Biogenic Silver Nanoparticles for Wound Healing: A Comprehensive Review of Green Synthesis, Characterization, and Future Perspectives

The skin, the largest organ in the human body, performs numerous vital functions, including maintaining hydration, protecting against chemicals and pathogens, initiating vitamin D synthesis, facilitating excretion, and regulating body temperature. Consequently, severe skin injuries can be life-threatening ^[1]. Wounds, which are frequently associated with increased morbidity and, in severe circumstances, significant mortality, are characterized by disruptions in the normal anatomical structure and function of the body most commonly affecting the skin. Depending on the severity, such injuries may extend beyond the skin to involve the underlying subcutaneous tissue, muscle, tendons, nerves, blood vessels, and bone^[2]. Wounds are often referred to as a “silent epidemic” because, if left untreated, they can lead to severe complications including limb amputation and mortality. Approximately 8.2 million Medicare beneficiaries are affected by chronic nonhealing wounds each year. The economic burden associated with wound management is substantial, with estimated annual costs ranging from $28.1 billion to $96.8 billion, including expenses related to infection management. Among these, surgical wounds and diabetic ulcers are some of the most costly conditions. Furthermore, outpatient wound care expenditures ($9.9–$35.8 billion) exceed inpatient costs ($5.0–$24.3 billion), likely reflecting the increasing shift toward outpatient treatment strategies ^[3].

Many researchers have reported that normal wound healing typically occurs within approximately 6–8 weeks. However, factors such as infection by foreign pathogens, continuous irritation, repeated trauma, and ischemia can delay this process and lead to chronic wounds. Unlike acute wounds, chronic wounds fail to progress through normal stages of healing in a timely manner. Several pathophysiological factors contribute to delayed healing, including hypoxia, changes in wound pH, and bacterial colonization. Chronic wounds affect a significant portion of the population and can severely reduce the patient’s quality of life ^[3].

Effective wound management is crucial for optimal healing and prevention of complications. Traditional treatments, such as topical antibiotics and wound dressings, have limitations, including frequent dressing changes, adhesion issues, and inconsistent drug delivery systems. These challenges highlight the need for innovative therapies that provide controlled drug release and deliver multiple active agents to the wound site, thereby enhancing healing and reducing healthcare burdens^[4]. Nanotechnology-based drug delivery, particularly using AgNPs, has gained attention owing to its strong antimicrobial, anti-inflammatory, and wound-healing properties. At the nanoscale (1–100 nm), AgNPs exhibit enhanced surface reactivity, enabling the effective inhibition of a broad spectrum of wound-infecting microorganisms, while reducing inflammation and promoting tissue regeneration. These properties make AgNPs highly valuable for advanced wound care applications ^[5].

Therefore, this review aims to provide a comprehensive overview of wound healing biology, wound dressing technologies, and the emerging role of green-synthesized AgNPs in wound management. Special emphasis is placed on synthesis strategies, antimicrobial mechanisms, characterization techniques, current limitations, and future research directions toward clinical translation.

Fig.1. Schematic representation of skin injury

1.1 OVERVIEW OF WOUND HEALING

Wound healing is a complex and highly organized biological process in which the body repairs damaged tissue through a sequence of overlapping cellular and molecular events^[6].

Wound healing, irrespective of the wound type, is a complex physiological and dynamic process that proceeds through four overlapping stages. These phases progress continuously and in a coordinated manner. Any interruption in one of these stages can impair the overall healing process, potentially resulting in delayed healing or the development of chronic wounds. The four main stages of wound healing include hemostasis, inflammation, proliferation, and remodeling^[7].These stages are shown in Fig.2.

1.1.1 Phases of wound healing

Hemostasis

Immediately after injury, damaged blood vessels constrict to reduce blood loss, and blood clots form to prevent excessive bleeding. Platelets activate upon contact with subendothelial extracellular matrix (ECM) proteins, such as collagen, fibronectin, and von Willebrand factor, via receptors such as glycoprotein VI. Thrombin further triggers platelet conformational changes, degranulation of alpha/dense granules, and release of chemokines, growth factors (e.g., PDGF), and antimicrobial peptides via Toll-like receptors (TLRs), aiding immune cell recruitment, bacterial defense, and stimulation of fibroblasts/ keratinocytes, which further promote coagulation and initiate the healing process. An insoluble fibrin-based eschar forms with fibronectin, vitronectin, and thrombospondin, plugging the wound to prevent exsanguination while providing a scaffold for immune cells, cytokines, and early repair^[6].

Inflammation

The inflammatory phase is the second stage of wound healing and typically lasts for up to 6 days. This phase serves as an essential defense mechanism to prevent infection, remove damaged tissue, and prepare the wound bed for tissue repair. Innate inflammation acts as the primary protective response against pathogenic invasion and is initiated by injury-induced signals such as damage-associated molecular patterns (DAMPs) released from necrotic cells and pathogen-associated molecular patterns (PAMPs) derived from microbial components, which activate resident immune cells such as mast cells and macrophages

These activated cells release pro-inflammatory cytokines, chemokines, and vasoactive mediators including histamine, which promote vasodilation and increase vascular permeability. Endothelial cells also express adhesion molecules, such as selectins, which facilitate leukocyte adhesion and migration to the wound site. Neutrophils are the first immune cells recruited to the injured area, typically within 24–48 h. They perform essential functions, including phagocytosis of pathogens and necrotic tissue, and release reactive oxygen species (ROS), proteolytic enzymes, and antimicrobial peptides to control infection.

Following neutrophil infiltration, circulating monocytes migrate into the wound and differentiate into macrophages within 48–72 h. Macrophages are key regulators of wound healing, initially exhibiting a proinflammatory phenotype that supports pathogen clearance and later transitioning to an anti-inflammatory phenotype that promotes tissue repair. These cells secrete growth factors, such as transforming growth factor-β (TGF-β), platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF), which stimulate fibroblast proliferation, angiogenesis, and extracellular matrix deposition.

The inflammatory response is tightly regulated, as excessive inflammation can delay healing and contribute to chronic wound formation, whereas insufficient inflammation can impair pathogen clearance. Inflammation resolution occurs through neutrophil apoptosis and macrophage-mediated clearance, allowing progression to the proliferative phase. Thus, a balanced inflammatory response is essential for effective wound healing and tissue regeneration ^[3,6]

Proliferation

The proliferative phase is characterized by tissue repair, ECM formation, and angiogenesis, and typically begins a few days after injury and continues until complete wound closure. Fibroblasts and endothelial cells are the predominant cell types involved in this phase. Angiogenesis, which is essential for granulation tissue formation, is primarily regulated by growth factors such as VEGF-A, fibroblast growth factor-2 (FGF-2), PDGF, and TGF-β. Pro-inflammatory cytokines including interleukin-1 (IL-1) and TNF-α further stimulate fibroblasts to secrete growth factors such as epidermal growth factor (EGF), keratinocyte growth factor (KGF), and hepatocyte growth factor (HGF), which promote keratinocyte migration and granulation tissue formation. Keratinocytes originating from the wound margins and skin appendages migrate to the wound site, where they proliferate and differentiate to restore the epithelial barrier. Concurrently, wound contraction occurs through the action of fibroblasts and their differentiated form, myofibroblasts, which generate contractile forces that draw the wound edges together, thereby facilitating wound closure ^[3].

Matrix remodelling

The remodelling phase, also known as the maturation phase, represents the final stage of wound healing and is responsible for restoring tissue strength and structural organization. This phase typically begins a few days after injury and may continue for several months to years, depending on the wound severity and physiological conditions. It is characterized by decreased cellular activity, regression of the neovasculature, and progressive reorganization of the ECM.

During this phase, fibroblasts play a central role in ECM remodelling by replacing the provisional matrix composed of fibrin, fibronectin, and proteoglycans with a more organized collagen-rich matrix. A major hallmark of this stage is the gradual replacement of collagen type III, which predominates in early granulation tissue, with collagen type I, the primary structural collagen in normal skin. This transition significantly improves the tensile strength of the repaired tissue, although scar tissue typically achieves only approximately 70–80% of the strength of uninjured skin. Unlike the normal dermis, where collagen fibers exhibit a basket-weave arrangement, scar tissue shows parallel collagen bundle organization, reflecting incomplete structural restoration.

Matrix turnover during remodelling is tightly regulated by matrix metalloproteinases (MMPs) and their inhibitors, which maintain a balance between collagen synthesis and degradation. Fibroblasts also produce lysyl oxidase, an enzyme responsible for collagen crosslinking, which enhances matrix stability and mechanical strength.

Myofibroblasts, differentiated from fibroblasts under the influence of TGF-β and mechanical tension, contribute to wound contraction by generating contractile forces that reduce the wound size. As remodelling progresses, unnecessary cells including fibroblasts, macrophages, and endothelial cells undergo apoptosis or migrate away from the wound site, resulting in reduced vascularity and metabolic activity^[3,6]

Fig .2. Stages of wound healing

1.1.2 Factors affecting wound healing

Wound healing can be delayed or impaired by various factors, as illustrated in Fig.3. These factors can be broadly classified into local and systemic factors. Local factors directly affect the wound environment and its physical characteristics, whereas systemic factors relate to the individual’s overall health status or underlying diseases that influence the body’s healing capacity. In many cases, these factors are interconnected, with systemic conditions often exerting their effects by altering the local wound environment, ultimately impacting the healing process ^[8]

Fig.3. Factors affecting wound healing

1.1.2.1 Local factors

Temperature: The thermal state of the skin is influenced by various internal and external factors, where fluctuations in ambient temperature, surface humidity, body orientation, and circulatory dynamics can significantly ffect the healing process. Temperature affects wound healing by modulating the local vascular supply and extravasation of lymphocytes. In vitro Studies have shown that hyperthermia can enhance polymorphonuclear leukocyte chemotaxis and phagocytosis, as well as stimulate fibroblast proliferation. Physiologically, higher temperatures in acute wounds may improve dermal blood circulation and increase subcutaneous oxygen levels, creating a more favorable environment for the healing process^[9].

Infection: Disruption of the skin barrier allows microorganisms present on the surface to infiltrate the underlying tissues^[9]. The classification of a wound as contaminated, colonized, locally infected (critical colonization), or as having a spreading invasive infection depends on the state of infection and  their replication status^[8]. Endotoxins and bacteria can both prolong the inflammatory phase by elevating pro-inflammatory cytokines, such as TNF-α and IL-1, which may eventually cause the wound to transition into a chronic state. This prolonged inflammation increases the levels of matrix metalloproteases (MMPs) that degrade the ECM while decreasing natural protease inhibitors. This imbalance can lead to the rapid degradation of growth factors in chronic wounds. In infected wounds, bacteria form biofilms, which are complex communities of bacteria in a self-secreted extracellular polysaccharide matrix (EPS) that create protected microenvironments, making them more resistant to conventional antibiotics. Staphylococcus aureus, Pseudomonas aeruginosa, and β-hemolytic streptococci are prevalent in both infected and noninfected wounds. P. aeruginosa and Staphylococcus are significant contributors to wound infections, with many chronic ulcers likely remaining unhealed due to biofilms formed by P. aeruginosa. These biofilms protect bacteria from the immune response of polymorphonuclear neutrophils, which may account for the ineffectiveness of antibiotics in treating chronic wounds^[8].

Oxygenation: Oxygen plays a crucial role in cell metabolism and wound healing, facilitating processes such as contraction, angiogenesis, keratinocyte differentiation, reepithelialization, increases fibroblast proliferation and collagen synthesis. Inadequate tissue oxygenation, often due to systemic disorders such as aging and diabetes, leads to hypoxic wounds, hindering recovery. While temporary hypoxia can initiate healing, chronic hypoxia impairs it. Hypoxia signals acute wounds and stimulates macrophages, keratinocytes, and fibroblasts to release cytokines and growth factors. Reactive oxygen species (ROS) serve as cellular messengers, promoting healing mechanisms, but excessive ROS can cause further tissue damage ^[9]

1.1.2.2 Systemic factors

Age: Older individuals are more prone to impaired or excessive healing due to age-related metabolic and systemic changes, including epidermal thinning. Aging also affects the inflammatory response, leading to delayed leukocyte migration, reduced macrophage function, lower growth factor and cytokine production, slower re-epithelialization, delayed angiogenesis, and reduced fibroblast activity and collagen remodelling. In contrast, fetal wound healing occurs through different regenerative mechanisms with less involvement of inflammatory cells, as neutrophil and macrophage migration occurs later than in adults. This altered response contributes to differences in scar formation between fetal and adult skin ^[9,10].

Diabetes mellitus: It is a systemic disease that impairs wound healing by affecting leukocyte activation and increasing proinflammatory cytokine levels, leading to chronic inflammation. It also alters the skin microvasculature, creating a hypoxic environment that reduces angiogenesis. Furthermore, diabetes modifies the proliferation and differentiation of keratinocytes and fibroblasts, delaying re-epithelialization and extracellular matrix remodeling ^[10].

Sex hormones: Estrogen serves as an anti-inflammatory agent by diminishing leukocyte infiltration and proinflammatory cytokine release. It promotes wound healing through its effects on keratinocyte and endothelial cell proliferation and migration, enhancing re-epithelialization, and angiogenesis. Conversely, androgen hormones, such as testosterone and 5α-dihydrotestosterone, have a chronic inflammatory impact on wounds, hindering the healing process by increasing inflammatory cytokines and leukocyte migration ^[10].

Stress: Stress negatively impacts wound healing and systemic diseases by disrupting endocrine hormone regulation. It activates the nervous system and hypothalamus, leading to increased secretion of epinephrine, norepinephrine, cortisol, and glucocorticoids. These hormones reduce cytokine release and the immune response of leukocytes, impairing inflammatory processes and delaying healing.^[10]

Obesity: Obese individuals are at a higher risk of serious conditions, such as delayed wound healing, primarily due to hypoperfusion and ischemic symptoms in the subcutaneous adipose tissue. Insufficient oxygen delivery to the surrounding tissues hampers oxygen-dependent cellular repair mechanisms. Hypovascularity, prevalent in obesity, results in inadequate perfusion and increases infection risk by limiting immune cell access to the wound area.^[9]

Smoking: Smoking negatively affects physiological wound healing by inducing vasoconstriction, which disrupts the microcirculation. It decreases inflammation by impairing white blood cell migration, reducing neutrophil activity, and lowering IL-1 production. Moreover, smoking hampers fibroblast migration and collagen synthesis, inhibits epithelial regeneration, and reduce ECM output. Consequently, smokers experience poorer wound healing and more complications compared to non-smokers.^[9]

Genetic factors: Hereditary disorders, such as Down syndrome and ataxia-telangiectasia, are known to cause non-physiologic and delayed healing. Genetic abnormalities, including mutations and chromosomal aberrations, can lead to immune system disorders such as vasculopathies and connective tissue diseases. Klinefelter syndrome, marked by an extra X-chromosome, results in a higher incidence of varicosis and thrombosis, with a 13% chance of developing venous ulcers due to post-thrombotic syndrome. Additionally, disorders affecting haemoglobin synthesis, such as sickle cell anemia resulting from hemoglobin gene mutations, lead to sickle-shaped erythrocytes that cause vascular occlusions and ischemic damage ^[9]

2. WOUND DRESSING

Selecting an appropriate wound dressing material for specific wound types is crucial for optimizing healing. Unlike bandages that hold dressings in place, dressings must be in direct contact with the wound to be effective. For acute wounds, dressings help maintain exposure to pro-healing cytokines and growth factors, reduce trauma, minimize infection, and support the electrical gradient necessary for healing ^[11]. The different types of wound dressings and their characteristics are summarized in Table 1

2.1 Traditional wound dressing

Traditional wound dressings have been widely utilized because they are inexpensive, simple to manufacture, and easy to apply. This group of dressings includes gauze, bandages, plaster, and lint. They shield the site from external infections but cannot regulate the wound's moisture absorption, leaving it too dry for prompt healing. Additionally, they may become adherent to the wound in the event of significant wound drainage, making dressing removal difficult and painful. Consequently, conventional dressings are typically applied as secondary dressings or to wounds with modest extrusion ^[12].

2.2 Advanced wound dressing

Wound healing is aided by modern dressings that maintain and create a moist environment around the wound. They are mainly classified into hydrocolloids, films, foams, hydrogels, and alginates^[11].

Table 1. Types of wound dressing

3. BIOACTIVE WOUND DRESSINGS WITH ANTIMICROBIAL ACTIVITY

Currently, high rates of morbidity and mortality are caused by bacterial contamination of skin wounds. Several laboratories worldwide have begun creating antimicrobial wound dressings to stop wound contamination and address this health concern ^[14]. To provide sufficient wound healing without bacterial infection, a variety of wound dressing materials have been produced with intrinsic antibacterial behavior or combined with antibacterial agents in hydrogels, films, sponges, or nanofiber structures. Some research teams have recently focused on creating wound dressings containing polymers with inherent antibacterial properties, such as polyethyleneimine (PEI), chitin, and chitosan (CS). However, the antibacterial properties of these materials are insufficient to prevent wound infections. For improved treatment of wound infections, a variety of antibacterial drugs (like gentamycin, vancomycin, and neomycin), nanoparticles (like silver, gold, and zinc oxide), and essential oils were added to polymeric materials^[15].

4. NANOTECHNOLOGY AND SILVER NANOPARTICLES

Nanotechnology offers innovative and cost-effective wound-healing solutions using metallic nanoparticles (MNPs) with diameters of 1–100 nm. MNPs, derived from inorganic sources, outperform organic nanomaterials because of their unique physicochemical properties, allowing deeper penetration and better interaction with biological components. Their high surface area-to-volume ratio enhances drug delivery, promotes accelerated healing, and exhibits mechanical strength. Additionally, MNPs can facilitate controlled drug release and are effective against fungi and bacteria, making them strong candidates for medical applications in wound healing ^[16].

AgNPs have drawn a lot of attention among the available NPs because of their wide inhibitory effect against approximately 650 kinds of microorganisms, and more significantly, against bacteria that are resistant to antibiotics ^[14]. AgNPs are promising for various applications, particularly in the medical field, owing to their antimicrobial properties. They are used in wound dressings, artificial implants, antitumor drug carriers, and coatings for medical devices to prevent infections and enhance wound healing ^[15]. Due to their nanometric size and increased surface area, they possess significant antimicrobial properties. They can disrupt bacterial membranes, penetrate microbial cells via electrostatic interactions, and induce intracellular damage. The mechanisms of action include oxidative stress, metal ion release, and interaction with cell membrane proteins, particularly sulfur and phosphorus-containing compounds such as DNA. AgNPs lead to cell death by interfering with the respiratory chain and cell division processes ^[17,18]

5. GREEN SYNTHESIS OF SILVER NANOPARTICLE

5.1 Synthesis of Nanoparticle

There are two approaches for synthesis of nanoparticle: top-down and bottom-up

Top-down approach

The top-down approach involves breaking down bulk materials into nanoparticles using physical and chemical processes. Common techniques include mechanical milling, lithography, and etching, where external forces are applied to reduce larger structures into nanoscale particles. While lithography produces nanoparticles by designing and selectively removing material from a substrate, mechanical milling uses high pressure to shatter materials. Although this method offers good control over particle size and uniformity, it frequently requires specialized equipment and may not be able to produce nanoparticles with a variety of surface characteristics.

Bottom-up approach

The bottom-up approach involves the formation of nanoparticles through the assembly of atoms and molecules. This method is generally divided into chemical and biological synthesis methods.

Chemical methods

Chemical synthesis typically involves the reduction of metal precursors using chemical reducing agents such as sodium borohydride, hydrazine, and citrate to convert silver ions into silver nanoparticles. Stabilizing agents, such as polymers and surfactants, are often added to prevent aggregation and control the particle size. Common chemical synthesis techniques include sol–gel, co-precipitation, and hydrothermal methods.

Biological methods

Biological synthesis (green synthesis) uses plant extracts, microorganisms, and other natural biomolecules as reducing and stabilizing agents. This method is considered eco-friendly, cost-effective, and sustainable because it reduces the use of toxic chemicals and harsh reaction conditions. Biological methods are increasingly preferred for biomedical applications due to their biocompatibility and environmental safety.

5.2 Importance of green synthesis

Hazardous compounds like hydrazine and sodium borohydride, which are dangerous to human health and the environment, are often used in chemical methods. These techniques also frequently require a lot of energy and produce a lot of waste and hazardous byproducts, all of which go against the fundamentals of green chemistry. Also, these methods are less accessible and sustainable because they frequently require expensive, complicated equipment.

Green synthesis uses biological resources such as plants, algae, fungi, and bacteria, making the process safer for both human health and the environment (Fig.4). One major advantage of green synthesis is its eco-friendly nature, as it minimizes the use of hazardous chemicals and reduces the generation of toxic by-products. It also supports the principles of green chemistry by using renewable resources and non-toxic solvents such as water and ethanol. The process is typically energy-efficient, as it can be carried out under mild reaction conditions such as room temperature, reducing energy consumption compared to chemical methods. Another important benefit is the biocompatibility of the synthesized nanoparticles. In green synthesis, biological molecules act as both reducing and stabilizing agents, which can improve the biological activity and safety of nanoparticles for biomedical applications. Furthermore, the method is often simple, requiring fewer steps and less expensive equipment, making it more economically feasible and accessible ^[19].

Fig.4. Schematic representation of green synthesis of silver nanoparticle from plants and microorganisms

5.3 Green synthesis of silver nanoparticle from plant extract

The antibacterial activity of AgNPs produced biogenically utilizing plant extract as a reducing agent has been extensively studied. AgNPs are produced by the reduction of Ag? to Ag? by various biomolecules present in plant extracts, such as flavonoids, ketones, aldehydes, tannins, phenolic compounds, carboxylic acids, and proteins, which act as natural reducing and stabilizing agents.

Different plant parts such as leaves, roots, flowers, fruits, and rhizomes have been effectively used for the synthesis of AgNPs. various plant parts are gathered from different sources and thoroughly cleaned with both ordinary and distilled water to remove the unwanted materials. A detailed list of plant-derived AgNPs is provided in Table 2. After that, the pieces are either used fresh to make the extract or dried and processed to make a powder. AgNPs are produced by adding plant extract with varying pH levels to solutions containing varying concentrations of Ag salt as a metal precursor and heating the mixture at various temperatures. Because the biomaterials in the extract serve as both a stabilizing and a reducing agent for the formation of AgNPs, this synthesis method removes the need for chemical stabilizers ^[20].

Table 2: Examples for plant derived AgNPs^[21]

5.4 Green synthesis of silver nanoparticle from microorganisms

As biological systems for the environmentally friendly synthesis of AgNPs, microorganisms like bacteria, fungus, yeast, and algae have drawn a lot of interest(Table 3). Because they can use proteins, enzymes, and other biomolecules to convert silver ions into metallic silver, these microbes function as natural biofactories. Microbial synthesis may have drawbacks, such as the possibility of culture contamination, longer processing times, and little control over the size and shape of nanoparticles, despite benefits like cost-effectiveness and environmental friendliness.

Algae also represent an important group of microorganisms used for AgNP synthesis. It has been found that both macroalgae and microalgae create nanoparticles because they contain bioactive metabolites that serve as stabilizing and reducing agents. Examples include microalgae such as Chaetoceros calcitrans, Isochrysis galbana, and Tetraselmis gracilis, as well as marine macroalgae like Cystophora moniliformis. Because of their high metal tolerance, bioaccumulation capacity , and high secretion of extracellular enzymes, fungi are considered for the large-scale generation of AgNP. Because of these characteristics, fungal synthesis is frequently more effective than bacterial techniques.

In microbial synthesis, a silver precursor, such as silver nitrate (AgNO3), is added after the chosen microbe has grown in a sterile growth medium. After that, the reaction mixture is continuously stirred and incubated under regulated conditions typically in the dark to avoid photoreactions. UV-visible spectroscopy is used to monitor the production of AgNPs. The nanoparticles are then collected by centrifugation (around 3000 rpm for 10–15 minutes) and purified for use in other applications ^[35].

Table 3: microorganisms based AgNPs

6. CHARACTERIZATION OF SILVER NANOPARTICLE

The behavior, bio-distribution, safety, and effectiveness of nanoparticles are greatly influenced by their physicochemical characteristics. In order to determine characteristics like shape, surface chemistry, surface area, and the intrinsic variability of AgNPs, characterization is a critical stage in the green synthesis of nanoparticles^[60,61]

Fig.5. Characterization of AgNPs