Au-S bonds) [26]. This results in the formation of a protein corona, which increases the hydrodynamic size of the NPs, as detected by UV–vis spectroscopy. Moreover, structural rearrangements of Br during adsorption onto AuNPs could lead to an apparent size increase due to partial unfolding or stretching of Br on the AuNP surface and multilayer formation if Br molecules stack on top of one another [27]. This effect contributes to an increase in the observed particle size, particularly in solution-based techniques like UV–vis. Interestingly, modification with Br may induce the aggregation or clustering of AuNPs due to changes in surface charge (e.g., from negatively charged unmodified AuNPs to a reduced charge after Br binding) [28]. Aggregation leads to a significant apparent size increase, visible in UV–vis. Br contains functional groups such as amino acids with thiol (-SH) or amine (-NH2) groups that can chemically reduce gold ions present in the solution [29]. This could lead to the formation of a thicker gold–protein layer, contributing to the observed size increase. Finally, Br adsorption increases the hydration layer around the NPs due to its hydrophilic nature by UV–vis [30].
FTIR analysis was used to determine the bioactive components in AuNPs, AuNPs-Br, and Br (Figure 3). The AuNPs present a peak at 3367 cm−1 for the O-H group. A peak was observed at 2924 cm−1 for the stretching vibration of C–H bond stretched alkane groups. These two bands, with their peaks at 1593 and 1396 cm−1, correspond to the symmetric COO- and are attributed to the C-O-H bending of carboxylic acid vibrations. In addition, absorption bands are observed between 1253 and 1076 cm−1, which correspond to the stretching vibration of the C-O bond and C-C bond.
The FTIR spectra for AuNPs-Br showed a peak at 3383 cm−1 for the O-H stretching vibration. A peak was observed at 2954 cm−1 for the stretching vibration of C-H bond-stretched alkane groups. On the other hand, their corresponding N-H bending vibration was seen at 1597 and 1562 cm−1, respectively. A sharp peak at 1396 cm−1 for the C-O-H bending was observed. Peaks at 1076 and 1033 cm−1 corresponded to the asymmetric and symmetric C-O-C stretching mode. The Br enzyme’s FTIR spectrum shows peaks at 3398.57 and 3321.42 cm−1, which are identified as representing amine compounds’ N-H bond groups, and peaks at 3545.16 and 3495.01 cm−1 that are identified as representing alcohol and phenol compounds’ O-H groups. The presence of the O-H group of phenolic and alcoholic compounds and an effective group of amino compounds was observed when the spectrum analysis findings for the AuNPs-Br and Br solutions were compared. For Br loaded on AuNPs, almost all of the peaks that define Br were also observed. These results are consistent with those of Kiroula et al. [31], who showed that the protonated amine group may form hydrogen bonds and interact electrostatically with the anionic AuNPs.
This could show that AuNPs-Br had naturally occurring Br solution components, proving that the bromelain-loading procedure on the AuNPs was successful.
XRD analysis was carried out to investigate the crystalline structure of the synthesized AuNPs (Figure 4). Multiple Bragg reflection peaks at 2θ values with ascending 77, 64, 44, and 38° are shown; these are indexed by planes 311, 220, 200, and 111, respectively, regarding the face-centered cubic alignment of gold and corresponding to the Joint Committee of Powder Diffraction Standards. By using the Scherrer formula, the average size of the AuNPs was determined; the peak intensity revealed a robust crystal structure in the AuNPs, and the samples had highly intense peaks at 111 and 200. The average crystallite size calculated with Scherrer’s equation using the width of the 38.20° (111) was 31.71 nm, while that for the peak at 44.43 (200) was 32.4 nm. For the AuNPs-Br diffraction peaks at 2θ values at 111, 200, 220, and 311, the size was 36 nm. Moreover, the AuNPs showed conjugation of all the diffraction peaks, proving that the AuNPs were structurally stable in the presence of bromelain. Because the Br-loading process did not affect the metallic core size, these results could be illuminated by the development of the organic layer. The crystallite size has an inverse relationship with the peak width. Consequently, the broadening of the diffraction peaks reduced as the crystallite size increased [32].

2.2. TEM

The transmission electron microscopy images showed that the AuNPs were formed with sizes between 35 and 44 nm, with a spherical shape (Figure 5). Spherical shapes are commonly observed in AuNPs synthesized through chemical reduction methods (e.g., citrate reduction in HAuCl4). The uniform spherical morphology is advantageous as it ensures consistent surface properties, optical behavior, and reactivity [33]. The 35–44 nm size range ensures good dispersibility in aqueous media and strong SPR in the visible spectrum, enhancing optical properties. Their moderate size and spherical shape enhance biocompatibility and cellular uptake efficiency. Consistent size ensures a predictable and sharp SPR response. Finally, the balance between size and stability allows for effective surface-mediated reactions. Bromelain, being a protein, forms a non-metallic shell that TEM may not resolve clearly, which may lead to a core size similar to unmodified AuNPs [34]. The size falls within the typical range for colloidal AuNPs used in biomedical applications, sensing, and catalysis.

2.3. Free Radical-Scavenging Activity

Antioxidants are essential for protecting tissues from oxidative stress caused by free radicals, which play a crucial role in damaging lipids, proteins, and DNA, leading to various health issues. In organic solvents, the DPPH free radical often produces a deep violet solution. According to a DPPH assay, AuNPs-Br demonstrated higher scavenging activity than AuNPs and Br alone compared to the control (vitamin C group). In particular, AuNPs-Br showed scavenging activity of 85.42 ± 1.5, 73.4 ± 0.71, 63.4 ± 0.9, and 48.57 ± 2.13% at 200, 100, 50, and 25 µg mL−1, respectively, while AuNPs showed activity of 77.14 ± 1.3, 61.45 ± 0.8, 58.93 ± 0.93, and 37.09 ± 2.01% and Br showed activity of 78.47 ± 1.04, 65.86 ± 0.42, 48.66 ± 0.21, and 40.9 ± 1.35% at the same doses, respectively (Figure 6). The results revealed significant scavenging activity at higher doses and indicated synergy in AuNPs-Br’s blocking of DPPH. The antioxidant function of AuNPs may be due to their chemical structure, as they contain a 4-hydroxyl group (-OH) that can supply an atom to the DPPH molecule [35]. According to several studies, pineapple peel may include bioactive substances such as flavonoids, phenol, carotenoid, and vitamin C, and these substances exhibit antioxidant activities [36,37,38,39,40]. Moreover, the presence of Br could have reduced free radicals due to the presence of a hydroxyl group decreasing the color intensity in the solution.

2.4. Serological and Hematological Analysis

As shown in Table 1, the results for the hematological parameters indicated significant increases in WBCs, RBCs, MCV, and platelets. However, there was a significant decrease in platelets in the treated group compared to the untreated group. AuNPs have antimicrobial and anti-inflammatory properties [41,42]. According to research, Br lowers platelet aggregation and inhibits platelets in a dose-dependent manner [43,44]. In addition, Br regulates the homeostasis of blood coagulation by decreasing the synthesis of new fibrin and increasing fibrinolytic activity. It has also been demonstrated that Br can influence blood coagulation by reducing prostaglandin E2 and thromboxane [45].
The serological analysis of ROS, TNF-α, and collagen III in the current study revealed another significant difference, as shown in Table 2: there was a significant decrease in ROS levels in the groups treated with AuNPs (107.94 ± 12.14 pg mL−1) and AuNPs-Br (202.02 ± 5.22 pg mL−1) compared to the negative control (365.46 ± 17.93 pg mL−1). There was also a decrease in TNF-α levels in the groups treated with AuNPs and AuNPs-Br (54.93 ± 16.35 and 71.96 ± 12.39 g mL−1, respectively) compared to the negative control group (315.19 ± 118.67 pg mL−1). The levels of collagen III showed a significant increase in both the AuNP and AuNP-Br groups at rates of 58.47 ± 6.39 and 39.60 ± 7.64 ng mL−1, respectively, compared to the control group, at 10.97 ± 3.28 ng mL−1.
Proteolytic enzymes improve the proliferation of endothelial fibroblast cells, their migration to the wound site, and their capacity to create and multiply DNA. They also digest the adhesion proteins, permitting cell migration [46,47]. These cells are involved in angiogenesis, the formation of extracellular matrix proteins (i.e., collagen III and fibronectin), and the expression of growth factors and cytokines essential for cell proliferation and tissue development [48].
Reactive oxygen species (ROS) have been demonstrated to impede the healing of wounds and exacerbate tissue damage by opposing the actions of some cytokines and TNF-α [49,50]. Researchers have investigated metal and oxide NPs, and their ability to remove ROS, with peroxidase activities, has significantly advanced research on the elimination of ROS. Bromelain reduces oxidative stress by increasing antioxidant expression and decreasing NADPH expression [51]. Thus, the anti-inflammatory and antioxidant properties of AuNPs and Br can hasten the healing of wounds [52].

2.5. Evolution of the In Vivo Wound Healing Activity

In vivo wound-healing activity in mice was detected by observing histologically stained microscopic images (Figure 7), from which the following main features were identified: new blood vessels (angiogenesis), the deposition of collagen, re-epithelialization, and inflammatory infiltration. The control image of tissue stained with H&E on the first day showed that re-epithelialization had begun, and newly generated granulation tissue had formed. Morphologically, the wound had a larger open size and had not been covered. The control group, compared to the other groups, showed slight improvement, with a smaller open wound size, less inflammatory cell aggregation, and more neovascularization. At the end of the experiment, the scar tissue was filled with a collagen matrix, almost covered with an epithelialization layer, and the completion of the inflammatory stage was accelerated.
The current results demonstrate significant variations in the wound closure rate. On day 5, the AuNP-Br group showed the highest closure percentage at 3.667 ± 0.33%, followed by the AuNP group at 4.967 ± 0.26%, and finally the Br group at 5.433 ± 0.29%, compared to the control group, where the wound closure rate was 5.433 ± 0.29% (Figure 8). Wound healing occurs in two main phases: First, starting with hemostasis, immediately after injury, blood vessels constrict and platelets aggregate to form a clot, preventing blood loss. The second phase is inflammation; in this phase, immune cells migrate to the wound site to prevent infection and clear debris. Signs include redness, heat, swelling, and pain. The third phase is proliferation, where new tissue forms as fibroblasts produce collagen and granulation tissue develops. Angiogenesis is the main key principle in this phase, and epithelial cells migrate to cover the wound. The last phase is maturation or remodeling, through which collagen is remodeled and strengthened, improving the tensile strength of the wound [53].
Nanomaterials can provide different functional platforms with antioxidant and antibacterial properties and allow for extended drug release and the delivery of signaling molecules to optimize wound healing [54]. Additionally, NPs can mimic the extracellular matrix; promote cell adhesion, migration, and proliferation; and alter signaling pathways related to tissue regeneration [55]. Moreover, collagen was deposited more regularly in the AuNP-Br group than in the other groups, indicating that Br in association with NPs promoted the development of new tissue. Several pathways, including the formation of a new blood route, a change in the membrane potential, the blockade of the ATP synthase enzyme, and the alteration of the ROS status inside cells, ultimately impair energy metabolism and wound healing. This study showed that the synergistic combination of Br and AuNPs successfully accelerated the wound-healing process, contributed to the closure of wounds, and facilitated the migration and proliferation of the key cells in the healing process.
Chick embryos are commonly utilized as a model for angiogenesis in the chorioallantoic membrane (CAM) experiment due to their sensitivity and economical advantage compared to other in vitro or in vivo models. The current study’s results are illustrated in Figure 9, showing that the vascular length density in the AuNP-Br group was higher than that in the AuNP and Br groups, which might be due to the synergistic work needed to block angiogenesis. However, the vascular density was significantly lower than that in the control group, in which a typical vascular model was observed. The CAM is considered an excellent model that bridges in vivo and in vitro experiments; this model allows for the rapid screening of NPs’ behavior under physiological conditions and offers a quick, practical, affordable, and morally acceptable way to qualify NPs. As NPs are trapped, the highly vascularized CAM resembles the converging and diverging vasculature of different organs (e.g., the lungs, spleen, and liver) [56]. Matrix metalloproteinases (MMPs) and serine proteases are the main two types of proteases involved in wound healing; several studies have reported that metal NPs can up-regulate MMPs. Also, increased VEGF expression and angiogenesis were associated with wound healing in research that used metal NPs as a drug delivery system [57]. It has been established that some plant extracts such as pineapple and green tea extracts contain bromelain and demonstrate anti-angiogenic effects through the suppression of phosphoinositide 3-kinase, protein kinase, and vascular endothelial growth factor receptor-2 (VEGFR2) [58].

3. Materials and Methods

3.1. Chemicals and Reagents

All the tissue culture media (RPMI-1640), fetal bovine serum, antimicrobial solution (penicillin and streptomycin), and EDTA–trypsin were purchased from Gibco, London, UK. Tetrachloroauric acid (HAuCl4,3H2O) was obtained from Fluka, Durham, NC, USA. 2-2-diphenylpicrylhydrazyl (DPPH) and bromelain (EC:3,4,22,32) were provided by Sigma Chemical, St. Louis, MO, USA. Collagen III (mouse), reactive oxygen species (ROS) (mouse), and tumor necrosis factor-alpha (TNF-α) (mouse) kits were obtained from Human, Hesse, Germany. The breast cancer cell line MCF-7 was a kind gift from Al-Nahrain University, Biotechnology Research Center, Baghdad, Iraq.

3.2. Preparation of the AuNPs and Immobilization of Bromelain

The adsorption of Br onto synthesized AuNPs typically involves several steps, as described below.

3.2.1. Synthesis of Gold Nanoparticles

The AuNPs were prepared using the citrate-reduction Turkevich standard method as described in [59]. A stock solution of HAuCl4 (10 mM) was first prepared by dissolving 1 g of HAuCl4 in 250 mL of distilled water, and the solution was kept in a dark brown bottle. Then, 25 mL of the stock solution was diluted to 250 mL to achieve a 1 mM final concentration, followed by heating to boiling point with stirring. A 2 mL volume of 1% sodium citrate solution (0.5 g in 50 mL of DW) was added with continuous stirring until a stable color change indicated nanoparticle formation.

3.2.2. Preparation of Bromelain Solution

Br was dissolved in deionized water at a final stock concentration of 10 mg mL−1 in a flask placed on a magnetic shaker in a room, followed by filtration and sterilization with filter paper [60].

3.2.3. Adsorption Process

One milliliter of Br solution was added drop-by-drop to the AuNPs at a ratio of 1:10 v/v for several hours following a protocol described by [24]. The resulting conjugated AuNPs-bromelain was purified using ultracentrifugation at 15,000 rpm for 15 min at 4 °C to remove unconjugated bromelain. The prepared solution was then wrapped in aluminum foil and kept at 4 °C for further use.

3.2.4. Characterization

The properties of the prepared NPs with and without Br were characterized using different techniques, including the use of a UV–vis spectrophotometer (Shimadzu 1900, Kyoto, Japan) with a range of 100–1100 nm, and X-ray diffraction (XRD) analysis was performed using a Philips PW1730 device (Amsterdam, The Netherlands) with a range of 20–80 degrees and 20 angles. Functional groups were identified using Fourier transform infrared (FTIR) spectroscopy, on a Shimadzu 1800 device (Kyoto, Japan), with a range of 400–4000 cm−1 and 4 cm−1 resolution. The NPs’ stability was evaluated through zeta potential analyses (Brookhaven Zeta PALS, Milton, UK), and a scanning and transmission electron microscope from Tescan Mira, Brno, The Czech Republic, was used to analyze the size and shape of the NPs [61].

3.3. Bromelain Standard Curve Preparation and Quantification Using Spectroscopy

Following Balakrishnan et al.’s [62] protocol, the quantity of Br attached to AuNPs was calculated using a Br standard curve (0–30 µg mL−1) via visible spectroscopy. Briefly, 2 mL of AuNPs-Br were centrifuged at 13,000 rpm for 15 min. Then, the supernatant was collected and observed by UV–vis spectroscopy.

3.4. Free Radical-Scavenging Activity

2,2-diphenyl-1-picrylhydrazy (DPPH) was used to measure the free radical-scavenging antioxidant activity [63]. To assess the capacity of AuNPs, Br, and AuNPs-Br to suppress free radicals, different concentrations of samples (25, 50, 100, and 200 µg mL−1) were mixed with a corresponding quantity (60 µM) of DPPH. Following one hour of incubation in the dark at 37 °C, the absorbance at 517 nm was spectrophotometrically determined. With L-ascorbic acid serving as the positive control, the scavenging activity was determined using the following formula:
Inhibition activity % = ((ABC − ABS)/ABC) × 100
where ABC is the control absorbance and ABS is the sample absorbance.

3.5. In Vivo Study

3.5.1. Experimental Design

Adult albino male mice were used in this study; 20 mice weighing 25 ± 5 g on average were maintained at the animal house/College of Education for Pure/Ibn Al-Haitham, University of Baghdad, Baghdad, Iraq. This protocol was approved by the committee of ethics of the University of Baghdad, DH/465, on 27/11/2022. All the animals were divided into 5 groups: Group one (G1) served as the negative untreated control group; group two (G2) was the positive control group treated with 1% silver sulfadiazine. Group three (G3) was treated with 1 mM AuNPs, the fourth group (G4) was treated with AuNPs-Br, and the fifth group (G5) was treated with 10 mg mL−1 Br. The mice were kept under conventional laboratory conditions and fed ad libitum.

3.5.2. Fabrication of Experimental Wounds

The wounds were created in mice after hair shaving on the dorsal side with an average area of 10 × 20 mm and about 5–8 mm away from the spinal cord and tail; 70% alcohol was used to sterilize the region. Then, a 13 mm wound was created using a blade for each animal. An identical procedure was used for all the animals in the center of the hair removal area, with one incision applied per animal [61].

3.5.3. Contraction of Wound Analysis

The materials used in this experiment included AuNPs, Br, AuNPs-Br, and silver sulfadiazine stabilizer (1%). They were applied daily to the affected area using a cotton swap. The wound’s healing progress was observed daily for 5 days based on naked-eye observation with a digital caliper and photography for the comparison of the apparent speed of recovery among the animal groups. The wound length (WL) proportion (speed of wound closure) was calculated according to the following equation:
WL (%) = (WL/day X mm2)/WL/day 0 (mm2) × 100
where X stands for a particular experiment day.

3.5.4. Biochemical Assays

After the experimental mice had been anesthetized with diethyl ether authorized for use in this study, a cardiac puncture was used to collect blood samples. About 0.5–1 mL of blood was transferred to gel tubes and then centrifuged at 4000 rpm for 15 min to separate the serum, which was then stored for 24 h before being used for the serological analysis. The levels of the inflammatory cytokine TNF-α, collagen III, and ROS were detected using ELISA kits provided by Invitrogen. Experiments were carried out according to the manufacturer’s instructions. Using an ELISA reader (VersaMaxTM, Molecular Devices, Sunnyvale, CA, USA), the absorbance at 450 nm was recorded (Figure 10). All the assays were performed in triplicate.

3.5.5. Histological Analysis

At the end of the in vivo wound healing experiment, the skin samples from the injured area were kept in a 10% formaldehyde fixative. They were then dehydrated with ascending grades of ethanol. After clearing the samples in xylene and embedding them in paraffin wax, thin slices (5 µm) were prepared for histological analysis through cutting, mounting, and staining with hematoxylin–eosin (H&E) [64]. The stained slides were examined and captured for further analysis using a digital camera and an Olympus BX41 microscope (Hamburg, Germany).

3.6. In Ovo Angiogenesis Study

Evaluation of Chorioallantoic Membrane (CAM)

According to Qaddoori and Al-Shmgani [59], the anti-angiogenic activities of the compounds of interest in this study were examined using a CAM assay. Ten fertilized chick eggs/group were incubated at 37 °C with 60–80% relative humidity, and a square window was opened in the shell after 72 h of incubation. The embryos were incubated for a further 24 h after the areas between the pre-existing vasculature had been covered with a disc of filter paper saturated with AuNPs, AuNPs-Br, and Br at varying concentrations around 100 µM, and PBS was used in the control group. The vascular length density and vascular density percentage were evaluated for each experimental group photographed using ImageJ software (Version 1.41).

3.7. Statistics

SPSS (V.23.0) was used to statistically analyze all the data, and one-way ANOVA and LSD were used to determine significant differences. The data are presented as means ± SEs; all values of p ≤ 0.05 were considered significant.

4. Conclusions

The current study investigated a simple, economical, and environmentally friendly method of immobilizing Br on AuNPs, and the products were examined using different techniques to confirm their stability and other characteristic properties. Bromelain’s enzymatic activity with AuNPs needs to be further investigated to determine the potential diverse applicability of Br and limitations on its use. Bromelain’s structural stabilization and increased specificity when combined with AuNPs are particularly advantageous for large-scale applications, significantly enhancing our comprehension of Br’s enzymatic activities.

Author Contributions

Conceptualization, H.S.A.-S. and W.B.A.; Methodology, A.A.A. (Amal Ahmed Ausaj), H.S.A.-S., W.B.A. and E.A.; Software, A.A.G. and M.A.K.; Validation, A.M.M. and A.A.A. (Abdullah A. Alamri) and E.A.; Formal analysis, A.A.A. (Amal Ahmed Ausaj), A.A.G., A.M.M. and E.A.; Investigation, A.A.A. (Amal Ahmed Ausaj), W.B.A., A.A.A. (Abdullah A. Alamri) and E.A.; Resources, M.A.K.; Data curation, A.A.G.; Writing—original draft, H.S.A.-S.; Writing—review & editing, M.A.K., A.A.A. (Abdullah A. Alamri) and E.A. All authors have read and agreed to the published version of the manuscript.

Funding

Deanship of Graduate Studies and Scientific Research, Jazan University, Saudi Arabia, through Project Number: GSSRD-24.

Data Availability Statement

All the data are presented within the article.

Acknowledgments

The authors gratefully acknowledge funding from the Deanship of Graduate Studies and Scientific Research, Jazan University, Saudi Arabia, through Project Number GSSRD-24.

Conflicts of Interest

All the authors declare no conflicts of interest.

References

  1. Jain, M.; Amera, G.M.; Muthukumaran, J.; Singh, A.K. Insights into biological role of plant defense proteins: A review. Biocatal. Agric. Biotechnol. 2022, 40, 102293. [Google Scholar] [CrossRef]
  2. Delimaris, I. Adverse Effects Associated with Protein Intake above the Recommended Dietary Allowance for Adults. ISRN Nutr. 2013, 2013, 26929. [Google Scholar] [CrossRef] [PubMed]
  3. Clemente-Suárez, V.J.; Martín-Rodríguez, A.; Redondo-Flórez, L.; López-Mora, C.; Yáñez-Sepúlveda, R.; Tornero-Aguilera, J.F. New Insights and Potential Therapeutic Interventions in Metabolic Diseases. Int. J. Mol. Sci. 2023, 24, 10672. [Google Scholar] [CrossRef] [PubMed]
  4. Mótyán, J.A.; Tóth, F.; Tőzsér, J. Research applications of proteolytic enzymes in molecular biology. Biomolecules 2013, 3, 923–942. [Google Scholar] [CrossRef]
  5. Contesini, F.J.; Melo, R.R.; Sato, H.H. An overview of Bacillus proteases: From production to application. Crit. Rev. Biotechnol. 2018, 38, 321–334. [Google Scholar] [CrossRef]
  6. Balakireva, A.V.; Kuznetsova, N.V.; Petushkova, A.I.; Savvateeva, L.V.; Zamyatnin, A.A., Jr. Trends and Prospects of Plant Proteases in Therapeutics. Curr. Med. Chem. 2019, 26, 465–486. [Google Scholar] [CrossRef]
  7. Singh, R.; Gautam, P.; Sharma, C.; Osmolovskiy, A. Fibrin and Fibrinolytic Enzyme Cascade in Thrombosis: Unravelling the Role. Life 2023, 13, 2196. [Google Scholar] [CrossRef]
  8. Majid, O.W.; Al-Mashhadani, B.A. Perioperative Bromelain reduces pain and swelling and improves quality of life measures after mandibular third molar surgery: A randomized, double-blind, placebo-controlled clinical trial. J. Oral. Maxillofac. Surg. 2014, 72, 1043–1048. [Google Scholar] [CrossRef]
  9. Colletti, A.; Procchio, C.; Pisano, M.; Martelli, A.; Pellizzato, M.; Cravotto, G. An Evaluation of the Effects of Pineapple-Extract and Bromelain-Based Treatment after Mandibular Third Molar Surgery: A Randomized Three-Arm Clinical Study. Nutrients 2024, 16, 784. [Google Scholar] [CrossRef]
  10. Dessale, M.; Mengistu, G.; Mengist, H.M. Nanotechnology: A Promising Approach for Cancer Diagnosis, Therapeutics and Theragnosis. Int. J. Nanomed. 2022, 17, 3735–3749. [Google Scholar] [CrossRef]
  11. Yusuf, A.; Almotairy, A.R.Z.; Henidi, H.; Alshehri, O.Y.; Aldughaim, M.S. Nanoparticles as Drug Delivery Systems: A Review of the Implication of Nanoparticles’ Physicochemical Properties on Responses in Biological Systems. Polymers 2023, 15, 1596. [Google Scholar] [CrossRef] [PubMed]
  12. Kim, D.; Jeong, Y.Y.; Jon, S.A. Drug-loaded aptamer−gold nanoparticle bioconjugate for combined CT imaging and therapy of prostate cancer. ACS Nano 2010, 4, 3689–3696. [Google Scholar] [CrossRef] [PubMed]
  13. Blau, R.; Krivitsky, A.; Epshtein, Y.; Satchi-Fainaro, R. Are nanotheranostics and nanodiagnostics-guided drug delivery stepping stones towards precision medicine? Drug Resist. Updates 2016, 27, 39–58. [Google Scholar] [CrossRef] [PubMed]
  14. Goddard, Z.R.; Marín, M.J.; Russell, D.A.; Searcey, M. Active targeting of gold nanoparticles as cancer therapeutics. Chem. Soc. Rev. 2020, 49, 8774–8789. [Google Scholar] [CrossRef]
  15. Wang, M.; Chang, M.; Chen, Q.; Wang, D.; Li, C.; Hou, Z.; Lin, J.; Jin, D.; Xing, B. Au2Pt-PEG-Ce6 nanoformulation with dual nanozyme activities for synergistic chemodynamic therapy/phototherapy. Biomaterials 2020, 252, 120093. [Google Scholar] [CrossRef]
  16. Kubis, N.; Levy, B.I. Vasculogenesis and angiogenesis: Molecular and cellular controls: Part 1: Growth factors. Interv. Neuroradiol. 2003, 9, 227–237. [Google Scholar] [CrossRef]
  17. Liu, Z.L.; Chen, H.H.; Zheng, L.L.; Sun, L.P.; Shi, L. Angiogenic signaling pathways and anti-angiogenic therapy for cancer. Signal Transduct. Target. Ther. 2023, 8, 198. [Google Scholar] [CrossRef]
  18. Pastar, I.; Stojadinovic, O.; Yin, N.C.; Ramirez, H.; Nusbaum, A.G.; Sawaya, A.; Patel, S.B.; Khalid, L.; Isseroff, R.R.; Tomic-Canic, M. Epithelialization in Wound Healing: A Comprehensive Review. Adv. Wound Care 2014, 3, 445–464. [Google Scholar] [CrossRef]
  19. Huang, X.; El-Sayed, M.A. Gold nanoparticles: Optical properties and implementations in cancer diagnosis and photothermal therapy. J. Adv. Res. 2010, 1, 13–28. [Google Scholar] [CrossRef]
  20. Mustafa, D.E.; Yang, T.; Xuan, Z.; Chen, S.; Tu, H.; Zhang, A. Surface Plasmon Coupling Effect of Gold Nanoparticles with Different Shape and Size on Conventional Surface Plasmon Resonance Signal. Plasmonics 2010, 5, 221–231. [Google Scholar] [CrossRef]
  21. Breausche, F.E.; Somerlot, A.; Walder, J.; Osei, K.; Okyem, S.; Driskell, J.D. Immobilization of Thiol-Modified Horseradish Peroxidase on Gold Nanoparticles Enhances Enzyme Stability and Prevents Proteolytic Digestion. Langmuir 2024, 40, 13957–13967. [Google Scholar] [CrossRef] [PubMed]
  22. Wu, C.S.; Wu, C.T.; Chen, C.; Huang, C.C.; Yeh, Y.L.; Yang, Y.S.; Ko, F.H. Catalytic behaviors in modulating enzymatic activity through different-sized gold nanoparticles. In Proceedings of the 2010 3rd International Nanoelectronics Conference (INEC), Hong Kong, China, 3–8 January 2010; pp. 334–335. [Google Scholar] [CrossRef]
  23. Rivera, R.B.P.; Unabia, R.B.; Reazo, R.L.D.; Lapening, M.A.; Lumod, R.M.; Ruda, A.G.; Omping, J.L.; Magdadaro, M.R.D.; Sayson, N.L.B.; Latayada, F.S.; et al. Influence of the Gold Nanoparticle Size on the Colorimetric Detection of Histamine. ACS Omega 2024, 9, 33652–33661. [Google Scholar] [CrossRef] [PubMed]
  24. Cedervall, T.; Lynch, I.; Lindman, S.; Berggård, T.; Thulin, E.; Nilsson, H.; Dawson, K.A.; Linse, S. Understanding the nanoparticle–protein corona using methods to quantify exchange rates and affinities of proteins for nanoparticles. Proc. Natl. Acad. Sci. USA 2007, 104, 2050–2055. [Google Scholar] [CrossRef]
  25. Shenoy, D.; Little, S.; Langer, R.; Amiji, M. Poly(ethylene oxide)-modified poly(β-amino ester) nanoparticles as a pH-sensitive system for tumor-targeted delivery of hydrophobic drugs. Biomaterials 2005, 26, 6583–6590. [Google Scholar] [CrossRef]
  26. Giri, K.; Trewyn, B.G.; Stellmaker, M.P.; Lin, V.S.Y. Stimuli-responsive controlled-release delivery system based on mesoporous silica nanorods capped with magnetic nanoparticles. Angew. Chem. Int. Ed. 2005, 44, 5038–5044. [Google Scholar] [CrossRef]
  27. Frens, G. Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions. Nat. Phys. Sci. 1973, 241, 20–22. [Google Scholar] [CrossRef]
  28. Murphy, C.J.; Gole, A.M.; Hunyadi, S.E.; Stone, J.W.; Sisco, P.N.; Alkilany, A.; Kinard, B.E.; Hankins, P. Chemical sensing and imaging with metallic nanorods. Chem. Commun. 2008, 5, 544–557. [Google Scholar] [CrossRef]
  29. Verma, A.; Stellacci, F. Effect of surface properties on nanoparticle-cell interactions. Small 2010, 6, 12–21. [Google Scholar] [CrossRef]
  30. Lynch, I.; Dawson, K.A. Protein-nanoparticle interactions. Nano Today 2008, 3, 40–47. [Google Scholar] [CrossRef]
  31. Kiroula, N.; Negi, J.S.; Singh, K.; Rawat, R.; Singh, B. Preparation and characterization of ganciclovir-loaded glutathione modified gold nanoparticles. Indian J. Pharm. Sci. 2016, 78, 313–319. [Google Scholar] [CrossRef]
  32. Oliveira, A.E.F.; Pereira, A.C.; Resende, M.A.C.; Ferreira, L.F. Gold Nanoparticles: A Didactic Step-by-Step of the Synthesis Using the Turkevich Method, Mechanisms, and Characterizations. Analytica 2023, 4, 250–263. [Google Scholar] [CrossRef]
  33. Eustis, S.; El-Sayed, M.A. Why gold nanoparticles are more precious than pretty gold: Noble metal surface plasmon resonance and its enhancement of the radiative and nonradiative properties of nanocrystals of different shapes. Chem. Soc. Rev. 2006, 35, 209–217. [Google Scholar] [CrossRef] [PubMed]
  34. Dykman, L.; Khlebtsov, N. Gold nanoparticles in biomedical applications: Recent advances and perspectives. Chem. Soc. Rev. 2012, 41, 2256–2282. [Google Scholar] [CrossRef] [PubMed]
  35. Romelle, F.D.; Rani, P.A.; Manohar, R.S. Chemical composition of some selected fruit peels. Eur. J. Food Sci. Technol. 2016, 4, 12–21. [Google Scholar]
  36. Saraswaty, V.; Risdian, C.; Primadona, I.; Andriyani, R.; Andayani, D.G.S.; Mozef, T. 1st International Symposium on Green Technology for Value Chains 2016; IOP Publishing: Bristol, UK, 2016; p. 012013. [Google Scholar]
  37. Saravanan, P.; Muthuvelayudham, R.; Viruthagiri, T. Enhanced Production of Cellulase from Pineapple Waste by Response Surface Methodology. J. Eng. 2013, 2013, 979547. [Google Scholar] [CrossRef]
  38. Ashij, M.A.; Al-Shmgani, H.S.; Khalil, A.A.; Mohammed, H.A. Synthesis and Characterization of Proteolytic Enzyme Loaded on Silver Nanoparticles. Ibn AL-Haitham J. Pure Appl. Sci. 2024, 37, 43–53. [Google Scholar] [CrossRef]
  39. Al-Rikabi, R.F.; Al-Shmgani, H.S. Evaluation of hesperidin protective effect on lipopolysaccharide-induced inflammation and lipid peroxidation in BALB/c male mice. Res. J. Pharm. Technol. 2018, 11, 5513–5516. [Google Scholar] [CrossRef]
  40. Abeed, B.S.; Al-Shmgani, H.S. Evaluation of the Potential Protective Role of Galangin Associated with Gold Nanoparticles in the Histological and Functional Structure of Testes of Adult Male Albino Mice Administrated with Carbon Tetrachloride. Baghdad Sci.J. 2024, 21, 2512–2521. [Google Scholar] [CrossRef]
  41. Pivodová, V.; Franková, J.; Galandáková, A.; Ulrichová, J. In Vitro AuNPs’ Cytotoxicity and Their Effect on Wound Healing. Nanobiomedicine 2015, 2, 7. [Google Scholar] [CrossRef]
  42. Sharma, M.; Gupta, N.; Pandey, E. Implications of nasal delivery of Bromelain on its pharmacokinetics, tissue distribution and pharmacodynamic profile—A preclinical study. PLoS ONE 2022, 17, e0277849. [Google Scholar] [CrossRef] [PubMed]
  43. Errasti, M.E.; Prospitti, A.; Viana, C.A.; Gonzalez, M.M.; Ramos, M.V.; Rotelli, A.E.; Caffini, N.O. Effects on fibrinogen, fibrin, and blood coagulation of proteolytic extracts from fruits of Pseudananas macrodontes, Bromelia balansae, and B. hieronymi (Bromeliaceae) in comparison with Bromelain. Blood Coagul. Fibrinolysis 2016, 27, 441–449. [Google Scholar] [CrossRef] [PubMed]
  44. Hikisz, P.; Bernasinska-Slomczewska, J. Beneficial properties of Bromelain. Nutrients 2021, 13, 4313. [Google Scholar] [CrossRef] [PubMed]
  45. Ausaj, A.A.; Abed, W.B.; Al-Shmgani, H.S. The Use of Proteolytic Enzymes in Inhibiting Cancer as Anti-Angiogenesis Agents: Mechanisms of Action. Opera Med. Physiol. 2024, 11, 24–31. [Google Scholar]
  46. Isabela Avila-Rodríguez, M.; Meléndez-Martínez, D.; Licona-Cassani, C.; Manuel Aguilar-Yañez, J.; Benavides, J.; Lorena Sánchez, M. Practical context of enzymatic treatment for wound healing: A secreted protease approach (Review). Biomed. Rep. 2020, 13, 3–14. [Google Scholar] [CrossRef]
  47. Rahayu, P.; Agustina, L.; Tjandrawinata, R.R. Tacorin, an extract from Ananas comosus stem, stimulates wound healing by modulating the expression of tumor necrosis factor α, transforming growth factor β and matrix metalloproteinase 2. FEBS Open Bio. 2017, 7, 1017–1025. [Google Scholar] [CrossRef]
  48. Lord, M.S.; Ellis, A.L.; Farrugia, B.L.; Whitelock, J.M.; Grenett, H.; Li, C.; O’Grady, R.L.; DeCarlo, A.A. Perlecan and vascular endothelial growth factor-encoding DNA-loaded chitosan scaffolds promote angiogenesis and wound healing. J. Control. Release 2017, 250, 48–61. [Google Scholar] [CrossRef]
  49. Al-Shmgani, H.S.; Kadri, Z.H.M.; Al-Halbosiy, M.M.; Dewir, Y.H. Phytochemical analysis, cytotoxicity and antioxidant activity of cuckoo pint (Arum maculatum) leaf extract. Acta Biol. Szeged. 2020, 63, 119–124. [Google Scholar] [CrossRef]
  50. Zhang, D.Y.; Tu, T.; Younis, M.R.; Zhu, K.S.; Liu, H.; Lei, S.; Qu, J.; Lin, J.; Huang, P. Clinically translatable gold nanozymes with broad spectrum antioxidant and anti-inflammatory activity for alleviating acute kidney injury. Theranostics 2021, 11, 9904–9917. [Google Scholar] [CrossRef]
  51. Seiwerth, S.; Rucman, R.; Turkovic, B.; Sever, M.; Klicek, R.; Radic, B.; Drmic, D.; Stupnisek, M.; Misic, M.; Vuletic, L.B.; et al. BPC 157 and standard angiogenic growth factors. Gastrointestinal tract healing, lessons from tendon, ligament, muscle and bone healing. Curr. Pharm. Des. 2018, 24, 1972–1989. [Google Scholar] [CrossRef]
  52. Chen, C.H.; Hsia, C.C.; Hu, P.A.; Yeh, C.H.; Chen, C.T.; Peng, C.L.; Wang, C.H.; Lee, T.S. Bromelain ameliorates atherosclerosis by activating the TFEB-mediated autophagy and antioxidant pathways. Antioxidants 2022, 12, 72. [Google Scholar] [CrossRef] [PubMed]
  53. Schultz, G.S.; Chin, G.A.; Moldawer, L.; Diegelmann, R.F. Principles of Wound Healing. In Mechanisms of Vascular Disease: A Reference Book for Vascular Specialists [Internet]; Fitridge, R., Thompson, M., Eds.; University of Adelaide Press: Adelaide, SA, Australia, 2011; p. 23. Available online: https://www.ncbi.nlm.nih.gov/books/NBK534261 (accessed on 26 March 2021).
  54. Leu, J.G.; Chen, S.A.; Chen, H.M.; Wu, W.M.; Hung, C.F.; Yao, Y.D.; Tu, C.S.; Liang, Y.J. The effects of gold nanoparticles in wound healing with antioxidant epigallocatechin gallate and α-lipoic acid. Nanomed. Nanotechnol. Biol. Med. 2012, 8, 767–775. [Google Scholar] [CrossRef] [PubMed]
  55. Ali, H.I.; Mutlak, B.H. Synthesis and Characterization of Gold Nanoparticles and Gold Nanoparticles Loaded with Bromelain. Iraqi J. Phys. 2024, 22, 37–49. [Google Scholar] [CrossRef]
  56. Butler, K.S.; Brinker, C.J.; Leong, H.S. Bridging the In Vitro to In Vivo gap: Using the Chick Embryo Model to Accelerate Nanoparticle Validation and Qualification for In Vivo studies. ACS Nano 2022, 16, 19626–19650. [Google Scholar] [CrossRef]
  57. Salvo, J.; Sandoval, C. Role of copper nanoparticles in wound healing for chronic wounds: Literature review. Burn. Trauma 2022, 10, tkab047. [Google Scholar] [CrossRef]
  58. Zhafira Mafaz, Z.; Hariono, A.M.; Nurhidayat, L. Antiangiogenic Activity of Pineapple (Ananas comosus) Stem Extract on Chicken Embryo’s Chorioallantois Membrane (CAM). BIO Web Conf. 2024, 94, 02007. Available online: https://api.semanticscholar.org/CorpusID:268758629 (accessed on 3 May 2021).
  59. Qaddoori, M.H.; Al-Shmgani, H.S. Galangin-Loaded Gold Nanoparticles: Molecular Mechanisms of Antiangiogenesis Properties in Breast Cancer. Int. J. Breast Cancer 2023, 2023, 3251211. [Google Scholar] [CrossRef]
  60. Bayat, S.; Rabbani Zabihi, A.; Amel Farzad, S.; Movaffagh, J.; Hashemi, E.; Arabzadeh, S.; Hahsemi, M. Evaluation of debridement effects of bromelain-loaded sodium alginate nanoparticles incorporated into chitosan hydrogel in animal models. Iran. J. Basic Med. Sci. 2021, 24, 1404–1412. [Google Scholar] [CrossRef]
  61. Ashij, M.A.; Al-Shmgani, H.S.; Sulaiman, G.M.; Mohammed, H.A.; Abdalrazaq, E.A.; Albukhaty, S. Investigation of Antibacterial Activity and Wound Healing Promotion Properties Induced by Bromelain-Loaded Silver Nanoparticles. Plasmonics 2024, 19, 1903–1916. [Google Scholar] [CrossRef]
  62. Balakrishnan, S.; Bhat, F.A.; Raja Singh, P.; Mukherjee, S.; Elumalai, P.; Das, S.; Patra, C.R.; Arunakaran, J. Gold nanoparticle-conjugated quercetin inhibits epithelial-mesenchymal transition, angiogenesis and invasiveness via EGFR/VEGFR-2-mediated pathway in breast cancer. Cell Prolif. 2016, 49, 678–697. [Google Scholar] [CrossRef]
  63. Al-Shmgani, H.S.A.; Mohammed, W.H.; Sulaiman, G.M.; Saadoon, A.H. Biosynthesis of silver nanoparticles from Catharanthus roseus leaf extract and assessing their antioxidant, antimicrobial, and wound-healing activities. Artif. Cells Nanomed. Biotechnol. 2017, 45, 1234–1240. [Google Scholar] [CrossRef] [PubMed]
  64. Bayram, E.N.; Al-Bakri, N.A.; A-Shmgani, H.S. Zinc chloride can mitigate the alterations in metallothionein and some apoptotic proteins induced by cadmium chloride in mice hepatocytes: A histological and immunohistochemical study. J. Toxicol. 2023, 2023, 2200539. [Google Scholar] [CrossRef] [PubMed]
Inorganics 12 00325 g001
Figure 1. Bromelain (Br)-conjugated gold nanoparticles (NPs) undergo color changes. (A) Bromelain, (B) AuNP-Br, (C) AuNPs, (D) citrate, and (E) gold salt.
Figure 1. Bromelain (Br)-conjugated gold nanoparticles (NPs) undergo color changes. (A) Bromelain, (B) AuNP-Br, (C) AuNPs, (D) citrate, and (E) gold salt.
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Figure 2. UV–vis absorption spectra of bromelain (blue line), AuNPs (black line), and AuNPs-Br (red line).
Figure 2. UV–vis absorption spectra of bromelain (blue line), AuNPs (black line), and AuNPs-Br (red line).
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Figure 3. FTIR spectra of bromelain (blue line), AuNPs (black line), and AuNPs-Br (red line).
Figure 3. FTIR spectra of bromelain (blue line), AuNPs (black line), and AuNPs-Br (red line).
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Figure 4. XRD analysis of synthesized AuNPs (black line) and AuNPs-Br (red line).
Figure 4. XRD analysis of synthesized AuNPs (black line) and AuNPs-Br (red line).
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Figure 5. Particle size of AuNPs according to transmission electron microscopy photographs (A) and a histogram of the particle size distribution (C). The particle size of the AuNPs-Br was determined using transmission electron microscopy photographs (B) and a histogram of the particle size distribution (D).
Figure 5. Particle size of AuNPs according to transmission electron microscopy photographs (A) and a histogram of the particle size distribution (C). The particle size of the AuNPs-Br was determined using transmission electron microscopy photographs (B) and a histogram of the particle size distribution (D).
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Figure 6. In vitro antioxidant activity of AuNPs-Br, AuNPs, and bromelain according to DPPH assay. Significantly difference at * p ≤ 0.05.
Figure 6. In vitro antioxidant activity of AuNPs-Br, AuNPs, and bromelain according to DPPH assay. Significantly difference at * p ≤ 0.05.
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Inorganics 12 00325 g007
Figure 7. A section of skin in (A) the control untreated group (G1) shows a thick fibrin colt with necrotic debris (asterisk), a line of inflammatory polymorphic nuclear leukocytes in the cell zone (black arrows), and the degeneration of dermal collagen fibers, with necrosis (red arrows). (B) The positive control group treated with silver sulfadiazine (G2); the dermis is composed of immature granulation tissue (asterisks), with angiogenesis and mononuclear leukocytes (red arrows). (C) The group treated with AuNPs (G3), showing epidermal re-epithelization (E) and dermis with a thick layer of immature granulation tissue (g). (D) The group treated with AuNPs-Br (G4) shows a mature epidermis with keratinized stratified squamous epithelium (E), a dermis with a thick layer of mature fibrous tissue with a meshwork of newly formed blood capillaries, fibroblasts (asterisk), and newly formed hair follicles (arrows) and fat cells (F). (E) The group treated with Br (G5): E: epidermis; F: fibroblast; HF: hair follicle; SeG: sebaceous gland. Staining was performed with H&E. Magnification: 40×.
Figure 7. A section of skin in (A) the control untreated group (G1) shows a thick fibrin colt with necrotic debris (asterisk), a line of inflammatory polymorphic nuclear leukocytes in the cell zone (black arrows), and the degeneration of dermal collagen fibers, with necrosis (red arrows). (B) The positive control group treated with silver sulfadiazine (G2); the dermis is composed of immature granulation tissue (asterisks), with angiogenesis and mononuclear leukocytes (red arrows). (C) The group treated with AuNPs (G3), showing epidermal re-epithelization (E) and dermis with a thick layer of immature granulation tissue (g). (D) The group treated with AuNPs-Br (G4) shows a mature epidermis with keratinized stratified squamous epithelium (E), a dermis with a thick layer of mature fibrous tissue with a meshwork of newly formed blood capillaries, fibroblasts (asterisk), and newly formed hair follicles (arrows) and fat cells (F). (E) The group treated with Br (G5): E: epidermis; F: fibroblast; HF: hair follicle; SeG: sebaceous gland. Staining was performed with H&E. Magnification: 40×.
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Figure 8. The proportion of wound length closure over each experiment day. Four independent experiments were carried out, and the data are the mean ± SD.
Figure 8. The proportion of wound length closure over each experiment day. Four independent experiments were carried out, and the data are the mean ± SD.
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Figure 9. The effects of adding AuNPs, AuNPs-Br, and bromelain on newly growing blood vessel branches after 0 and 24 h of treatment in the chick embryo chorioallantoic membrane (CAM). NS = Non-Significant.
Figure 9. The effects of adding AuNPs, AuNPs-Br, and bromelain on newly growing blood vessel branches after 0 and 24 h of treatment in the chick embryo chorioallantoic membrane (CAM). NS = Non-Significant.
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Figure 10. Bromelain standard curve.
Figure 10. Bromelain standard curve.
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Table 1. Results of complete blood counts.
Table 1. Results of complete blood counts.
GroupWBCsRBCsPlatelets
(Mean ± S.E.)
Control (−ve)8.18 ± 0.17 a8.81 ± 0.01 a1204.5 ± 20.5 a
Control (+ve)8.51 ± 0.14 b8.54 ± 0.08 ab914.5 ± 1.5 ab
Bromelain10.50 ± 0.17 abc8.90 ± 0.07 bc1038.5 ± 15.5 abc
AuNPs8.68 ± 0.05 cd7.53 ± 0.01 abcd1157.5 ± 6.5 abcd
AuNPs-Br10.90 ± 0.33 abd9.11 ± 0.11 bd907.00 ± 10 acd
p value<0.001 **<0.001 **<0.001 **
Different letters represent significant differences at p < 0.05 between groups; Significantly difference at ** p ≤ 0.001.
Table 2. The serum levels of collagen III, ROS, and TNF-α at the end of wound closure.
Table 2. The serum levels of collagen III, ROS, and TNF-α at the end of wound closure.
GroupsROS
(pg mL−1)		TNF-α
(pg mL−1)		Collagen III
(ng mL−1)
(Mean ± S.E.)
Control (−ve)365.46 ± 17.93 a315.19 ± 118.67 a10.97 ± 3.28 a
Control (+ve)378.97 ± 76.31 b110.61 ± 1.38 a25.07 ± 1.46 ab
AuNPs107.94 ± 12.14 abc54.93 ± 16.35 a58.47 ± 6.39 abc
AuNPs-Br202.02 ± 5.22 b71.96 ± 12.39 a39.60 ± 7.64 abc
Bromelain455.46 ± 96.64 c216.62 ± 80.2424.21 ± 0.44 ac
p value0.008 **0.05 *<0.001 **
Different letters represent significant differences at p < 0.05 between groups; Significantly difference at * p ≤ 0.05, ** p ≤ 0.001.
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Ausaj, A.A.; Al-Shmgani, H.S.; Abid, W.B.; Gadallah, A.A.; Mashlawi, A.M.; Khormi, M.A.; Alamri, A.A.; Abada, E. Immobilization of Bromelain on Gold Nanoparticles for Comprehensive Detection of Their Antioxidant, Anti-Angiogenic, and Wound-Healing Potentials. Inorganics 2024, 12, 325. https://doi.org/10.3390/inorganics12120325

AMA Style

Ausaj AA, Al-Shmgani HS, Abid WB, Gadallah AA, Mashlawi AM, Khormi MA, Alamri AA, Abada E. Immobilization of Bromelain on Gold Nanoparticles for Comprehensive Detection of Their Antioxidant, Anti-Angiogenic, and Wound-Healing Potentials. Inorganics. 2024; 12(12):325. https://doi.org/10.3390/inorganics12120325

Chicago/Turabian Style

Ausaj, Amal Ahmed, Hanady S. Al-Shmgani, Wijdan Basheer Abid, Abdelalim A. Gadallah, Abadi M. Mashlawi, Mohsen A. Khormi, Abdullah Ali Alamri, and Emad Abada. 2024. "Immobilization of Bromelain on Gold Nanoparticles for Comprehensive Detection of Their Antioxidant, Anti-Angiogenic, and Wound-Healing Potentials" Inorganics 12, no. 12: 325. https://doi.org/10.3390/inorganics12120325

APA Style

Ausaj, A. A., Al-Shmgani, H. S., Abid, W. B., Gadallah, A. A., Mashlawi, A. M., Khormi, M. A., Alamri, A. A., & Abada, E. (2024). Immobilization of Bromelain on Gold Nanoparticles for Comprehensive Detection of Their Antioxidant, Anti-Angiogenic, and Wound-Healing Potentials. Inorganics, 12(12), 325. https://doi.org/10.3390/inorganics12120325

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