Joint replacement surgery has become a boon for patients with bone diseases by restoring joint function. There are several benefits of arthroplasty surgeries including good quality life, symptomatic relief, regaining physical activity, improving mobility, and regaining everyday routine freedom. There is a greater demand for orthopedic implants as more than 1.8 million fracture cases (Liu L. et al., 2013). And over 1 million knee and hip implants are performed annually in the United States (Etkin and Springer, 2017). A majority of surgeries are performed in osteoarthritis or rheumatoid arthritis cases over elderly patients who suffer from joint deterioration (Zhang et al., 2014). It is estimated that by 2050 the elderly population of 65 years or above will increase two-fold from 40.2 million in 2010 to 88.5 million in the United States alone (Vincent and Velkoff, 2010). The incidence of joint arthroplasties has significantly risen over the last several years on a global scale and it has benefitted worldwide patients with bone ailments (Cataldo et al., 2010). A successful joint replacement surgery reduces pain and discomfort, reclaims independence and physical activities, and enhances joint function and the patient’s life quality (Osmon et al., 2013a). Approximately, 800,000 joint arthroplasties are performed yearly in the United Kingdom and the United States, with estimates that the number will rise to more than 4 million by 2030 (Kapadia et al., 2016).

Biomaterials used in prosthetic medical devices for joint arthroplasties include metals, ceramics, composites, polymers, and natural materials and other foreign bodies that are extremely vulnerable to bacterial infections causing implant failure, out of which up to 15% of infection-associated hip implant failures require revision surgery (Khatoon et al., 2018; Zimmerli, 2014). Implant-Related Infections (IRIs) limit the efficacy of antibiotics, generate resistance, and allow infection to recur. IRIs may be divided into Prosthetic Joint Infection (PJIs) and Fracture-Related Infections (FRIs), both posing greater clinical and surgical challenges. Specifically, Prosthetic Joint Infection (PJI) has proven to be a very devastating postoperative consequence of implant failures (Lamagni, 2014). Being the most significant consequence of joint prosthesis, PJI occurs in 1.5–2.5 percent in terms of first treatments and even to 20 percent for further revision interventions. Moreover, the PJI is one of the most frequent causes (23%–25%) of revision surgery after knee arthroplasty, according to data from the United States and the United Kingdom (Osmon et al., 2013b). The PJI expenses are even up to 24 times more expensive including extended hospital stays, numerous surgeries, and extensive medical supplements (Osmon et al., 2013b). Among other factors that lead to orthopaedic implant failures are aseptic loosening and infection. Approximately, 18% of implant failures are attributable to aseptic loosening and 20% are related to implant-related infections (Raphel et al., 2016).

The primary factor causing implant-related infections or orthopaedic implant failure is biofilm formation on implants, which has been reported in patients who had undergone joint replacement surgery over 1–2 years (Kurtz et al., 2012). Among the most common biofilm formation is caused by the Staphylococci bacteria, whose one type, Staphylococcus aureus, is the most prevalent pathogen linked to total arthroplasty in the United States. Its other type, Staphylococcus epidermidis, is marginally more prevalent in Europe (Arciola et al., 2015; Montanaro et al., 2010). The Staphylococci can enter and remain inside host cells, enabling long-term retention of the microbes in bone by evading the immune system and antibiotic responses. As a result, bacteria form a biofilm, clustering into microorganisms and adhering to substrates and encasing in a sophisticated multicellular matrix (Aggarwal et al., 2013). Such complications need to be addressed on priority by finding out novel and effective measures to reduce such infections.

During the last 1 decade, several strategies and alternatives have been explored to resolve the issue of biomaterial formation in implant surgeries. These strategies have helped in modifying the surfaces of biomaterials, enhancing the delivery systems and creation of a novel and more effective antimicrobial substance. However, there is still a great need to initiate containment of implant-related infections and demonstrate an efficient antibacterial coating. Unfortunately, very limited technologies are available in the field of orthopedics and trauma that might offer a long-term solution.

Nanoparticles (NPs) are currently deemed to be an effective replacement for antibiotics which can potentially prevent infection on orthopedic implants (Sahoo et al., 2022). Metal-based nanoparticles, such as silver (AgNPs) and zinc oxide (ZnO NPs), particularly act as versatile tools to successfully resist bacteria. They disrupt bacterial cell membranes and generate ROS that damages bacterial deoxyribonucleic acid (DNA) and proteins, leading to cell death (Mondal et al., 2024; Gao et al., 2018). Thin-film coatings on medical devices, such as Ti₃Au–Ag/Cu alloys, have demonstrated significant reductions in biofilm formation, offering a promising strategy to prevent infections. Antimicrobial peptides (AMPs), like murepavadin, disrupt bacterial membranes and show efficacy against multidrug-resistant strains, presenting a novel approach to combat antibiotic resistance. These innovations contribute to reducing infection risks and limiting the development of resistance in healthcare settings (Sahoo et al., 2022; Savvidou et al., 2020).

There are several other novel initiatives like silver nanoparticles coated exterior fixation pins, proximal femur or tibia mega-prostheses, and bone cement containing silver nanoparticles (AgNPs), that have revolutionized orthopedic science, in general and arthroplasty, in particular (Savvidou et al., 2020). All these novel inventions exhibit the characteristic inhibition of infections. Green synthesis of silver nanoparticles, for example, is an effective method to reduce the toxicity and enhanced activity using natural plant extracts for implants (Vincent and Velkoff, 2010; Arciola et al., 2015). Another method used for checking biofilm formation on implants is the use of natural antimicrobial peptides (AMPs), which are proteins or peptides that are the component of the innate immune response (Manobala, 2024). The AMPs are found in all forms of life like plants, animals, algae, fungi, archaea, and protists. These AMPs have been proven to be alternatives to diverse antibiotics that inhibit the growth of bacteria, viruses, fungi, and malignant cells (Brown and Hancock, 2006). Among the common AMPs, the mollusks are a diverse class of invertebrates that comprises snails, mussels, slugs, and octopuses, with which several members reside in marine and estuaries (Zhong et al., 2013). Thus, mollusks can be a valuable source of AMPs since they have intrinsic defenses to combat harmful pathogens (Zannella et al., 2017).

The study premised that a conjugation with NPs is beneficial to the treatment of infection because of their antimicrobial properties. It is argued in this study that AMPs prove to be a novel antibiotic alternative, which can be conjugated with biomaterial to predict an improvement in biomaterial performance. Conjugating AMPs with nanoparticles enhances the availability of the peptide at the targeting site. Such a conjugation either immobilizes AMPs or incorporate them into other material. Hence, when AgNPs are combined with AMP, their conjugation builds a potentially high synergy which leads to an effective therapeutic solution. Moreover, the conjugation of AMPs with nanoparticles also improves stability, antimicrobial potency, and also reduces toxicity (Gakiya-Teruya et al., 2020; Pal et al., 2016). This requires adoption of novel strategies to design peptides with enhanced antimicrobial activities (Pal et al., 2016). A need was thus felt to synthesize AgNPs at nanoscales and judge the ensuing enhanced efficacies, and devise a novel, stable, and potent AgNPs@AMPs agent that can stabilize and reduce the antibacterial potential over implant surfaces. Hence, the study premised that conjugation of Ag Nanoparticles and AMP can provide a more potent therapeutic approach for prosthetic joint infection (PJI) because of their synergistic action. Peptide-nanoparticle conjugates (PNCs) are a recently developed technology for biomedical purposes. The synergy between the two potential classes of substances enables greater regulation over their biological functions, circumventing each substance’s intrinsic constraints (Liu M. et al., 2013).

In this study, we have investigated the potential of AMP-AgNPs conjugate coated implants (Polypropylene, titanium, and Stainless steel) in combating micro-organisms responsible for causing Prosthetic Joint Infection. This occurs because alloys of certain characteristics such as superior mechanical form, resistance to fatigue and anti-corrosion properties, are preferred to metallic biomaterials over other materials such as polymers and ceramics to produce implants (Burduşel et al., 2018). A significant objective of this study, therefore, lies in finding alternative biomaterial coating agents, improvising that such novel agents should be used as a substitute to enhance the inherent organic properties of coating materials that can improve its processability and help in retaining an adequate level of bioactivity within tissues (Shekhawat et al., 2021). In order to accommodate these novel coating materials, it is important to ascertain how implants should have a design that can fabricate an implant in accordance with the choice of potential biomaterial. The study aimed at finding the best biosynthesized silver nanoparticle conjugates for the use in joint replacement. It also examined such potent antimicrobial agents (AMP-Np conjugates) for the coating on joint replacement biomaterials that can inhibit the biofilm formation and prevent the PJI. Specifically, the study framed the following objectives:

To synthesize Ag nanoparticles by biosynthesis methods and determine its antibacterial efficacy against S. aureus and Staphylococcus epidermidis.

Due to longevity of life and increased rates of obesity, and accessibility to modern medical advancements, total knee arthroplasty has become a very common treatment. However, the greater is the rate of arthroplasties, the larger are the complications and number of failures and the greater is also the number of revision and repetitions of arthroplasties. The combination of AgNPs and AMPs to prepare the implant coatings would significantly enhance the antimicrobial properties of the implants and improve the success rate. It is hypothesized in this study that the AgNPs@AMPs will not only improve the functionality of the implant surface but will also enhance the state of fixation and monitor the bacterial resistance.

This study examined how AMP-AgNPs conjugate-coated implants can result in antibacterial synergism of NPs; with the highest level of such synergism was tested against S. epidermidis. Studies have reported the ability of AgNPs interaction of AMPs within their active groups (Shafagh et al., 2015; Naqvi et al., 2013), thus predicting a synergistic effect. Moreover, the AgNPs conjugates exhibited a more potent antibacterial effect against P. aeruginosa showing the same synergy when they interacted with different types of antibiotics after the conjugation of AgNPs to counter pathogenic microorganisms (Shahbandeh et al., 2021). It was also noted in this study that a higher synergy affected the NPs by enhancing the activity of AMP and cell penetration. The AgNPs and AMPs were less interactive but NPs alone facilitated the AMP penetration within the cell wall where the bactericidal action took place after the suppression of cell wall synthesis often ending with cell death (Masri et al., 2018). It was noted that the activity of AgNPs@AMPs was higher than that for biosynthesized AgNPs and AMP indicating the same trend against the tested microbes related to their large surface area and small size failing the easy penetration of the bacterial cell walls (Prabhu and Poulose, 2012).

In the end, a significant reduction of the antibacterial effect of the AgNPs@AMPs conjugate in relation to NPs activity was noted which requires. Further screening and tests in future studies.

This study thus accepted and validated the conjugation between biosynthesized AgNPs and AMP on bacteria. It also accepted the hypothesis that AgNPs@AMPs could induce only the membrane damage of bacteria; however, the pathways of AgNPs for the eradication of bacteria to treat bacterial infections still remain uncertain (Din et al., 2015). The silver nanoparticles (AgNPs) have been regarded as extremely promising for the development of new antimicrobials, which will provide a novel strategy for addressing a broad spectrum of bacterial infections. AgNPs can disrupt the bacterial cell wall, and inert the bacteria by attaching to its membrane proteins, and genetic material. Moreover, AgNPs can potentially interact with a multitude of enzymes, notably respiratory enzymes (López-Heras et al., 2015). The Hemigraphis colorata leaves-based AgNPs exhibited a maximum absorption peak at 415 nm, which is within the normal wavelength range of silver nanoparticles, i.e., 390–450 nm (Bethu et al., 2018), which corresponds with our findings.

The spherical-shaped nanoparticle with a size of 68 nm showed an absorption band at 418 nm (Reetha et al., 2020). The stability and surface charge of the AgNPs were examined using the zeta potential. Negatively charged surfaces help in maintaining the size and shape of nanoparticles by preventing them from aggregating and adhering together (Gakiya-Teruya et al., 2018). The surface area of the nanoparticles is one of the crucial factors determining antimicrobial potency. The AgNPs with the largest surface area illustrated the maximum release of Ag + against the bacterial cells, resulting in a significant bactericidal property (Paosen et al., 2017). These findings align with recent studies. For instance, a 2024 study by Nayak et al., demonstrated that PEG-grafted AgNPs formed stable assemblies, with short-chain PEG leading to dense, well-ordered films, while longer chains resulted in more complex structures (Zawadzka et al., 2014). Similarly, a 2024 study by Ogungbesan et al. reported that chitosan–silver nanocomposite films exhibited well-defined spherical AgNPs with uniform dispersion, as confirmed by SEM and TEM analyses (Nayak et al., 2025).

The potential interactions between AgNPs and the AMP conjugate as well as the functional groups found in the AgNPs were investigated using FTIR analysis. Similarly, the findings of (Ogungbesan et al., 2024). Reported the existence of a band at 1646.98 cm-1, which was determined to be the -NH2 stretch vibrations of amide. The broad peak at 3425.10 cm1 is made up of secondary amides. The current study reported that spherical-shaped nanoparticles with clear edges were observed in the SEM, and TEM images of AgNPs, whereas the addition of AMP leads to the aggregation of nanoparticles. The TEM analysis of AgNPs conjugated with antimicrobial Peptide Ubiquicidin (29–41) exhibited an increase in the average diameter as a result of peptide conjugation (Parasuraman et al., 2020), Morales-Avila et al., which is highly compromised with our findings.

AgNPs are typically utilized to transform the surface of orthopaedic implants in order to gain antibacterial and possible osteointegration properties (Morales-Avila et al., 2017). Retention of antibacterial activity for a long period of time to prevent the formation of biofilm is another crucial characteristic of implants. Biofilms are microbial populations that are adhered to a solid surface. Once the biofilm has developed on the implant surface, it prevents microorganisms from being treated with antibiotics and creates detrimental effect (Cheng et al., 2018). The MIC is commonly utilized as a significant indicator of antibacterial effectiveness. Our findings concluded that the extracted antimicrobial peptide from the Freshwater snail Bellamya bengalensis conjugated with biosynthesized AgNPs showed excellent antibacterial properties. Results from earlier studies suggested that conjugated peptides often kill microorganisms by rupturing the bacterial permeability barrier (Stoodley et al., 2002).

The conjugation of AMPs resulted in antibacterial synergism of NPs; the highest level of such synergism was against S. epidermidis. Studies have reported the ability of AgNPs interaction of AMPs within their active groups (Shafagh et al., 2015; Naqvi et al., 2013), thus predicting a synergistic effect. Moreover, the AgNPs conjugates exhibit a more potent antibacterial effect against P. aeruginosa (Shahbandeh et al., 2021), showing the same synergy when they interacted with different types of antibiotics after the conjugation of AgNPs to counter pathogenic microorganisms.

It was also noted in this study that a higher synergy affected the NPs by enhancing the activity of AMP and cell penetration. The AgNPs and AMP were less interactive but NPs alone facilitated the AMP penetration within the cell wall where the bactericidal action took place after the suppression of cell wall synthesis often ending with cell death (Masri et al., 2018). In the end, a significant reduction of the antibacterial effect of the Ag-NPs@AMPs conjugate in relation to NPs activity was noted which requires. Further screening and tests in future studies. A previous study done by (Hilpert et al., 2009), reported the significant anti-bacterial activity of the AgNPs synthesized using the aqueous extract of H. colorata leaves against the Staphylococcus aureus. Likewise, the AgNPs@AMPs complex exhibited higher anti-bacterial action against the S. aureus than the AgNPs or AMPs alone (Santhosh et al., 2018). AgNPs immobilized on the surface of titanium prevented bacterial adherence and icaAD transcription, which ultimately decreased the formation of bacterial biofilm. The immobilized AgNPs provide effective resistance against repeated bacterial attacks in vitro. Moreover, it lessens implant-related periprosthetic infection in-vivo (Zhou et al., 2023). The lowest MIC was recorded for the AgNPs-PEG-CIP, which demonstrated strong antibacterial activity with a considerable reduction in the MIC when compared to CIP and AgNPs alone (Qin et al., 2014). Likewise, the study of van (Ibraheem et al., 2022), Hengel et al., also disclosed the anti-biofilm activity of the AgNPs inserted into porous titanium implants against methicillin-resistant S. aureus.

SYTO9 dyes cannot cross undamaged membranes, however, they can attach to DNA when antimicrobial agents break the cell membrane and exhibit green fluorescence (van Hengel et al., 2017). The AMP-AgNPs coated in the implants disrupted the bacterial cell membrane and so intensified green fluorescence was observed in the microscopic investigation. A 2024 study by Lee et al. (2017). Reported that silver nanoparticles stabilized with C-pyocyanin significantly decreased biofilm formation in multidrug-resistant Pseudomonas aeruginosa and S. aureus, with CLSM images showing disrupted biofilm structures (Hussain et al., 2019). Similarly, a 2024 study by Manobala T et al. demonstrated that hydrogels containing biosynthesized AgNPs effectively inhibited biofilm formation by S. aureus and S. epidermidis, as evidenced by CLSM imaging (Chegini et al., 2024).

These studies corroborate our findings, highlighting the potent antibacterial and anti-biofilm properties of AMP-AgNPs conjugates. The biocompatibility of Nanoparticle Antimicrobial Peptide Conjugate with mammalian red cells and other nucleated cell types, primarily fibroblasts, osteoblast-like cell lines, and bone marrow-derived mesenchymal stem cells from human, mouse, rat, and rabbit, can be studied in vitro (Manobala, 2024). Red blood cells (erythrocytes) rupture during hemolysis, which is a sign that the red blood cells are being subjected to cytotoxic effects (Skerlavaj and Boix-Lemonche, 2023). The test samples are considered harmful to erythrocytes if there is more than 30% hemolysis (Velika and Kron, 2012), Table: 3 represents the RBC hemolysis activity of the AMP-AgNPs, which revealed that the 2x MIC of AMP-AgNPs exhibited a hemolysis percentage of 28.73 (less than 30%). Thus, it is considered to be safe, and non-toxic to humans. The limitation of hemcompatibility and enhanced mammalian cell toxicity were primary factors for the insignificant activity of silver-based antimicrobial coating on implants (Rasmussen et al., 2011).

Antimicrobial Peptides (AMPs) play a pivotal role in the innate immune system of invertebrates exhibiting a wide range of antimicrobial efficacy against bacteria, fungi, and viruses. AMPs have been viewed as prospective prospects for therapeutic purposes for the management of health ailments in humans, plants, and animals since the 1980s. AMPs have been regarded as a possible source for the development of new therapeutic compounds to treat infectious disorders for more than 30 years due to their considerable affinity for microorganisms and minimal toxicity for human cells. They are regarded as promising alternatives to traditional antibiotics to combat MDR bacterial infections (Ramstedt et al., 2009). The nanoparticles have a high promise for combating the problem associated with the multidrug resistance of bacteria (Manohar et al., 2019).

Nanoparticles conjugated with the antimicrobial agent can be used to lessen the toxicity of both substances toward human cells. Therefore, the combined antibacterial actions are boosted synergistically and the need for high dosages seems to be decreased (Lakkim et al., 2020). Hence, the use of nanoparticles is considered to be another effect. Active strategy for treating diseases caused by multidrug-resistant microorganisms. The antimicrobial properties of nanoparticles, particularly silver nanoparticles, are widely reported, and so conjugating them with AMPs has been suggested as a way to increase the activity (Allahverdiyev et al., 2011).

The current study demonstrated the potential of the AMP-AgNPs conjugate to inhibit the formation of biofilm by S. aureus and S. epidermidis. It showed how the antimicrobial activity of the AMP extracted from B. bengalensis can enhance significantly after the conjugation with AgNPs synthesized from H. colorata. Moreover, the 2X concentration of AMP-AgNPs does not exhibit any significant cytotoxic effects on the L929 cell line. However, it completely inhibited the biofilm formation of S. aureus and S. epidermidis. Hence, the AMP-AgNPs coated orthopedic implants will be the novel, non-toxic, and biocompatible approach for preventing Prosthetic Joint Infection.

The utilization of AgNPs as a pharmaceutical agent is restricted because of their susceptibility to cytotoxic effects against mammalian cells, despite their significant antibacterial properties and extensive pharmacological applications (Ramesh et al., 2016), But, our study demonstrated that the cytotoxicity of biosynthesized AgNPs conjugated with AMP against L929 cells was assessed using the MTT assay, which depends on the fact that viable cells convert MTT to purple formazan. As a result, the intensity of the color recorded at 550 nm was precisely related to the number of live cells. So, the AMP-AgNPs conjugate had no noticeable cytotoxic effects at 2X concentration.

Hence, the orthopedic implants could be modified with coatings with therapeutic substances to act as an antimicrobial defense, preventing infections from causing osteolysis and implant loosening. It is essential to prevent bacteria from adhering to the implant surface, to maintain a good interface between bone tissue and the substance, and to reduce the risk of infection while encouraging osseointegration (Mohanty et al., 2012). Last, but not the least, AMP-AgNPs have tremendous potential to be developed for commercial production. Future studies may determine what optimum conditions can be negotiated with AMP-AgNPs, and how the intensity of AgNPs can be increased within its limitations of small size and still retaining the stability. Such new studies may also examine any possible inhibitory mechanism of AgNPs in similar or different environment.