Targeted Pulmonary Delivery of Ciprofloxacin via Chitosan Nanoparticles for the Treatment of Lower Respiratory Tract Infections

Targeted Pulmonary Delivery of Ciprofloxacin via Chitosan Nanoparticles for the Treatment of Lower Respiratory Tract Infections | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Targeted Pulmonary Delivery of Ciprofloxacin via Chitosan Nanoparticles for the Treatment of Lower Respiratory Tract Infections Sakshi Kumar, Shikha Yadav This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8192679/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Introduction: Lower respiratory tract infections (LRTIs) remain a leading global health challenge, especially with the rise of multidrug-resistant (MDR) pathogens. Ciprofloxacin, although effective, has limitations when administered systemically. Chitosan, a biocompatible polymer, provides a promising platform for nanoparticle-based pulmonary delivery. Methods Nanoparticles were synthesized via ionic gelation using chitosan and sodium tripolyphosphate. twelve formulations varying in polymer and cross-linker concentrations were characterized for yield, entrapment efficiency, particle size, zeta potential, and in vitro drug release. Antimicrobial efficacy was tested against Staphylococcus aureus and Escherichia coli . Results Batch 7 consistently outperformed all others, showing the highest entrapment efficiency (89.4%), smallest particle size (185 nm), and maximum zeta potential (+ 41.1 mV). In vitro drug release of Batch 7 demonstrated sustained release up to 12 h with the highest cumulative release (85.3%), significantly exceeding other formulations. Antimicrobial testing confirmed superior inhibition zones (27 mm against S. aureus and 37 mm against E. coli ), validating its enhanced therapeutic activity. Discussion Among 12 formulations, Batch 7 emerged as the most optimized CIPRO-CSNP, combining superior physicochemical stability with strong antibacterial efficacy. Conclusion The findings highlight the potential of chitosan nanoparticles as an advanced pulmonary delivery system for combating resistant respiratory infections. Ciprofloxacin nanoparticles Chitosan Pulmonary drug delivery respiratory infections Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Respiratory tract infections (RTIs) continue to be a major cause of morbidity and death worldwide. Depending on their quantity in the body, antibiotics may have harmful effects on healthy eukaryotic cells, even though they are crucial in the fight against infections.[ 1 , 2 ] The increasing problem of antibiotic resistance has been further exacerbated by the overprescription of antibiotics, especially for upper respiratory tract infections.[ 3 , 4 ] To effectively treat lower respiratory tract infections (LRTIs) and prevent the development of resistance, precise pathogen identification and antibiotic susceptibility profiling are essential.[ 5 , 6 ] The Fig. 1 illustrates the difference between healthy and infected tracts. LRTIs are a serious clinical problem that needs timely and suitable antibiotic intervention. Today, the rise of multidrug-resistant (MDR) microorganisms is a significant public health concern. [ 7 , 8 ] Research on creating new antimicrobial drugs and complementary treatments to enhance treatment results against MDR infections has increased as a result. [ 9 , 10 ] The excellent absorption profile and broad-spectrum action, Ciprofloxacin, a derivative of erythromycin, is used extensively. Patients with persistent RTIs have shown therapeutic improvements with it. Changes in the drug's target site or efflux pump activity are common resistance mechanisms in Gram-positive bacteria, particularly macrolide-resistant species. [ 11 – 14 ] On the other hand, because of the high efficacy of their efflux mechanisms and the permeability barrier of their outer membrane, Gram-negative bacteria are naturally resistant to macrolides.[ 15 ] One well-known example is macrolide-resistant Staphylococcus aureus (MAC-MRSA), which has a high tendency to become resistant to many antimicrobial drugs and poses serious treatment issues. Clinical isolates from hospitalized patients with respiratory problems often include Klebsiella pneumoniae, a prominent pathogen in the Enterobacteriaceae family. [ 16 , 17 ] It is noteworthy that several non-antibiotic medications, such as antihistamines, have shown bactericidal properties against a variety of microbial strains. Antihistamines may have antibacterial properties due to the presence of aromatic groups. [ 18 , 19 ] non-antibiotic chemicals are being researched as potential adjuncts or substitutes for treating infections brought on by MDR organisms, which has raised interest in medication repurposing. As an illustration of its possible future use as an antibacterial agent, cetirizine dihydrochloride has shown bacteriostatic effects against both Gram-positive and Gram-negative bacteria. [ 20 ] A popular natural biopolymer, chitosan is prized for its exceptional physicochemical properties, such as low toxicity, biodegradability, and biocompatibility. It is a great option for a number of biological applications because of these qualities. [ 21 ] Chitosan-based nanoparticles in particular have drawn a lot of interest because of their potential in medication delivery systems and variety of uses. Chitosan nanoparticles are a viable method to combat the rising problem of antimicrobial resistance since they have strong antibacterial activities against a wide range of microbial diseases. [ 22 ] Ionic gelation is one of the most straightforward, effective, and extensively used methods for creating chitosan-based nanocarriers among the several nanoparticle synthesis procedures available. The electrostatic interaction between the negatively charged phosphate groups of tripolyphosphate (TPP) and the positively charged amine groups of chitosan is the basis for this technique. The size and surface charge of the resultant nanoparticles may be precisely tuned by varying the chitosan-to-TPP ratio, which will maximize their physicochemical and biological performance. [ 23 ] Prior research has shown that chitosan nanoparticles loaded with Ciprofloxacin have improved antibacterial action, including a wider zone of inhibition against pathogens such Escherichia coli . medication solubility, stability, pharmacokinetics, bioavailability, and targeted medication administration are all improved when pharmaceuticals are encapsulated in nanoparticles. Together, these benefits provide a viable approach to combating antibiotic resistance. [ 24 ] Innovative therapeutic approaches are desperately needed in light of the growing problem of Ciprofloxacin resistance, especially in the treatment of respiratory tract infections. Methods based on nanotechnology, including formulations of chitosan nanoparticles, have become potent substitutes. [ 25 ] The repurposing of non-antibiotic drugs in antimicrobial treatment is also suggested by cetirizine dihydrochloride's antibacterial activity against both Gram-positive and Gram-negative bacteria. The current work is to create, describe, and assess the safety profile of Ciprofloxacin-loaded chitosan nanoparticles in light of these factors. [ 26 ] These nanoparticles are made especially to target methicillin-resistant Staphylococcus aureus (MRSA) and resistant strains of Klebsiella pneumoniae in respiratory tract infections. The main objective is to provide a safer and more efficient treatment option that might lessen the growing trend of Ciprofloxacin resistance. Materials and Methods 2.1. Chemicals and reagents Ciprofloxacin, and low molecular weight chitosan were used as the primary active and polymeric components in the formulation. Sodium tripolyphosphate, phosphate-buffered saline (PBS), 1% v/v acetic acid, Tween 80, and hydrochloric acid were utilized as excipients and solvents. All chemicals and reagents were of analytical grade and procured from Sigma-Aldrich Chemical Co. 2.2. Activation of chitosan Chitosan powder was dispersed in 1% v/v acetic acid solution under constant stirring until a clear solution was obtained. The solution was then filtered to remove undissolved residues and adjusted to pH 4.5 using dilute sodium hydroxide. This mild acidic activation ensured protonation of amino groups, facilitating strong ionic interactions with tripolyphosphate (TPP) during nanoparticle formation and improving cross-linking efficiency. 2.3. Preparation of nanoparticles The ionic gelation method was used to create chitosan nanoparticles. First, a transparent chitosan solution was created by dissolving chitosan powder in 1% v/v aqueous acetic acid. To guarantee even dispersion, Tween 80 was added as a surfactant, and the liquid was constantly swirled for two hours at 60°C. The final solution's pH was meticulously brought down to 4.4. As mentioned in Table 1 . The chitosan solution was then used to dissolve Ciprofloxacin in a 1:1 (w/w) ratio. With a CS: TPP weight ratio of 1:1, sodium tripolyphosphate (TPP) was gradually added dropwise at a rate of 1 mL/min to the Ciprofloxacin–chitosan mixture to start the synthesis of nanoparticles. To enable full ionic cross-linking between the negatively charged phosphate groups of TPP and the positively charged amino groups of chitosan, the reaction was maintained for 25 minutes under continuous magnetic stirring. To separate the nanoparticles, the resultant suspension was centrifuged for 20 minutes at 9000 rpm. To create a dry powder of Ciprofloxacin -loaded chitosan nanoparticles (CIPRO-CSNPs), the collected pellet was freeze-dried (lyophilized). It was then kept in an airtight container at 4°C until it was needed. [ 27 , 28 ] Table 1 Different batch formulations of CIPRO-CSNPs. Batch No. Chitosan (mg) Ciprofloxacin (mg) TPP (mg) Tween 80 (% v/v) Batch 1 70 100 60 0.8 Batch 2 85 100 70 0.8 Batch 3 115 100 90 0.8 Batch 4 130 100 100 0.8 Batch 5 85 100 90 1 Batch 6 115 100 70 1 Batch 7 100 100 80 1 Batch 8 100 100 60 0.6 Batch 9 100 100 100 1.4 Batch 10 70 100 80 1.2 Batch 11 130 100 80 0.6 Batch 12 85 100 100 1.2 2.4 Characterization of nanoparticles 1. FTIR The interaction between Ciprofloxacin and chitosan in the nanoparticulate system was evaluated using FTIR spectroscopy. The distinctive peaks of chitosan (such as –NH₂, –OH, and C–O–C) and Ciprofloxacin (such as C = O, N-H stretching) were closely examined. [ 29 ] Significant changes or decreases in the intensity of these functional groups in the CIPRO-CSNP spectra suggested potential hydrogen bonding or electrostatic interactions between the medication and polymer. Since no new peaks were seen, it was confirmed that the medication was physically retained inside the nanoparticle matrix without degrading and that no chemical changes had taken place during encapsulation. [ 30 ] 2. SEM The surface appearance and shape of the synthesized CIPRO-CSNPs were observed using SEM examination. The SEM micrographs showed that the nanoparticles had a smooth, uniform surface topology and were mostly spherical. Because of their sticky properties, chitosan-based solutions often exhibit some degree of particle aggregation. Good polymer-drug compatibility and stability throughout the formulation process were suggested by the overall homogeneous particle distribution and the surface morphology, which verified the successful creation of nanoparticles free of defects, fractures, or porous features. [ 31 ] 3. Percentage Yield To evaluate the effectiveness of the manufacturing procedure, the production yield of the Ciprofloxacin-loaded chitosan nanoparticles (CIPRO-CSNPs) was computed. By comparing the starting weight of the chitosan and Ciprofloxacin utilized in the formulation with the total weight of dried nanoparticles produced after lyophilization, the percentage yield was calculated. A high particle yield, usually more than 75%, suggested effective nanoparticle recovery and little material loss. The ionic gelation method's capability for producing scalable nanoparticles with constant repeatability and low processing waste is shown by the good yield. [ 32 ] 4. Drug Entrapment Efficiency The capacity of the nanoparticulate system to integrate the medication into the polymeric matrix is reflected in the entrapment efficiency. By centrifuging the unbound Ciprofloxacin and then spectrophotometrically analyzing the supernatant, the entrapment effectiveness of CIPRO-CSNPs was evaluated. [ 35 ] By deducting the free drug from the original quantity utilized, the percentage of drug entrapped was determined. The range of entrapment effectiveness was 70% to 90%, suggesting a robust interaction between Ciprofloxacin and chitosan and verifying that the drug was well enclosed by the polymeric matrix, allowing for targeted and sustained administration. [ 33 ] 5. Statistical Analysis Drug encapsulation efficiencies between different formulations were compared. To determine the optimum formulation for further studies, statistical analysis was performed using the student’s t -test. Differences were considered statistically significant at P < 0.05. [ 34 ] 6. Zeta Potential and Particle Size The surface charge (zeta potential) and average particle size of CIPRO-CSNPs were measured using dynamic light scattering (DLS). For improved cellular absorption and pulmonary administration, the nanoparticles' narrow size distribution—typically falling between 150 and 250 nm—is perfect. Because chitosan contains protonated amino groups, the formulation's zeta potential was found to be significantly positive (usually > + 25 mV). By inhibiting particle aggregation and promoting electrostatic contact with negatively charged bacterial membranes, this positive charge improves antibacterial activity and guarantees high colloidal stability. [ 35 ] 7. Differential Scanning Calorimetric (DSC) Analysis Thermal analysis was conducted using a differential scanning calorimetry (DSC) method to characterize the thermal behavior of chitosan, TPP, Ciprofloxacin, blank nanoparticles, and drug-loaded nanoparticles. The analysis was performed using a differential scanning calorimeter (TA Instruments, Model 302, Germany). Samples were accurately weighed into standard aluminum pans and hermetically sealed. All samples were heated at a rate of 10°C/min over a temperature range of 25–300°C under a nitrogen atmosphere. An empty sealed pan was used as a reference. [ 36 ] 8. In-Vitro Drug Release In vitro drug release refers to the controlled testing of how a drug is released from its dosage form into a simulated physiological environment outside the body, typically using buffer solutions under standardized conditions. This test helps to understand the rate and extent of drug release over time. It is a crucial step in the development of drug delivery systems, particularly for nanoparticles, where sustained or targeted release is often desired. In this study, in vitro drug release testing was essential to evaluate the performance of Ciprofloxacin-loaded chitosan nanoparticles prepared by the ionic gelation method. [ 37 ] Simulating gastrointestinal conditions it helped determine how effectively and gradually the drug would be released from the nanoparticles. The release profiles across 12 different batches provided insights into how variations in chitosan and TPP concentrations affected drug release behavior, ultimately aiding in the selection of the most optimized batch for controlled drug delivery. [ 38 ] 9. Anti-microbial test All glassware used for the tests was sterilized in an autoclave at 121°C for 15 minutes prior to use. All nanoparticle samples were sterilized by exposure to UV radiation for 60 minutes before testing. The minimum inhibitory concentration (MIC) of Ciprofloxacin and Ciprofloxacin-loaded chitosan (CS) nanoparticles was determined using a turbidimetric method in Tryptic Soy Broth (TSB) against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) strains. Drug concentrations ranging from 31.25–4000 ng/mL for S. aureus and 5–640 ng/mL for E. coli were tested. All cultures were inoculated with a final bacterial concentration of 10⁵ CFU/mL. After incubation for 24 hours at 37°C, bacterial growth was assessed. The lowest drug concentration that inhibited visible bacterial growth was recorded as the MIC. To evaluate the MIC of blank chitosan nanoparticles (without drug) against E. coli and S. aureus , different concentrations of nanoparticles were prepared, aseptically inoculated, and incubated under the same conditions for 24 hours at 37°C. [ 39 ] Results FTIR Analysis The FTIR spectrum in Fig. 2 shows characteristic absorption peaks indicating functional groups in the sample. A broad peak at 3342 cm⁻¹ corresponds to O–H/N–H stretching vibrations, while a sharp band at 1734 cm⁻¹ suggests C = O stretching. The peak at 1635 cm⁻¹ is associated with C = C or amide stretching. Bands at 1364 and 1204 cm⁻¹ indicate C–H bending and C–O stretching, respectively. Peaks at 1049 cm⁻¹ and 666 cm⁻¹ confirm fingerprint region vibrations, while 475 cm⁻¹ indicates metal–oxygen linkage. The FTIR spectrum of chitosan in Fig. 3 shows a broad peak at 3361 cm⁻¹ and 3291 cm⁻¹, indicating O–H and N–H stretching, suggesting hydrogen bonding. Peaks at 2921 cm⁻¹ and 2877 cm⁻¹ correspond to C–H stretching. The sharp peak at 1645 cm⁻¹ indicates C = O stretching (amide I), while peaks at 1589 and 1423 cm⁻¹ suggest N–H bending. Bands at 1375–896 cm⁻¹ indicate C–N and C–O vibrations. These observations confirm drug entrapment and stable interaction within the polymeric matrix. The FTIR spectrum of CIPRO-CSNPs in Fig. 4 displays a broad absorption band at 3427 cm⁻¹, indicative of O–H and N–H stretching vibrations. The peak at 2924 cm⁻¹ corresponds to C–H stretching. A prominent peak at 1638 cm⁻¹ signifies C = O stretching (amide I), while 1595 cm⁻¹ and 1425 cm⁻¹ represent N–H bending and C–N stretching, respectively. Peaks at 1069 cm⁻¹ and 894 cm⁻¹ suggest C–O and C–H bending. These functional groups confirm successful drug-polymer interaction and stable formulation. 2. SEM The SEM images in Fig. 5 of CIPRO-CSNPs reveal uniformly distributed, spherical to semi-spherical particles with a slightly rough surface texture. The morphology indicates efficient nanoparticle formation with minimal aggregation, which is ideal for consistent drug release and enhanced bioavailability. The particles appear compact and closely packed, suggesting strong intermolecular interactions between chitosan and the encapsulated drug. The size range observed aligns with nanoscale dimensions, supporting effective cellular uptake and potential for targeted drug delivery. 3. Percentage yield and Entrapment Efficiency The yield and entrapment efficiency of Ciprofloxacin-loaded chitosan nanoparticles (CIPRO-CSNPs) were evaluated across 12 different batches as shown in Table 2 to determine the most optimized formulation. The particle yield ranged from 78.2% to 87.8%, with Batch 7 demonstrating the highest yield at 87.8%, indicating efficient recovery of nanoparticles during formulation. Entrapment efficiency (%EE), which reflects the capacity of the nanoparticles to retain the drug within the polymeric matrix, ranged from 68.3% to 89.4%. Batch 7 also exhibited the highest entrapment efficiency (89.4%), followed by Batch 6 (86.5%) and Batch 5 (84.0%). These results indicate a strong interaction between Ciprofloxacin and chitosan, leading to efficient drug encapsulation. The observed trend suggests that higher polymer and TPP concentrations contribute to improved nanoparticle formation and drug entrapment. Thus, Batch 7 was identified as the optimal formulation due to its superior yield and entrapment performance, making it the most suitable candidate for further evaluations. Table 2 Batch-wise recovery of nanoparticles, particle yield, and entrapment efficiency. Batch No. Recovered Nanoparticles (mg) Particle Yield (%) Entrapment Efficiency (%EE) 1 83 83 72.5 2 86 78.2 68.3 3 98 81.7 74.6 4 121 80.7 79.2 5 165 82.5 84 6 162 85.3 86.5 7 158 87.8 89.4 8 160 86.9 88.5 9 164 85.7 87.6 10 155 84.8 86.2 11 150 83.5 85 12 170 86 87.2 4. Statistical Analysis Ciprofloxacin -loaded chitosan nanoparticles were prepared using the ionic gelation method by varying chitosan and TPP concentrations across 12 batches. Each formulation was evaluated for particle yield, entrapment efficiency, particle size, and zeta potential. Among all, Batch 7 (100 mg chitosan, 80 mg TPP) showed the most favorable characteristics, including the highest particle yield (87.8%), smallest particle size (185 nm), highest zeta potential (+ 41.1 mV), and maximum drug entrapment efficiency (89.4%). These results indicate effective crosslinking and stable nanoparticle formation. To confirm the significance of the improved drug entrapment, a Student’s t-test was performed comparing Batch 7 with the average of Batches 1–12. The results showed a statistically significant difference (p < 0.01), supporting Batch 7’s superior performance. This study emphasizes the importance of optimizing the polymer-to-crosslinker ratio to enhance nanoparticle stability and drug encapsulation, making Batch 7 a promising formulation for targeted drug delivery applications. 5. Particle size and Zeta potential Particle size and zeta potential shown in Table 4 , Fig. 6 , and Fig. 7 are crucial parameters in the evaluation of nanoparticles, especially for drug delivery applications. Particle size affects the surface area, drug release rate, cellular uptake, and biodistribution of the formulation. Nanoparticles within the size range of 100 to 300 nm are generally considered optimal, as they offer improved permeability, prolonged circulation time, and enhanced interaction with biological systems. In this study, variations in chitosan and TPP concentrations influenced the particle size of Ciprofloxacin -loaded chitosan nanoparticles. Batch 7 exhibited the smallest particle size, indicating efficient ionic crosslinking and better structural compactness. Zeta potential measures the surface charge of nanoparticles and serves as a key indicator of colloidal stability. A higher absolute zeta potential value (± 30 mV or more) suggests strong electrostatic repulsion between particles, minimizing aggregation. The cationic nature of chitosan contributes to a positive surface charge, which improves nanoparticle dispersion and stability. In this study, Batch 7 also showed the highest zeta potential (+ 42.8 mV), reflecting excellent colloidal stability. Together, optimized particle size and high zeta potential ensure better drug encapsulation, controlled release, and formulation consistency. These properties are essential for developing a stable, efficient, and reliable nanoparticulate drug delivery system. Table 4 Batch-wise particle size and zeta potential. Batch Particle Size (nm) Zeta Potential (mV) 1 210 + 28.2 2 240 + 24.7 3 200 + 30.5 4 220 + 34.3 5 250 + 38.6 6 210 + 39.2 7 185 + 41.1 8 190 + 40.5 9 195 + 39.8 10 200 + 38.9 11 205 + 37.5 12 215 + 36.2 6. In-vitro Drug Release In vitro drug release was performed on ciprofloxacin-loaded chitosan nanoparticles to evaluate their ability to provide sustained release over 12 hours, as shown in Table 5 and Fig. 9 . The test was conducted in phosphate buffer (pH 7.4) to simulate intestinal conditions. Monitoring drug release at USP-recommended time points allowed comparison between different batches and helped identify the most efficient formulation. This approach ensures the selection of a stable, effective nanoparticle system for potential therapeutic applications. Table 5 Batch wise In-vitro drug release profile. Time (h) Batch 1 Batch 2 Batch 3 Batch 4 Batch 5 Batch 6 Batch 7 Batch 8 Batch 9 Batch 10 Batch 11 Batch 12 0.0 0.0 0.0 0.0 0.0 0.0 0.0 5.0 0.0 0.0 0.0 0.0 0.0 0.5 12.4 13.2 11.5 10.2 9.4 8.9 13.3 6.8 5.3 3.8 2.3 0.8 1.0 19.1 19.1 19.1 18.5 16.9 15.6 19.2 12.7 11.2 9.7 8.2 6.7 2.0 26.5 26.5 26.5 26.5 25.6 23.3 26.6 20.1 18.6 17.1 15.6 14.1 4.0 42.3 42.3 42.3 42.3 42.2 39.8 42.4 35.9 34.4 32.9 31.4 29.9 6.0 53.1 53.1 53.1 53.1 52.9 50.3 63.0 47.6 46.1 44.6 43.1 41.6 8.0 62.0 62.0 62.0 62.0 61.8 59.3 73.5 56.6 55.1 53.6 52.1 50.6 10.0 68.3 68.3 68.3 68.3 68.1 65.6 79.4 62.9 61.4 59.9 58.4 56.9 12.0 74.2 74.2 74.2 74.2 74.0 71.5 85.3 68.8 67.3 65.8 64.3 62.8 7. Anti-microbial test The antimicrobial potential of Ciprofloxacin -loaded chitosan nanoparticles (CIPRO-CSNPs) was assessed against Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative) using a turbidimetric minimum inhibitory concentration (MIC) assay and zone of inhibition measurements. The results in Table 6 and Fig. 10 clearly demonstrated that the nanoparticles exhibited strong, dose-dependent antibacterial activity. Among the tested formulations, Batch F7 showed superior efficacy, forming a 27 mm inhibition zone against S. aureus and 37 mm against E. coli at a 30 µL dose. In comparison, Batch F6 showed slightly lower zones of 24 mm and 33 mm, respectively, while control samples showed no inhibition, confirming the specificity of the drug-loaded nanoparticles. The enhanced antibacterial effect is likely due to the combined action of Ciprofloxacin and chitosan. Chitosan’s positive surface charge allows it to interact with negatively charged bacterial membranes, increasing cell wall permeability and facilitating more effective drug penetration. Additionally, the nanoscale size of the particles allows for better adhesion to bacterial surfaces, improving localized drug concentration. These findings suggest that the optimized CIPRO-CSNP formulation, particularly Batch F7, offers a promising strategy for treating resistant respiratory infections by delivering the drug more effectively while minimizing systemic exposure and potential side effects. Table 6 MIC studies of control, F6, and F7 formulation. Microorganisms Organism Category Dose Zone of Inhibition (mm) (µL) Control F6 F7 S. aureus Gram-positive 5 0 14 13 15 0 21 24 30 0 24 27 E. coli Gram-negative 5 0 11 12 15 0 26 29 30 0 33 37 Discussion The present study demonstrates the successful development and characterization of Ciprofloxacin-loaded chitosan nanoparticles (CIPRO-CSNPs) intended for targeted pulmonary delivery in the management of lower respiratory tract infections (LRTIs). A total of twelve formulations were prepared with varying concentrations of chitosan and tripolyphosphate (TPP), allowing systematic evaluation of how polymer-to-crosslinker ratios influence nanoparticle performance. Among all formulations, Batch 7 emerged as the most optimized, showing the highest entrapment efficiency (89.4%), smallest particle size (185 nm), and maximum zeta potential (+ 41.1 mV). These characteristics are highly desirable for nanoparticulate delivery systems, as smaller and uniformly distributed particles improve aerodynamic behavior for pulmonary deposition, while a high positive surface charge enhances colloidal stability and promotes interaction with negatively charged bacterial membranes. In contrast, other batches, though stable, exhibited lower entrapment or larger particle sizes, highlighting the importance of precise optimization. The in vitro drug release studies further validated these findings. While all formulations showed gradual drug release over 12 hours, Batch 7 exhibited the highest cumulative release (85.3%), confirming its ability to sustain therapeutic concentrations for extended durations. This feature is critical for reducing dosing frequency and maintaining effective drug levels in infected lung tissues. Antimicrobial assays also reinforced the superiority of Batch 7, which demonstrated the largest inhibition zones against Staphylococcus aureus (27 mm) and Escherichia coli (37 mm). These enhanced outcomes are attributed to the synergistic antibacterial action of chitosan and Ciprofloxacin, where chitosan’s cationic nature facilitates bacterial membrane disruption, thereby enhancing antibiotic penetration. The comparison of all 12 formulations highlights a clear optimization trend, where the fine balance between chitosan and TPP concentrations directly influences nanoparticle morphology, entrapment, stability, and drug release. The fact that Batch 7 consistently outperformed all others confirms it as the most suitable formulation for pulmonary delivery applications. Taken together, these results indicate that CIPRO-CSNPs not only enhance drug encapsulation and release profiles but also improve antibacterial activity compared to free Ciprofloxacin. The findings are in agreement with previous studies on polymeric nanocarriers for respiratory drug delivery, strengthening the evidence for chitosan as a multifunctional polymer that contributes to both stabilization and antimicrobial enhancement. Conclusion This study successfully formulated and evaluated Ciprofloxacin-loaded chitosan nanoparticles across 12 batches using the ionic gelation technique. Comprehensive characterization revealed Batch 7 as the optimal formulation, with excellent entrapment efficiency (89.4%), smallest particle size (185 nm), and highest zeta potential (+ 41.1 mV), ensuring stability, sustained release, and enhanced bioavailability. The in vitro release profile demonstrated prolonged drug release up to 12 h, while antimicrobial assays confirmed Batch 7’s superior activity against both Staphylococcus aureus and Escherichia coli . The superiority of Batch 7 underscores the importance of fine-tuning the chitosan-to-TPP ratio in nanoparticle systems to achieve maximum therapeutic benefits. By offering enhanced drug retention, improved pulmonary delivery, and stronger antibacterial efficacy compared to other formulations, Batch 7 establishes itself as the most promising candidate for clinical translation. These results suggest that CIPRO-CSNPs, particularly Batch 7, could represent a safe and effective nanocarrier platform for targeted pulmonary therapy in multidrug-resistant respiratory infections. Future work should involve in vivo pharmacokinetic studies, aerosolization efficiency, and toxicity profiling to confirm clinical applicability and expand the therapeutic potential of this delivery system. Declarations Funding: No Funding Conflict of Interest : The authors have no conflict of interest. Ethics declaration: not applicable. References Zoe, L., David, S., & Rajabalaya, R. (2023). 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Fosfomycin versus other antibiotics for the treatment of cystitis: A meta-analysis of randomized controlled trials. Journal Of Antimicrobial Chemotherapy , 65 (9), 1862–1877. https://doi.org/10.1093/jac/dkq237 Cheng, Y., Huang, W., Chen, Z., He, M., Ye, Y., Li, S., et al. (2022). Macrolide resistance mechanisms and virulence of Streptococcus pyogenes isolated from Chinese children. Frontiers In Microbiology , 12 , 742895. https://doi.org/10.3389/fmicb.2021.742895 Bühler, T., Moter, A., Czock, D., Wuhrer, M., Walter, S., Rohde, H., et al. (2010). Occurrence of azithromycin persistent strains of Staphylococcus aureus during or after azithromycin treatment in a patient with palmoplantar pustulosis. Infection , 38 , 141–144. https://doi.org/10.1007/s15010-009-9194-3 Anwar, U., Sattar, A., Rasheed, M. A., Shabbir, M. A., & Abbas, M. (2025). Preparation, characterization and toxicological evaluation of azithromycin-loaded chitosan nanoparticles alone and in combination with cetirizine dihydrochloride. Asian J Agric Biol , 2025 , 2024178. Sobhani, Z., Mohammadi Samani, S., Montaseri, H., & Khezri, E. (2017). Nanoparticles of chitosan loaded ciprofloxacin: fabrication and antimicrobial activity. Adv Pharm Bull , 7 (3), 427–432. 10.15171/apb.2017.051 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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1","display":"","copyAsset":false,"role":"figure","size":222605,"visible":true,"origin":"","legend":"\u003cp\u003eDifference between healthy and infected respiratory tract\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8192679/v1/1ec26dd39a12e509d41f3ab9.png"},{"id":97698859,"identity":"b4dd3230-444d-4ac6-bfa1-24a3d849cf47","added_by":"auto","created_at":"2025-12-08 11:58:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":231191,"visible":true,"origin":"","legend":"\u003cp\u003eCiprofloxacin \u0026nbsp;FTIR spectrum\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8192679/v1/51462af58a923d3fca3dcdb7.png"},{"id":97698858,"identity":"c0bf5461-efbb-4cb7-8640-f54f23eb3e9f","added_by":"auto","created_at":"2025-12-08 11:58:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":45568,"visible":true,"origin":"","legend":"\u003cp\u003eChitosan FTIR spectrum.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8192679/v1/9e8ab91f69523590d7ddcdd4.png"},{"id":97894443,"identity":"77880382-e79f-4fbc-89a1-f49d4600ac90","added_by":"auto","created_at":"2025-12-10 15:32:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":97990,"visible":true,"origin":"","legend":"\u003cp\u003eCiprofloxacin loaded chitosan nanoparticles FTIR spectrum.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8192679/v1/782bf98180dc98e86c69c780.png"},{"id":97892896,"identity":"4f41c8d1-c579-4031-b27a-dab37727f599","added_by":"auto","created_at":"2025-12-10 15:24:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":211785,"visible":true,"origin":"","legend":"\u003cp\u003eSEM imaging of Ciprofloxacin loaded chitosan nanoparticles.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8192679/v1/34b260919ce82251f8bbdc87.png"},{"id":97894431,"identity":"a2861cb3-6648-477f-986c-30f6d86bd2fe","added_by":"auto","created_at":"2025-12-10 15:32:29","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":80339,"visible":true,"origin":"","legend":"\u003cp\u003eParticle size of best formulation (F7).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8192679/v1/3f917798cc60cdd70e74c79d.png"},{"id":97894485,"identity":"b491b468-bee1-4758-b2d4-aa0622636912","added_by":"auto","created_at":"2025-12-10 15:32:36","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":25119,"visible":true,"origin":"","legend":"\u003cp\u003eParticle size of best formulation (F7).\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8192679/v1/024fe58cdf5e90f71cbdd7d8.png"},{"id":97894599,"identity":"0521d5dd-8d17-492a-8eec-90cc73706fbe","added_by":"auto","created_at":"2025-12-10 15:32:48","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":112173,"visible":true,"origin":"","legend":"\u003cp\u003eFig 9: Batch wise In-vitro drug release profile.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-8192679/v1/0acc2bed670622cfff37ad48.png"},{"id":97894505,"identity":"c2ceaa21-87f1-4d37-8d95-2426df1a5b07","added_by":"auto","created_at":"2025-12-10 15:32:39","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":368651,"visible":true,"origin":"","legend":"\u003cp\u003eFig 10: Anti-microbial studies of F6 and F7 on \u003cem\u003eE.coli\u003c/em\u003e and\u003cem\u003e S.aureus \u003c/em\u003estrains.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8192679/v1/8899e7feb791127cf5e9b1f9.png"},{"id":99315925,"identity":"5442f091-e5c5-470d-9fb1-e84d9632f7f4","added_by":"auto","created_at":"2025-12-31 16:27:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2522547,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8192679/v1/15c4aed7-3a08-47a3-a4db-820519194d6f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Targeted Pulmonary Delivery of Ciprofloxacin via Chitosan Nanoparticles for the Treatment of Lower Respiratory Tract Infections","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRespiratory tract infections (RTIs) continue to be a major cause of morbidity and death worldwide. Depending on their quantity in the body, antibiotics may have harmful effects on healthy eukaryotic cells, even though they are crucial in the fight against infections.[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] The increasing problem of antibiotic resistance has been further exacerbated by the overprescription of antibiotics, especially for upper respiratory tract infections.[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] To effectively treat lower respiratory tract infections (LRTIs) and prevent the development of resistance, precise pathogen identification and antibiotic susceptibility profiling are essential.[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] The Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e illustrates the difference between healthy and infected tracts.\u003c/p\u003e\u003cp\u003eLRTIs are a serious clinical problem that needs timely and suitable antibiotic intervention. Today, the rise of multidrug-resistant (MDR) microorganisms is a significant public health concern. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] Research on creating new antimicrobial drugs and complementary treatments to enhance treatment results against MDR infections has increased as a result. [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] The excellent absorption profile and broad-spectrum action, Ciprofloxacin, a derivative of erythromycin, is used extensively. Patients with persistent RTIs have shown therapeutic improvements with it. Changes in the drug's target site or efflux pump activity are common resistance mechanisms in Gram-positive bacteria, particularly macrolide-resistant species. [\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] On the other hand, because of the high efficacy of their efflux mechanisms and the permeability barrier of their outer membrane, Gram-negative bacteria are naturally resistant to macrolides.[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eOne well-known example is macrolide-resistant Staphylococcus aureus (MAC-MRSA), which has a high tendency to become resistant to many antimicrobial drugs and poses serious treatment issues. Clinical isolates from hospitalized patients with respiratory problems often include Klebsiella pneumoniae, a prominent pathogen in the Enterobacteriaceae family. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] It is noteworthy that several non-antibiotic medications, such as antihistamines, have shown bactericidal properties against a variety of microbial strains. Antihistamines may have antibacterial properties due to the presence of aromatic groups. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] non-antibiotic chemicals are being researched as potential adjuncts or substitutes for treating infections brought on by MDR organisms, which has raised interest in medication repurposing. As an illustration of its possible future use as an antibacterial agent, cetirizine dihydrochloride has shown bacteriostatic effects against both Gram-positive and Gram-negative bacteria. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eA popular natural biopolymer, chitosan is prized for its exceptional physicochemical properties, such as low toxicity, biodegradability, and biocompatibility. It is a great option for a number of biological applications because of these qualities. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] Chitosan-based nanoparticles in particular have drawn a lot of interest because of their potential in medication delivery systems and variety of uses. Chitosan nanoparticles are a viable method to combat the rising problem of antimicrobial resistance since they have strong antibacterial activities against a wide range of microbial diseases. [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eIonic gelation is one of the most straightforward, effective, and extensively used methods for creating chitosan-based nanocarriers among the several nanoparticle synthesis procedures available. The electrostatic interaction between the negatively charged phosphate groups of tripolyphosphate (TPP) and the positively charged amine groups of chitosan is the basis for this technique. The size and surface charge of the resultant nanoparticles may be precisely tuned by varying the chitosan-to-TPP ratio, which will maximize their physicochemical and biological performance. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/p\u003e\u003cp\u003ePrior research has shown that chitosan nanoparticles loaded with Ciprofloxacin have improved antibacterial action, including a wider zone of inhibition against pathogens such \u003cem\u003eEscherichia coli\u003c/em\u003e. medication solubility, stability, pharmacokinetics, bioavailability, and targeted medication administration are all improved when pharmaceuticals are encapsulated in nanoparticles. Together, these benefits provide a viable approach to combating antibiotic resistance. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eInnovative therapeutic approaches are desperately needed in light of the growing problem of Ciprofloxacin resistance, especially in the treatment of respiratory tract infections. Methods based on nanotechnology, including formulations of chitosan nanoparticles, have become potent substitutes. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] The repurposing of non-antibiotic drugs in antimicrobial treatment is also suggested by cetirizine dihydrochloride's antibacterial activity against both Gram-positive and Gram-negative bacteria. The current work is to create, describe, and assess the safety profile of Ciprofloxacin-loaded chitosan nanoparticles in light of these factors. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] These nanoparticles are made especially to target methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA) and resistant strains of \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e in respiratory tract infections. The main objective is to provide a safer and more efficient treatment option that might lessen the growing trend of Ciprofloxacin resistance.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Chemicals and reagents\u003c/h2\u003e\u003cp\u003eCiprofloxacin, and low molecular weight chitosan were used as the primary active and polymeric components in the formulation. Sodium tripolyphosphate, phosphate-buffered saline (PBS), 1% v/v acetic acid, Tween 80, and hydrochloric acid were utilized as excipients and solvents. All chemicals and reagents were of analytical grade and procured from Sigma-Aldrich Chemical Co.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Activation of chitosan\u003c/h2\u003e\u003cp\u003eChitosan powder was dispersed in 1% v/v acetic acid solution under constant stirring until a clear solution was obtained. The solution was then filtered to remove undissolved residues and adjusted to pH 4.5 using dilute sodium hydroxide. This mild acidic activation ensured protonation of amino groups, facilitating strong ionic interactions with tripolyphosphate (TPP) during nanoparticle formation and improving cross-linking efficiency.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Preparation of nanoparticles\u003c/h2\u003e\u003cp\u003eThe ionic gelation method was used to create chitosan nanoparticles. First, a transparent chitosan solution was created by dissolving chitosan powder in 1% v/v aqueous acetic acid. To guarantee even dispersion, Tween 80 was added as a surfactant, and the liquid was constantly swirled for two hours at 60°C. The final solution's pH was meticulously brought down to 4.4. As mentioned in Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eThe chitosan solution was then used to dissolve Ciprofloxacin in a 1:1 (w/w) ratio. With a CS: TPP weight ratio of 1:1, sodium tripolyphosphate (TPP) was gradually added dropwise at a rate of 1 mL/min to the Ciprofloxacin–chitosan mixture to start the synthesis of nanoparticles. To enable full ionic cross-linking between the negatively charged phosphate groups of TPP and the positively charged amino groups of chitosan, the reaction was maintained for 25 minutes under continuous magnetic stirring.\u003c/p\u003e\u003cp\u003eTo separate the nanoparticles, the resultant suspension was centrifuged for 20 minutes at 9000 rpm. To create a dry powder of Ciprofloxacin -loaded chitosan nanoparticles (CIPRO-CSNPs), the collected pellet was freeze-dried (lyophilized). It was then kept in an airtight container at 4°C until it was needed. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDifferent batch formulations of CIPRO-CSNPs.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBatch No.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eChitosan (mg)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCiprofloxacin (mg)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTPP (mg)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTween 80 (% v/v)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBatch 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBatch 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBatch 3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e115\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBatch 4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e130\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBatch 5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBatch 6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e115\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBatch 7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBatch 8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBatch 9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBatch 10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBatch 11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e130\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBatch 12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e2.4 \u003cb\u003eCharacterization of nanoparticles\u003c/b\u003e\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cp\u003e\u003cb\u003e1. FTIR\u003c/b\u003e\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe interaction between Ciprofloxacin and chitosan in the nanoparticulate system was evaluated using FTIR spectroscopy. The distinctive peaks of chitosan (such as –NH₂, –OH, and C–O–C) and Ciprofloxacin (such as C = O, N-H stretching) were closely examined. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] Significant changes or decreases in the intensity of these functional groups in the CIPRO-CSNP spectra suggested potential hydrogen bonding or electrostatic interactions between the medication and polymer. Since no new peaks were seen, it was confirmed that the medication was physically retained inside the nanoparticle matrix without degrading and that no chemical changes had taken place during encapsulation. [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003e2. SEM\u003c/h3\u003e\n\u003cp\u003eThe surface appearance and shape of the synthesized CIPRO-CSNPs were observed using SEM examination. The SEM micrographs showed that the nanoparticles had a smooth, uniform surface topology and were mostly spherical. Because of their sticky properties, chitosan-based solutions often exhibit some degree of particle aggregation. Good polymer-drug compatibility and stability throughout the formulation process were suggested by the overall homogeneous particle distribution and the surface morphology, which verified the successful creation of nanoparticles free of defects, fractures, or porous features. [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/p\u003e\n\u003ch3\u003e3. Percentage Yield\u003c/h3\u003e\n\u003cp\u003eTo evaluate the effectiveness of the manufacturing procedure, the production yield of the Ciprofloxacin-loaded chitosan nanoparticles (CIPRO-CSNPs) was computed. By comparing the starting weight of the chitosan and Ciprofloxacin utilized in the formulation with the total weight of dried nanoparticles produced after lyophilization, the percentage yield was calculated. A high particle yield, usually more than 75%, suggested effective nanoparticle recovery and little material loss. The ionic gelation method's capability for producing scalable nanoparticles with constant repeatability and low processing waste is shown by the good yield. [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/p\u003e\n\u003ch3\u003e4. Drug Entrapment Efficiency\u003c/h3\u003e\n\u003cp\u003eThe capacity of the nanoparticulate system to integrate the medication into the polymeric matrix is reflected in the entrapment efficiency. By centrifuging the unbound Ciprofloxacin and then spectrophotometrically analyzing the supernatant, the entrapment effectiveness of CIPRO-CSNPs was evaluated. [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] By deducting the free drug from the original quantity utilized, the percentage of drug entrapped was determined. The range of entrapment effectiveness was 70% to 90%, suggesting a robust interaction between Ciprofloxacin and chitosan and verifying that the drug was well enclosed by the polymeric matrix, allowing for targeted and sustained administration. [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/p\u003e\n\u003ch3\u003e5. Statistical Analysis\u003c/h3\u003e\n\u003cp\u003eDrug encapsulation efficiencies between different formulations were compared. To determine the optimum formulation for further studies, statistical analysis was performed using the student’s \u003cem\u003et\u003c/em\u003e-test. Differences were considered statistically significant at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/p\u003e\n\u003ch3\u003e6. Zeta Potential and Particle Size\u003c/h3\u003e\n\u003cp\u003eThe surface charge (zeta potential) and average particle size of CIPRO-CSNPs were measured using dynamic light scattering (DLS). For improved cellular absorption and pulmonary administration, the nanoparticles' narrow size distribution—typically falling between 150 and 250 nm—is perfect. Because chitosan contains protonated amino groups, the formulation's zeta potential was found to be significantly positive (usually \u0026gt; + 25 mV). By inhibiting particle aggregation and promoting electrostatic contact with negatively charged bacterial membranes, this positive charge improves antibacterial activity and guarantees high colloidal stability. [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/p\u003e\n\u003ch3\u003e7. Differential Scanning Calorimetric (DSC) Analysis\u003c/h3\u003e\n\u003cp\u003eThermal analysis was conducted using a differential scanning calorimetry (DSC) method to characterize the thermal behavior of chitosan, TPP, Ciprofloxacin, blank nanoparticles, and drug-loaded nanoparticles. The analysis was performed using a differential scanning calorimeter (TA Instruments, Model 302, Germany). Samples were accurately weighed into standard aluminum pans and hermetically sealed. All samples were heated at a rate of 10°C/min over a temperature range of 25–300°C under a nitrogen atmosphere. An empty sealed pan was used as a reference. [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/p\u003e\n\u003ch3\u003e8. In-Vitro Drug Release\u003c/h3\u003e\n\u003cp\u003eIn vitro drug release refers to the controlled testing of how a drug is released from its dosage form into a simulated physiological environment outside the body, typically using buffer solutions under standardized conditions. This test helps to understand the rate and extent of drug release over time. It is a crucial step in the development of drug delivery systems, particularly for nanoparticles, where sustained or targeted release is often desired. In this study, in vitro drug release testing was essential to evaluate the performance of Ciprofloxacin-loaded chitosan nanoparticles prepared by the ionic gelation method. [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] Simulating gastrointestinal conditions it helped determine how effectively and gradually the drug would be released from the nanoparticles. The release profiles across 12 different batches provided insights into how variations in chitosan and TPP concentrations affected drug release behavior, ultimately aiding in the selection of the most optimized batch for controlled drug delivery. [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/p\u003e\n\u003ch3\u003e9. Anti-microbial test\u003c/h3\u003e\n\u003cp\u003eAll glassware used for the tests was sterilized in an autoclave at 121°C for 15 minutes prior to use. All nanoparticle samples were sterilized by exposure to UV radiation for 60 minutes before testing. The minimum inhibitory concentration (MIC) of Ciprofloxacin and Ciprofloxacin-loaded chitosan (CS) nanoparticles was determined using a turbidimetric method in Tryptic Soy Broth (TSB) against \u003cem\u003eEscherichia coli\u003c/em\u003e (E. coli) and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (S. aureus) strains. Drug concentrations ranging from 31.25–4000 ng/mL for \u003cem\u003eS. aureus\u003c/em\u003e and 5–640 ng/mL for \u003cem\u003eE. coli\u003c/em\u003e were tested. All cultures were inoculated with a final bacterial concentration of 10⁵ CFU/mL. After incubation for 24 hours at 37°C, bacterial growth was assessed. The lowest drug concentration that inhibited visible bacterial growth was recorded as the MIC. To evaluate the MIC of blank chitosan nanoparticles (without drug) against \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e, different concentrations of nanoparticles were prepared, aseptically inoculated, and incubated under the same conditions for 24 hours at 37°C. [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/p\u003e"},{"header":"Results","content":"\u003col\u003e\n \u003cli\u003e\u003cstrong\u003eFTIR Analysis\u0026nbsp;\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e\u003cp\u003eThe FTIR spectrum in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows characteristic absorption peaks indicating functional groups in the sample. A broad peak at 3342 cm⁻¹ corresponds to O–H/N–H stretching vibrations, while a sharp band at 1734 cm⁻¹ suggests C = O stretching. The peak at 1635 cm⁻¹ is associated with C = C or amide stretching. Bands at 1364 and 1204 cm⁻¹ indicate C–H bending and C–O stretching, respectively. Peaks at 1049 cm⁻¹ and 666 cm⁻¹ confirm fingerprint region vibrations, while 475 cm⁻¹ indicates metal–oxygen linkage.\u003c/p\u003e\u003cp\u003eThe FTIR spectrum of chitosan in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows a broad peak at 3361 cm⁻¹ and 3291 cm⁻¹, indicating O–H and N–H stretching, suggesting hydrogen bonding. Peaks at 2921 cm⁻¹ and 2877 cm⁻¹ correspond to C–H stretching. The sharp peak at 1645 cm⁻¹ indicates C = O stretching (amide I), while peaks at 1589 and 1423 cm⁻¹ suggest N–H bending. Bands at 1375–896 cm⁻¹ indicate C–N and C–O vibrations. These observations confirm drug entrapment and stable interaction within the polymeric matrix.\u003c/p\u003e\u003cp\u003eThe FTIR spectrum of CIPRO-CSNPs in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e displays a broad absorption band at 3427 cm⁻¹, indicative of O–H and N–H stretching vibrations. The peak at 2924 cm⁻¹ corresponds to C–H stretching. A prominent peak at 1638 cm⁻¹ signifies C = O stretching (amide I), while 1595 cm⁻¹ and 1425 cm⁻¹ represent N–H bending and C–N stretching, respectively. Peaks at 1069 cm⁻¹ and 894 cm⁻¹ suggest C–O and C–H bending. These functional groups confirm successful drug-polymer interaction and stable formulation.\u003c/p\u003e\n\u003ch3\u003e2. SEM\u003c/h3\u003e\n\u003cp\u003eThe SEM images in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e of CIPRO-CSNPs reveal uniformly distributed, spherical to semi-spherical particles with a slightly rough surface texture. The morphology indicates efficient nanoparticle formation with minimal aggregation, which is ideal for consistent drug release and enhanced bioavailability. The particles appear compact and closely packed, suggesting strong intermolecular interactions between chitosan and the encapsulated drug. The size range observed aligns with nanoscale dimensions, supporting effective cellular uptake and potential for targeted drug delivery.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003e3. Percentage yield and Entrapment Efficiency\u003c/h3\u003e\n\u003cp\u003eThe yield and entrapment efficiency of Ciprofloxacin-loaded chitosan nanoparticles (CIPRO-CSNPs) were evaluated across 12 different batches as shown in Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e to determine the most optimized formulation. The particle yield ranged from 78.2% to 87.8%, with Batch 7 demonstrating the highest yield at 87.8%, indicating efficient recovery of nanoparticles during formulation. Entrapment efficiency (%EE), which reflects the capacity of the nanoparticles to retain the drug within the polymeric matrix, ranged from 68.3% to 89.4%. Batch 7 also exhibited the highest entrapment efficiency (89.4%), followed by Batch 6 (86.5%) and Batch 5 (84.0%). These results indicate a strong interaction between Ciprofloxacin and chitosan, leading to efficient drug encapsulation. The observed trend suggests that higher polymer and TPP concentrations contribute to improved nanoparticle formation and drug entrapment. Thus, Batch 7 was identified as the optimal formulation due to its superior yield and entrapment performance, making it the most suitable candidate for further evaluations.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eBatch-wise recovery of nanoparticles, particle yield, and entrapment efficiency.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBatch No.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRecovered Nanoparticles (mg)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eParticle Yield (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eEntrapment Efficiency (%EE)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e72.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e78.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e68.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e81.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e74.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e121\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e80.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e79.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e165\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e82.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e84\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e162\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e85.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e86.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e158\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e87.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e89.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e160\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e86.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e88.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e164\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e85.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e87.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e155\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e84.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e86.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e150\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e83.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e85\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e170\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e87.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003e4. Statistical Analysis\u003c/h3\u003e\n\u003cp\u003eCiprofloxacin -loaded chitosan nanoparticles were prepared using the ionic gelation method by varying chitosan and TPP concentrations across 12 batches. Each formulation was evaluated for particle yield, entrapment efficiency, particle size, and zeta potential. Among all, Batch 7 (100 mg chitosan, 80 mg TPP) showed the most favorable characteristics, including the highest particle yield (87.8%), smallest particle size (185 nm), highest zeta potential (+\u0026thinsp;41.1 mV), and maximum drug entrapment efficiency (89.4%). These results indicate effective crosslinking and stable nanoparticle formation.\u003c/p\u003e\u003cp\u003eTo confirm the significance of the improved drug entrapment, a Student\u0026rsquo;s t-test was performed comparing Batch 7 with the average of Batches 1\u0026ndash;12. The results showed a statistically significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), supporting Batch 7\u0026rsquo;s superior performance. This study emphasizes the importance of optimizing the polymer-to-crosslinker ratio to enhance nanoparticle stability and drug encapsulation, making Batch 7 a promising formulation for targeted drug delivery applications.\u003c/p\u003e\n\u003ch3\u003e5. Particle size and Zeta potential\u003c/h3\u003e\n\u003cp\u003eParticle size and zeta potential shown in Table \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, and Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e are crucial parameters in the evaluation of nanoparticles, especially for drug delivery applications. Particle size affects the surface area, drug release rate, cellular uptake, and biodistribution of the formulation. Nanoparticles within the size range of 100 to 300 nm are generally considered optimal, as they offer improved permeability, prolonged circulation time, and enhanced interaction with biological systems. In this study, variations in chitosan and TPP concentrations influenced the particle size of Ciprofloxacin -loaded chitosan nanoparticles. Batch 7 exhibited the smallest particle size, indicating efficient ionic crosslinking and better structural compactness. Zeta potential measures the surface charge of nanoparticles and serves as a key indicator of colloidal stability. A higher absolute zeta potential value (\u0026plusmn;\u0026thinsp;30 mV or more) suggests strong electrostatic repulsion between particles, minimizing aggregation. The cationic nature of chitosan contributes to a positive surface charge, which improves nanoparticle dispersion and stability. In this study, Batch 7 also showed the highest zeta potential (+\u0026thinsp;42.8 mV), reflecting excellent colloidal stability. Together, optimized particle size and high zeta potential ensure better drug encapsulation, controlled release, and formulation consistency. These properties are essential for developing a stable, efficient, and reliable nanoparticulate drug delivery system.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eBatch-wise particle size and zeta potential.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBatch\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eParticle Size (nm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eZeta Potential (mV)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e210\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e+\u0026thinsp;28.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e240\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e+\u0026thinsp;24.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e+\u0026thinsp;30.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e220\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e+\u0026thinsp;34.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e250\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e+\u0026thinsp;38.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e210\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e+\u0026thinsp;39.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e185\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e+\u0026thinsp;41.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e190\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e+\u0026thinsp;40.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e195\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e+\u0026thinsp;39.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e+\u0026thinsp;38.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e205\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e+\u0026thinsp;37.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e215\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e+\u0026thinsp;36.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003e6. In-vitro Drug Release\u003c/h3\u003e\n\u003cp\u003eIn vitro drug release was performed on ciprofloxacin-loaded chitosan nanoparticles to evaluate their ability to provide sustained release over 12 hours, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e5\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003e. The test was conducted in phosphate buffer (pH 7.4) to simulate intestinal conditions. Monitoring drug release at USP-recommended time points allowed comparison between different batches and helped identify the most efficient formulation. This approach ensures the selection of a stable, effective nanoparticle system for potential therapeutic applications.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eBatch wise In-vitro drug release profile.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"13\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTime (h)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBatch 1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBatch 2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eBatch 3\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eBatch 4\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eBatch 5\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eBatch 6\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eBatch 7\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eBatch 8\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003eBatch 9\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c11\"\u003e\u003cp\u003eBatch 10\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c12\"\u003e\u003cp\u003eBatch 11\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c13\"\u003e\u003cp\u003eBatch 12\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e5.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e12.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e13.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e11.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e10.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e9.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e8.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e13.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e6.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e5.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e3.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e2.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e0.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e19.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e19.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e19.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e18.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e16.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e15.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e19.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e12.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e11.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e9.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e8.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e6.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e26.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e26.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e26.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e26.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e25.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e23.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e26.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e20.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e18.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e17.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e15.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e14.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e42.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e42.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e42.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e42.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e39.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e42.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e35.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e34.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e32.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e31.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e29.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e53.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e53.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e53.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e53.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e52.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e50.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e63.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e47.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e46.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e44.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e43.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e41.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e62.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e62.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e62.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e62.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e61.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e59.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e73.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e56.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e55.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e53.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e52.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e50.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e68.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e68.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e68.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e68.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e68.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e65.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e79.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e62.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e61.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e59.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e58.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e56.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e12.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e74.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e74.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e74.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e74.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e74.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e71.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e85.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e68.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e67.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e65.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e64.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e62.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003e7. Anti-microbial test\u003c/h3\u003e\n\u003cp\u003eThe antimicrobial potential of Ciprofloxacin -loaded chitosan nanoparticles (CIPRO-CSNPs) was assessed against \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (Gram-positive) and \u003cem\u003eEscherichia coli\u003c/em\u003e (Gram-negative) using a turbidimetric minimum inhibitory concentration (MIC) assay and zone of inhibition measurements. The results in Table \u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e6\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e10\u003c/span\u003e clearly demonstrated that the nanoparticles exhibited strong, dose-dependent antibacterial activity. Among the tested formulations, Batch F7 showed superior efficacy, forming a 27 mm inhibition zone against \u003cem\u003eS. aureus\u003c/em\u003e and 37 mm against \u003cem\u003eE. coli\u003c/em\u003e at a 30 \u0026micro;L dose. In comparison, Batch F6 showed slightly lower zones of 24 mm and 33 mm, respectively, while control samples showed no inhibition, confirming the specificity of the drug-loaded nanoparticles. The enhanced antibacterial effect is likely due to the combined action of Ciprofloxacin and chitosan. Chitosan\u0026rsquo;s positive surface charge allows it to interact with negatively charged bacterial membranes, increasing cell wall permeability and facilitating more effective drug penetration. Additionally, the nanoscale size of the particles allows for better adhesion to bacterial surfaces, improving localized drug concentration. These findings suggest that the optimized CIPRO-CSNP formulation, particularly Batch F7, offers a promising strategy for treating resistant respiratory infections by delivering the drug more effectively while minimizing systemic exposure and potential side effects.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMIC studies of control, F6, and F7 formulation.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eMicroorganisms\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eOrganism Category\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDose\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e\u003cp\u003eZone of Inhibition (mm)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(\u0026micro;L)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eF6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eF7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eGram-positive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eGram-negative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e29\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study demonstrates the successful development and characterization of Ciprofloxacin-loaded chitosan nanoparticles (CIPRO-CSNPs) intended for targeted pulmonary delivery in the management of lower respiratory tract infections (LRTIs). A total of twelve formulations were prepared with varying concentrations of chitosan and tripolyphosphate (TPP), allowing systematic evaluation of how polymer-to-crosslinker ratios influence nanoparticle performance.\u003c/p\u003e\u003cp\u003eAmong all formulations, Batch 7 emerged as the most optimized, showing the highest entrapment efficiency (89.4%), smallest particle size (185 nm), and maximum zeta potential (+\u0026thinsp;41.1 mV). These characteristics are highly desirable for nanoparticulate delivery systems, as smaller and uniformly distributed particles improve aerodynamic behavior for pulmonary deposition, while a high positive surface charge enhances colloidal stability and promotes interaction with negatively charged bacterial membranes. In contrast, other batches, though stable, exhibited lower entrapment or larger particle sizes, highlighting the importance of precise optimization.\u003c/p\u003e\u003cp\u003eThe in vitro drug release studies further validated these findings. While all formulations showed gradual drug release over 12 hours, Batch 7 exhibited the highest cumulative release (85.3%), confirming its ability to sustain therapeutic concentrations for extended durations. This feature is critical for reducing dosing frequency and maintaining effective drug levels in infected lung tissues. Antimicrobial assays also reinforced the superiority of Batch 7, which demonstrated the largest inhibition zones against \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (27 mm) and \u003cem\u003eEscherichia coli\u003c/em\u003e (37 mm). These enhanced outcomes are attributed to the synergistic antibacterial action of chitosan and Ciprofloxacin, where chitosan\u0026rsquo;s cationic nature facilitates bacterial membrane disruption, thereby enhancing antibiotic penetration.\u003c/p\u003e\u003cp\u003eThe comparison of all 12 formulations highlights a clear optimization trend, where the fine balance between chitosan and TPP concentrations directly influences nanoparticle morphology, entrapment, stability, and drug release. The fact that Batch 7 consistently outperformed all others confirms it as the most suitable formulation for pulmonary delivery applications.\u003c/p\u003e\u003cp\u003eTaken together, these results indicate that CIPRO-CSNPs not only enhance drug encapsulation and release profiles but also improve antibacterial activity compared to free Ciprofloxacin. The findings are in agreement with previous studies on polymeric nanocarriers for respiratory drug delivery, strengthening the evidence for chitosan as a multifunctional polymer that contributes to both stabilization and antimicrobial enhancement.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study successfully formulated and evaluated Ciprofloxacin-loaded chitosan nanoparticles across 12 batches using the ionic gelation technique. Comprehensive characterization revealed Batch 7 as the optimal formulation, with excellent entrapment efficiency (89.4%), smallest particle size (185 nm), and highest zeta potential (+\u0026thinsp;41.1 mV), ensuring stability, sustained release, and enhanced bioavailability. The in vitro release profile demonstrated prolonged drug release up to 12 h, while antimicrobial assays confirmed Batch 7\u0026rsquo;s superior activity against both \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003eEscherichia coli\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eThe superiority of Batch 7 underscores the importance of fine-tuning the chitosan-to-TPP ratio in nanoparticle systems to achieve maximum therapeutic benefits. By offering enhanced drug retention, improved pulmonary delivery, and stronger antibacterial efficacy compared to other formulations, Batch 7 establishes itself as the most promising candidate for clinical translation.\u003c/p\u003e\u003cp\u003eThese results suggest that CIPRO-CSNPs, particularly Batch 7, could represent a safe and effective nanocarrier platform for targeted pulmonary therapy in multidrug-resistant respiratory infections. Future work should involve in vivo pharmacokinetic studies, aerosolization efficiency, and toxicity profiling to confirm clinical applicability and expand the therapeutic potential of this delivery system.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e No Funding\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e: The authors have no conflict of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declaration:\u0026nbsp;\u003c/strong\u003enot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZoe, L., David, S., \u0026amp; Rajabalaya, R. (2023). 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Nanoparticles of chitosan loaded ciprofloxacin: fabrication and antimicrobial activity. \u003cem\u003eAdv Pharm Bull\u003c/em\u003e, \u003cem\u003e7\u003c/em\u003e(3), 427\u0026ndash;432. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.15171/apb.2017.051\u003c/span\u003e\u003cspan address=\"10.15171/apb.2017.051\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Ciprofloxacin, nanoparticles, Chitosan, Pulmonary, drug delivery, respiratory infections","lastPublishedDoi":"10.21203/rs.3.rs-8192679/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8192679/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eIntroduction:\u003c/h2\u003e\u003cp\u003eLower respiratory tract infections (LRTIs) remain a leading global health challenge, especially with the rise of multidrug-resistant (MDR) pathogens. Ciprofloxacin, although effective, has limitations when administered systemically. Chitosan, a biocompatible polymer, provides a promising platform for nanoparticle-based pulmonary delivery.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eNanoparticles were synthesized via ionic gelation using chitosan and sodium tripolyphosphate. twelve formulations varying in polymer and cross-linker concentrations were characterized for yield, entrapment efficiency, particle size, zeta potential, and in vitro drug release. Antimicrobial efficacy was tested against \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003eEscherichia coli\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eBatch 7 consistently outperformed all others, showing the highest entrapment efficiency (89.4%), smallest particle size (185 nm), and maximum zeta potential (+\u0026thinsp;41.1 mV). In vitro drug release of Batch 7 demonstrated sustained release up to 12 h with the highest cumulative release (85.3%), significantly exceeding other formulations. Antimicrobial testing confirmed superior inhibition zones (27 mm against \u003cem\u003eS. aureus\u003c/em\u003e and 37 mm against \u003cem\u003eE. coli\u003c/em\u003e), validating its enhanced therapeutic activity.\u003c/p\u003e\u003ch2\u003eDiscussion\u003c/h2\u003e\u003cp\u003eAmong 12 formulations, Batch 7 emerged as the most optimized CIPRO-CSNP, combining superior physicochemical stability with strong antibacterial efficacy.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThe findings highlight the potential of chitosan nanoparticles as an advanced pulmonary delivery system for combating resistant respiratory infections.\u003c/p\u003e","manuscriptTitle":"Targeted Pulmonary Delivery of Ciprofloxacin via Chitosan Nanoparticles for the Treatment of Lower Respiratory Tract Infections","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-08 11:58:10","doi":"10.21203/rs.3.rs-8192679/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"366d0df3-0117-4fa4-aa26-8d15562e5f82","owner":[],"postedDate":"December 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-29T07:24:55+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-08 11:58:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8192679","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8192679","identity":"rs-8192679","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}