1 © 2025 The Author(s). Published by College of Education for Pure Science (Ibn Al-Haitham), University of Baghdad. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License Development and Characterization of Chitosan Nanoparticles Loaded with Amoxicillin as Advanced Drug Delivery Systems against Streptococcus Mutans Abdullah J Jasem1* and Maha A Mahmood2 1,2Department of Basic Sciences, College of Dentistry, University of Baghdad, Baghdad, Iraq. *Corresponding Author. Received: 17 May 2023 Accepted: 1 August 2023 Published: 20 January 2025 doi.org/10.30526/38.1.3501 Abstract This study's primary objective was to create nanotechnology-based, precisely regulated drug delivery devices. Antibiotic Amoxicillin was chosen, and chitosan nanoparticles (CSNPs) were chosen to transport the medicine. Using the ionic gelation process, chitosan solution NPs were synthesized using tripolyphosphate (TPP). Antibiotic-loaded chitosan nanoparticles (CSNPs) were then used to create a nanocomposite with good performance. The resultant nanocomposite can be put to use as an effective, non-toxic antimicrobial agent. CMCS has a considerably wider range of uses as an anti-bacterial agent than chitosan since it is soluble in a wide pH range. Due to its water solubility, Ten S. mutans isolates were purified in two mediums, making it easy to use and elicit a variety of biological activities of CMCS in pharma and cosmetics. Tryptone yeast extract cysteine sucrose mitis salivarius bacitracin agar. MS colonies on MSBA plates were blue, spherical or ovoid, 1-2 mm in diameter, with elevated surfaces that stuck well to the agar. Rough colonies had a rough or frosted glass surface. Samples treated with chitosan, carboxymethyl chitosan, chitosan nanoparticles, and their nanocomposite containing antibiotics (Amoxicillin) stymied the growth of Streptococcus mutans. Chitosan nanoparticles and their loaded antibiotics were discovered by scanning Electron Microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). In addition, the cytotoxicity of both the nanocomposite and amoxicillin loaded on nano-chitosan were compared to that of amoxicillin alone. PH 4.5 with a drug/polymer ratio of 1:2 (w/v) resulted in 89.33% entrapment and 53% loading efficiency for Amoxicillin in terms of their particle size, surface charge, bond interaction, and shape, respectively. A zeta potential of +24.5 mV and an average particle size of 258 nm were found through analysis. The results showed the superior antibacterial efficacy of the AMX-CSNPs. It can be concluded that AMX-CSNPs and CSNPs displayed acceptable physicochemical characterizations, and effective antimicrobial activities https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ https://doi.org/10.30526/38.1.3501 https://orcid.org/0009-0008-9210-1277 mailto:abdalsudani1@gmail.com https://orcid.org/0000-0003-3516-1309 mailto:adelmaha70@codental.uobaghdad.edu.iq IHJPAS. 2025, 38 (1) 2 against Streptococcus mutans. These formulations could enhance drug delivery for treating cariogenic bacteria causing dental caries. Keywords: Chitosan nanoparticles CSNP, carboxymethyl chitosan CMC, Fourier-transform infrared spectroscopy FTIR, Polydispersity Index PDI, ζ –average Zeta potential. 1. Introduction Dental caries (dental decay) can inflame, kill vital pulp tissue, and transmit infection to the tooth's periapex. Acidogenic plaque bacteria cause sickness (1). S. mutans, a crucial component of dental plaque, is often considered the foundation of the oral microbiota. Tooth decay (also known as "thetal caries") can cause inflammation and death of the pulp tissue, spreading infection to the tooth's periapex. Acidogenic plaque bacteria can lead to various illnesses (2). S. mutans, a crucial component of dental plaque, is often considered the foundation of the oral microbiota. Cariogenic bacteria like Streptococcus and Lactobacillus produce lactic acid during carbohydrate fermentation, lowering the local pH below the threshold value and demineralizing the tooth surface(47,48). Nanomaterials' compact size and high surface area per unit mass improves solution binding and dispersibility. Its antibacterial properties come from binding with proteins, plaque, and harmful bacteria. Nanoparticles' size and high surface-to-volume ratio may make them antimicrobial (46). They should interact closely with bacterial membranes to produce an antibiotic effect (3). Metallic and other nanoparticles combined with polymers and other base materials can be placed onto surfaces for antibacterial and anti-adhesive purposes (4, 5). Biomedical applications use chitosan because it is nontoxic, biocompatible, and antimicrobial. Deacetylating crustacean exoskeleton chitin produces the biopolymer chitosan. Positively charged chitosan dissolves in acidic to neutral liquids and sticks to mucosal surfaces. Regional medicine distribution may use chitosan nanoparticles (6). Chitosan is a cationic polymer made up of N-acetylglucosamine and glucose amine in a repeating structure. Protein carriers, drug administration, and wound healing are only some of the biological uses that could benefit from this material's biocompatibility, biodegradability, and nontoxicity (7-8). CSNPs excel in these areas due to their size, shape, and zeta potential. CSNPs can transport drugs (9, 10), vaccinations (11, 12), and even genes (13, 14). Studies have shown that chitosan nanoparticles are more effective than chitosan itself at eliminating bacteria and viruses (19). Slowly releasing medicines from chitosan nanoparticles may increase their solubility, stability, effectiveness, and toxicity (15, 16). Chitosan nanoparticles can be made in a number of ways (17). These include molecular self-assembly, template polymerization, reverse micelle, and emulsion cross-linking. When deciding on a preparation process, it is important to consider particle size, heat and chemical stability, as well as the stability of the finished product (18). The study's objective is to investigate whether chitosan-based nanoparticles could improve antimicrobial treatment. Many antimicrobial treatments are less effective when they can't get into cells. Some antibiotics, like amoxicillin, can be used without side effects to treat cariogenic bacterial infections better. IHJPAS. 2025, 38 (1) 3 2. Materials and Methods Chemicals used as analytical reagents were not purified. Merck Chemical Co. provided chitosan (MW = 60-120 kDa, deacetylation degree 85%), TPP, glacial acetic acid, and amoxicillin. Absorbance was measured using a PerkinElmer Lambda 25 spectrometer (Waltham, MA, USA). Morphological examination using SEM (TESCAN, MIRA III) was performed. Spectra were obtained using an FTIR spectrophotometer (Termo, AVATAR). The size distribution of NPs was determined using a Zetasizer Nano ZS dynamic light scattering spectrophotometer (Malvern Instruments, Malvern, UK). Solar energy produces Carboxymethyl Chitosan. Weight 220, Formula: C8H14NO6 Around 80% of the time, you can substitute. By letting 100 mg of CS dissolve in 100 mL of 1% acetic acid at room temperature overnight, the mixture was filtered using a 0.45 millimeter syringe filter. The addition of the ice cold TPP solution to the heating CS solution mixture resulted in the spontaneous generation of CSNPs. The synthesis of CSNPs was evidenced by opalescence in cloudy solutions. CSNPs were synthesized at pH 3.5, 4.5, 5.0, and 5.5 with CS to TPP mass ratios of 3 to 1, 4 to 1, and 5:1, respectively, and then analyzed by DLS. AMX was added to a solution of CSNPs at various drug/polymer ratios (1:1, 1:2, 1:3, 2:1, and 3:1) to create drug-loaded nanoparticles. The nanoparticle suspensions were centrifuged at 18,000 rpm for 30 minutes after being agitated for 1 hour. Pellets were lyophilized and stored to evaluate drug loading efficiency. AMX-CSNPs solution free Amoxi was calculated by monitoring absorbance at 317 nm. We compared two AMX standard curves. The comparison of two AMX standard curves was performed to ensure accuracy in determining free AMX concentration in the CSNPs solution, as monitored by absorbance at 317 nm (19). 2.1 AMX+CSNPs: A Characterization Zetasizer Nano ZS (Malvern Instruments, UK) was used to determine particle size and zeta potential. The He-Ne 633 nm laser was used to take the measurements at room temperature (25 °C). Overnight at room temperature, stabilization and sonication were performed on samples before measurements (19). DLS analysis was used to determine the size of the particles and surface charge of CSNPs synthesized at several pH levels (3.5, 4.5, 5.0, and 5.5) using CS to TPP mass ratios of 3:1, 4:1, and 5:1. 2.2 Fourier-transform infrared spectroscopy (FTIR): FTIR of intact CLM, NPs, and physical mixture were obtained from FTIR spectrophotometer: model (Teramo, AVATAR) Spectra were obtained by ATR technique. The scanning range was 4000-400 cm-1 (19). 2.3 Scanning Electron Microscopy (SEM): The nanoparticles' morphology was studied with scanning electron microscopy (SEM). After being air-dried at ambient temperature, the NPs were sputter coated with gold, mounted on metal stubs with adhesive tape, and examined using a scanning electron microscope (TESCAN, MIRA III) under high vacuum at an acceleration voltage of 20 kV. 2.4 Drug entrapment study Nanoparticle yield is the theoretical weight of the polymer and medicine used minus the nanoparticle weight. After dispersing nanoparticles by weight in 10 ml of PBS (pH 7.4), incubating IHJPAS. 2025, 38 (1) 4 for 25 minutes, and centrifuging at 19000 rpm for 20 minutes, the encapsulation efficiency was calculated. Amoxicillin absorbance of 247 nm was found in the supernatant. Amoxicillin loading capacity (L.C.) and amoxicillin encapsulation efficiency (A.E.E.) of the nanoparticles were determined using the following formulas. (19) Entrapment efficiency = (Drug Total − Drug free)/Drug total × 100 Loading capacity = (Drug Total − Drug free)/Weight of the nanoparticles × 100 2.5 Assay Cytotoxicity for Nano chitosan, amoxicillin and amoxicillin loaded chitosan nano particles The study investigates the growth and extracts of MCF-7 breast cancer cells. MTT assesses cell viability. A 24-hour 96-well plate experiment seeded 10,000 MCF-7 cells. Cells received different extracts after 24 hours. Cell viability was assessed after medium removal using MTT (5 g/L in PBS, pH 7.4) for 4 hours. Active cells precipitate MTT-soluble DMSO-soluble formazan. After gently shaking the plate to disperse the precipitate, absorbance was 570 nm instead of 630. Stable absorbance showed cell viability. Linear regression determined the extracts' 50% median lethal concentration (LC50), which halves cell viability. MCF-7 cells are seeded into a 96-well plate, treated with different extracts, assessed for viability using the MTT assay, and estimated for LC50s using linear regression analysis (20). 3. Results Amoxicillin-CSNPs had their drug loading and encapsulation efficiency maximized out using spectrophotometry. To quantify the amoxicillin loaded into CSNPs, the derivative o- Phthaldialdehyde Reagent was utilized to observe a shift in absorbance at 340 nm. Maximum encapsulation (89.33 2.7%) and loading efficiency (53.2 2.4) were observed in the formulation containing amoxicillin-CSNPs at a ratio of 1:2. Using dynamic light scattering, the hydrodynamic size and surface charge of CSNPs/AMOXICILLIN CSNPs in an aqueous media were determined. The poly-dispersity index for AMX-CSNPs was 0.19, and their average particle size was 258 nm (Figure 2a). For efficient medication distribution, it is crucial to pay attention to particle size and surface charge. Zeta analysis reveals the size distribution of the produced nanoparticles in Figure 3(A). AMX-NP's mean particle size is 258 nm. Table 1. UV-visible spectrophotometer analysis of AMX-CSNPs encapsulation and loading effectiveness. Mean standard deviation (n = 3). CSNPs: AMX ratio Encapsulation efficiency (%) wt Drug loading (%) wt Particle size Polydispersity Index PDI Zeta potential 1:1 69.05 ± 1.35 27 ± 2.2 237 ± 33.1 0.65 10.5 ± 4.4 1:2 89.33 ± 2.7 53 ± 2.4 258 ± 46.5 0.19 24.5 ± 4.8 1:3 85.28 ± 3.1 42 ± 1.9 314 ± 45.9 0.38 18.3 ± 4.8 2:1 38.54 ± 2.8 29 ± 2.3 368 ± 23.9 0.32 15.8 ± 4.8 3:1 39.39 ± 6.5 26 ± 1.6 289 ± 5.8 0.44 14.4 ± 4.9 IHJPAS. 2025, 38 (1) 5 Figure 1. Employing the Zetasizer Nano ZS, we determined the particle size distribution and Zeta potential value of various CSNPs to characterize their size and surface charges. A 2:1 Amoxi ratio 3.1 Fourier Transform Infrared (FT-IR) analysis The interaction between CS and Amoxicillin which confirms the encapsulation of Amoxicillin into the Nano carrier. Was identified by using FT-IR Technique: In the image below, we can see the FTIR spectra of CSNPs, AMX, and AMX-CSNPs. CSNPs showed a peak at 3500 cm1, consistent with the NH vibrational frequency, while AMX-CSNPs red-shifted to 3446 cm1, possibly due to the hydroxyl stretch (-OH). It is normal for C-H stretch vibrations to exhibit a peak at 2925 cm1. N-H bending in the NP causes a peak at 1627 cm1 that is red-shifted to 1634 cm1 in drug-loaded nanoparticles. We observed peaks at 1540 and 1558 cm1 due to the N-H bending of the amine in amoxicillin-specific nuclear peptides (-CSNPs). The drug's strong peak at 1053 cm1 and red shifts to 1098 cm1 were both caused by C-O stretching. Finally, we observed an 897 cm1 change in C-H out-of-plane bending. As a result, we know that CSNPs are loaded with AMX. Figure2: FTIR spectra of CSNPs (green), Amoxicillin (red), AMX-CSNPs (black) 3.2 Morphology Figure 3 displays the SEM of amoxicillin-loaded chitosan nanoparticles. The size of the particles fell within the nanoscale range, exhibiting a relatively spherical and regular form. We confirmed that the particles fall within the nanoscale range, possessing a smooth surface texture and a regular spherical shape. IHJPAS. 2025, 38 (1) 6 Figure 3: SEM showing chitosan nanoparticles loaded with chitosan nanoparticles 3.3 The MTT cell viability assay for cytotoxicity testing The statistical analysis demonstrated no significant cell inhibition with varied concentrations of chitosan nanoparticles, amoxicillin, and amoxicillin-loaded Nano Chitosan with TPP. The study materials are safe and non-toxic to MCF-7 breast cancer cells at various doses. Table 2 and Figure 3 a,b,c showMCF-7 cell cytotoxicity of chitosan nanoparticles, amoxicillin, and amoxicillin-loaded Nano Chitosan. The graphic shows that cell viability remained unaffected at all concentrations. This suggests that these materials are safe for cancer treatment and do not harm breast cancer cells. Figures 3 (B, C, D, and E) exhibit blue fluorescence staining (Hochst) of active nucleus cells. Image (b) shows breast cancer cell viability before therapy. Images (C-E) show MCF-7 cells that survived treatment with chitosan nanoparticles and amoxicillin-loaded Nano Chitosan with TPP at 16 to 250 μg/ml. Using pictures b to e, chitosan nanoparticles and amoxicillin-loaded Nano Chitosan with TPP were non-cytotoxic. Thus, they may be used as antibacterial and antifungal drugs in organs without harming healthy or sick cells. They can also fight oral infection-causing germs. (a) Control (no. treatment) (b) 16 (µg/ml) Concentration (c) 62(µg/ml) Concentration (d) 125 (µg/ml) Concentration (e) 250 (µg/ml) Concentration (f) 500 (µg/ml) Concentration Figure 4. Relative cell viability of Amoxicillin loaded chitosan nanoparticles (a) Control (no. treatment), (b) 16 (µg/ml), (C) 62(µg/ml), (d) 125 (µg/ml), (e) 250 (µg/ml), (f) 500 (µg/ml). IHJPAS. 2025, 38 (1) 7 Table 2. Show The MTT cell viability assay for cytotoxicity testing 3.4 Isolation of S.mutans Ten S.mutans isolates were purified in two mediums. Tryptone yeast extract cysteine sucrose mitis salivarius bacitracin agar Bacitracin Agar. The blue, spherical or ovoid, 1-2 mm in diameter MS colonies on MSBA plates had raised surfaces that adhered well to the agar. The surface of rough colonies was rough or frosted glass. While smooth colonies were spherical. Most MS colonies had a dip in the middle with a polysaccharide drop, although others were completely immersed (Figure 5). Figure 5. (a) S.mutans and colonies on Mitis Salivarius Bacitracin agar (b) S.mutans colonies on TYCSB selective agar. 3.5 The Anti-Bacterial Activity of different chitosan compounds against S. mutans By measuring the mean inhibition zone diameters of the ten bacterial isolates in mm using a ruler, we were able to estimate the antibacterial activities of chitosan, carboxymethyl chitosan, chitosan nanoparticles, amoxicillin loaded on chitosan nanoparticles, and amoxicillin. The MTT cell viability assay for cytotoxicity testing co n c. (µ g /m l) Amoxicillin Amoxicillin + CSNP CSNP z S D V ia b ility S D A v era g e S D V ia b ility S D A v era g e S D V ia b ility S D 500 1.412 0.078 259 101.2 791 5.613 285 1.392 667 0.049 298 96.99 362 3.433 388 1.4091 67 0.0268 95 101.075 9 1.9291 44 250 1.428 333 0.035 887 102.4 507 2.574 072 1.383 0.073 935 96.32 037 5.149 282 1.3833 33 0.0222 68 99.2229 5 1.5972 31 125 1.404 5 0.038 986 100.7 412 2.796 358 1.350 833 0.044 486 94.08 009 3.098 241 1.3443 33 0.0268 96.4255 8 1.9223 31 62 1.354 667 0.030 395 97.16 677 2.180 168 1.320 667 0.024 736 91.97 911 1.722 758 1.3103 33 0.0335 96 93.9868 5 2.4097 29 31 1.414 0.036 721 101.4 226 2.633 872 1.301 167 0.028 806 90.62 101 2.006 199 1.304 0.0292 0274 93.5325 7621 2.0946 37628 16 1.435 667 0.023 209 102.9 767 1.664 736 1.306 667 0.023 754 91.00 406 1.654 391 1.3871 66667 0.0235 06737 99.4979 0795 1.6860 77941 Con trol 1.394 167 0.038 118 100 2.734 092 1.435 833 0.058 69 100 4.087 552 1.3941 67 0.0381 18 100 2.7340 92 IHJPAS. 2025, 38 (1) 8 Table 3. Values of inhibition zone for the five groups of compounds against Streptococcus mutans. Figure 6. The inhibition zone (IZ) of S. mutans isolates against five compound (CS, CSNPs CMC, Amoxicillin loaded on CSNPs, Amoxicillin) Compound Conc. (Mg/ml) Min Max Mean SD F-Test P-Value Cs 10 15 17 15.86 0.76 304.672 0 5 12 13.65 12.74 0.54 2.5 10.5 12.32 10.93 0.66 1.25 8.5 10.5 9.05 0.69 0.625 6 7.59 6.69 0.5 CMC 10 15.5 18.32 16.49 0.96 219.907 0 5 13 14.44 13.34 0.51 2.5 10 12.5 11.5 0.78 1.25 9 11.86 9.69 0.94 0.625 6 7.88 6.82 0.59 CSNPs 10 18 21.5 19.14 1.12 298.81 0 5 15 16.6 15.63 0.53 2.5 12 14.39 13.28 0.75 1.25 9.5 11.85 10.59 0.91 0.625 7 8.5 7.74 0.56 AMX + CSNPs 10 24 29 26.41 1.79 131.759 0 5 20.1 26 22.9 2.33 2.5 16.32 20.3 18.12 1.33 1.25 13 17 15 1.13 0.625 9.5 12.63 11.66 1.3 AMX 10 19 24.7 22.17 1.86 157.139 0 5 15.4 20.5 18.59 1.46 2.5 12.8 16.5 15.72 1.14 1.25 10.18 13.6 12.64 0.97 0.625 8 10 8.93 0.76 IHJPAS. 2025, 38 (1) 9 Table 4. Values of MIC and MBC for the five groups of compounds against Streptococcus mutans. 4. Discussion Chitosan, a cationic biocide used for external cleaning, was used in our study because it attacks bacterial cell membranes. Sudarshan et al. found that chitosan's amino group binds to bacterial surface components and limits their growth. At lower concentrations, chitosan may have adhered to the negatively charged bacterial surface, causing the cell membrane to rupture and release internal components, killing the cell. It's possible that a chitosan coating on the bacteria prevented them from leaking their internal ingredients and stopped the transfer of large amounts of mass across the cell membrane. Partially deacetylating chitin produces chitosan, a biopolymer with applications in the food, pharmaceutical, and chemical industries (21, 22). Antimicrobial chitosan prevents cavities (22). Chitosan is a nanoscale antibacterial, medication, gene, vaccine, and anti- tumor agent. Modified chitosan derivatives like CMC have increased antibacterial activity (23– 24). Because chitosan is insoluble above pH 6.5, it is only bactericidal in acidic environments. Water-soluble, acidic-, and basic-soluble chitosan derivatives may be acceptable for the polycationic biocide (25). Because CMCS is pH-soluble, it is more antimicrobial than chitosan. Water solubility makes CMCS an appealing complexation agent for medicinal and cosmetic purposes. Water-soluble chitosan compounds were harmful to cancer cells but not to normal cells (26, 27). Since dentists recommend amoxicillin for mouth infections, we put it on nanochitosan. Parameter compound No Minimum Maximum Median p-value MIC Chitosan 10 2.5 2.5 2.5 0.000 CMC 10 2.5 2.5 2.5 CSNPs 10 1.25 2.2 2.5 AMX 10 1.25 1.25 1.25 AMX+CSNPs 10 0.625 0.625 0.625 MBC Chitosan 10 5 5 5 0.000 CMC 10 5 5 5 CSNPs 10 2.5 5 5 AMX 10 2.5 2.5 2.5 AMX+ CSNPs 10 0.625 0.625 0.625 MBC\MIC Chitosan 10 2 2 2 0.000 CMC 10 2 2 2 AMX 10 2 4 2 CSNPs 10 2 2 2 AMX+ CSNPs 10 1 1 1 IHJPAS. 2025, 38 (1) 10 Amoxicillin enhances oral microbiota resistance. We observed an increase in oral streptococci resistant to amoxicillin (28). The CS/TPP ratio has an impact on the size and biology of CS nanoparticles (37). This study found that ion-charge interactions resulted in the best size distribution at 4:1 CS/TPP. Drug-loaded nanoparticles were produced. PH reduced the average chitosan NP particle size. PH and ionic strength (38). Tsai et al. studied CS/TPP nanoparticle size and storage solution ph. Nanoparticles shrank as pH increased. Based on the findings, the optimal size distribution was achieved at a CS/TPP ratio of 4:1 due to ion-charge interactions. The nanoparticles produced were loaded with drugs. The average size of chitosan nanoparticles was decreased by pH and ionic strength. Tsai and colleagues researched the effect of nanoparticle size and storage solution pH in CS/TPP. Tsai and colleagues examined the effect of storage solution pH on the size of CS/TPP nanoparticles. They found that nanoparticles decreased in size, leading to a reduction in the overall increase. Additionally, high drug concentrations during gel formation resulted in decreased interaction between CS and TPP, leading to the formation of larger particles. AMOXI-CSNPs are less homogeneous than interfacial depositions. All formulations showed a low PDI (0.4) and size variation. From 0 to 1, PDI measures mixture molecule or particle size heterogeneity. PDI values that are above 0.5 indicate significant heterogeneity (39). Zeta potential has an impact on nanoparticle stability and mucoadhesivity (40). Gel-formed protonated amine groups increased zeta potential. Studies show that nanoparticles with higher zeta potentials are more stable. Electrostatic repulsion disperses charged particles (40). Long-chain amino groups in CS make a big electrical double layer that soaks up TPP anionic groups and stops them from sticking together (22). Increasing formulation CS/D ratios does not improve loading efficiency. Nanoparticle size, without aggregation, improved amoxicillin loading in CSNP solutions at pH 4.5. Precipitation occurred as the CS solution pH rose over 5.0, notably around 6.5. If its pH is below pH 4.2, chitosan's hydrogen ions partially neutralize TPP's hydroxide ions. Hydroxide ions do not deprotonate chitosan. Tripolyphosphoric ions connect protonated amino groups despite intramolecular electrostatic repulsion (30). PH impacts nanoparticle size. Table 2 demonstrates the spectrophotometrically calculated maximum drug loading and encapsulation effectiveness of Amoxicillin-CSNPs. The 1:2 Amoxicillin-CSNP formulation achieved the highest encapsulation and loading efficiencies, 89.33 2.7% and 53 2.4%, respectively. Increased amoxicillin or CSNP ratios reduce drug loading and encapsulation efficiency. Research supports these conclusions. (41, 42) The hydrodynamic size and surface charge. As drug loading increases the particle size, the amoxicillin amine group may cross-link with CSNPs. Shielding widens the particle size range, thereby reducing electrostatic attraction and elevating electrolyte ions, ultimately leading to a decrease in the water layer on the surface (30). Shielding particles reduces electrostatic contact, increases electrolyte ions, and thins the surface hydration layer (30). AM's amino group boosted amoxicillin-CSNPs' zeta potential to +24.5 mV. Residual amine groups boosted gentamicin-loaded CSNPs' zeta potential (43). Positive ions increased amoxicillin-CSNPs' zeta potential, stability, and antibacterial activity. Amoxicillin-CSNPs' stability showed that their surface has a large electric charge, which repels aggregation. Zhang et al. (44) believe that a big IHJPAS. 2025, 38 (1) 11 positive potential minimizes particle agglomeration. SEM investigated amoxicillin-CSNP morphology. The amoxicillin-CSNPs were spherical at 200–300 nm. These may have influenced grouping. Drying lowers particle size, raises specific surface energy, and boosts CS hydrogen bond interaction, which may have promoted amoxicillin-CSNP clustering. In amoxicillin-CSNPs (30), the hydrogen bond interaction dominates drying. Particles gravitate without resistance. The zeta potential is constant above -30 mV or +30 mV (23). CSNPs are cationic because of the biopolymer's amino groups. Concentration increased chitosan viscosity, lowering drug encapsulation. Ionic drug-polymer interactions may capture nanoparticle drugs. 5. Conclusion The AMX-CSNP preparation method is used. The CSNPs were made using a 4:1 CS to TPP ratio at a pH of 4.5. The M 1:2 formulations of CSNPs demonstrated maximum treatment effectiveness. Drug capsules. FTIR was used to detect AMX-CSNP bonding. Based on zeta potential measurements of +24.5 mV, DLS studies found that the average size of AMX-CSNPs was 258 nm. AMX-CSNPs exhibited both elemental analyses and dispersed spherical nanoparticles in experiments with S. mutans, AMX-CSNP demonstrated greater effectiveness than AMX alone. By enhancing AMX's antibacterial properties, CSNPs help AMX to better penetrate the bacterial cell membrane despite the presence of exotoxins. Finally, AMX-CSNPs did not kill MCF-7 cells in vitro. These findings suggest that AMX-CSNPs can enhance the therapeutic efficacy of AMX and facilitate the delivery of medications against cariogenic infections. Acknowledgment Many thanks to the Department of Basic Sciences, College of Dentistry, University of Baghdad, for their invaluable assistance in facilitating the practice sections of this article. Also, I would like to sincerely thank and express my great appreciation and heartfelt gratitude to “ALzahraa Jabbar Jasiem” for her scientific guidance, recommendations, advice, and support. Conflict of Interest The authors declare that they have no conflicts of interest.” Funding There is no funding for the article. Ethical Clearance The study was conducted in accordance with the ethical principles. It was carried out with patients verbal and analytical approval before the sample was taken. The study protocol and the subject information and consent form were reviewed and approved by Baghdad University, College of dentistry a local ethics committee according to the document number Ref. Number 404 and project No. 404821 on 27/December/2021 to get this approval. 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